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    Interferon-beta signaling in retinal mononuclear phagocytes attenuates pathological neovascularization. Lückoff Anika,Caramoy Albert,Scholz Rebecca,Prinz Marco,Kalinke Ulrich,Langmann Thomas EMBO molecular medicine Age-related macular degeneration (AMD) is a leading cause of vision loss among the elderly. AMD pathogenesis involves chronic activation of the innate immune system including complement factors and microglia/macrophage reactivity in the retina. Here, we show that lack of interferon-β signaling in the retina accelerates mononuclear phagocyte reactivity and promotes choroidal neovascularization (CNV) in the laser model of neovascular AMD Complete deletion of interferon-α/β receptor (Ifnar) using Ifnar1(-/-) mice significantly enhanced early microglia and macrophage activation in lesion areas. This triggered subsequent vascular leakage and CNV at later stages. Similar findings were obtained in laser-treated Cx3cr1(Cre) (ER):Ifnar1(fl/fl) animals that allowed the tamoxifen-induced conditional depletion of Ifnar in resident mononuclear phagocytes only. Conversely, systemic IFN-β therapy of laser-treated wild-type animals effectively attenuated microgliosis and macrophage responses in the early stage of disease and significantly reduced CNV size in the late phase. Our results reveal a protective role of Ifnar signaling in retinal immune homeostasis and highlight a potential use for IFN-β therapy in the eye to limit chronic inflammation and pathological angiogenesis in AMD. 10.15252/emmm.201505994
    Anti-Angiogenic and Anti-Scarring Dual Action of an Anti-Fibroblast Growth Factor 2 Aptamer in Animal Models of Retinal Disease. Matsuda Yusaku,Nonaka Yosuke,Futakawa Satoshi,Imai Hirotaka,Akita Kazumasa,Nishihata Toshiaki,Fujiwara Masatoshi,Ali Yusuf,Bhisitkul Robert B,Nakamura Yoshikazu Molecular therapy. Nucleic acids Currently approved therapies for age-related macular degeneration (AMD) are inhibitors against vascular endothelial growth factor (VEGF), which is a major contributor to the pathogenesis of neovascular AMD (nAMD). Intravitreal injections of anti-VEGF drugs have shown dramatic visual benefits for AMD patients. However, a significant portion of AMD patients exhibit an incomplete response to therapy and, over the extended management course, can lose vision, with the formation of submacular fibrosis as one risk factor. We investigated a novel target for AMD treatments, fibroblast growth factor 2 (FGF2), which has been implicated in the pathophysiology of both angiogenesis and fibrosis in a variety of tissue and organ systems. The anti-FGF2 aptamer, RBM-007, was examined for treatment of nAMD in animal models. In in vivo studies conducted in mice and rats, RBM-007 was able to inhibit FGF2-induced angiogenesis, laser-induced choroidal neovascularization (CNV), and CNV with fibrosis. Pharmacokinetic studies of RBM-007 in the rabbit vitreous revealed high and relatively long-lasting profiles that are superior to other approved anti-VEGF drugs. The anti-angiogenic and anti-scarring dual action of RBM-007 holds promise as an additive or alternative therapy to anti-VEGF treatments for nAMD. 10.1016/j.omtn.2019.07.018
    Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor, and Pigment Epithelium-Derived Factor Expression in the Neovascular Iris in Retinal Diseases. Miao Heng,Hou Xianru,Hwang De-Kuang,Tao Yong Journal of ophthalmology Objective:To determine the expression of cytokines in the iris of patients with neovascular glaucoma (NVG). Methods:Patients with NVG associated with proliferative diabetic retinopathy (PDR, group 1) or central retinal vein occlusion (CRVO, group 2) who had undergone surgical treatment were enrolled. Patients with primary open-angle glaucoma requiring surgical treatment were included in the control group (group 3). All iris specimens were obtained during trabeculectomy, 7 days after intravitreal injections of ranibizumab. The messenger RNA (mRNA) and protein levels of three target cytokines-vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF)-in the iris were analyzed and compared. Results:We included 39 eyes from 39 patients (12, 15, and 12 in groups 1, 2, and 3, resp.). The protein and mRNA levels of PEDF were higher in two NVG groups. The protein levels, but not mRNA level, of bFGF were higher in the two NVG groups. The protein and mRNA levels of VEGF were similar in the three groups. Conclusions:The protein level of bFGF increased in the irises of the NVG patients was not expressed by the iris itself, whereas PEDF may be expressed by the iris tissue in these patients. 10.1155/2018/8025951
    Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo. Ribatti D,Presta M,Vacca A,Ria R,Giuliani R,Dell'Era P,Nico B,Roncali L,Dammacco F Blood Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.
    Vascular endothelial growth factor a in intraocular vascular disease. Miller Joan W,Le Couter Jennifer,Strauss Erich C,Ferrara Napoleone Ophthalmology UNLABELLED:The vascular beds supplying the retina may sustain injury as a result of underlying disease such as diabetes, and/or the interaction of genetic predisposition, environmental insults, and age. The vascular pathologic features observed in different intraocular vascular diseases can be categorized broadly as proliferation, exemplified by proliferative diabetic retinopathy, leakage such as macular edema secondary to retinal vein occlusion, or a combination of proliferation and leakage, as seen in neovascular age-related macular degeneration (AMD). The World Health Organization has identified diabetic retinopathy and AMD as priority eye diseases for the prevention of vision loss in developed countries. The pathologic transformations of the retinal vasculature seen in intraocular vascular disease are associated with increased expression of vascular endothelial growth factor A (VEGF), a potent endothelial-specific mitogen. Furthermore, in model systems, VEGF alone is sufficient to trigger intraocular neovascularization, and its inhibition is associated with functional and anatomic improvements in the affected eye. Therapeutic interventions with effect on VEGF include intraocular capture and neutralization by engineered antibodies or chimeric receptors, downregulation of its expression with steroids, or alleviation of retinal ischemia, a major stimulus for VEGF expression, with retinal ablation by laser treatment. Data from prospective randomized clinical trials indicate that VEGF inhibition is a potent therapeutic strategy for intraocular vascular disease. These findings are changing clinical practice and are stimuli for further study of the basic mechanisms controlling intraocular angiogenesis. FINANCIAL DISCLOSURE(S):Proprietary or commercial disclosure may be found after the references. 10.1016/j.ophtha.2012.07.038
    Vascular endothelial growth factor and age-related macular degeneration: from basic science to therapy. Ferrara Napoleone Nature medicine 10.1038/nm1010-1107
    Auranofin Inhibits Retinal Pigment Epithelium Cell Survival through Reactive Oxygen Species-Dependent Epidermal Growth Factor Receptor/ Mitogen-Activated Protein Kinase Signaling Pathway. Chen Xiaodong,Tzekov Radouil,Su Mingyang,Hong Haiyan,Min Wang,Han Aidong,Li Wensheng PloS one Abnormal survival of retinal pigment epithelium (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR), a sight-threatening disease. In this study, we explored the effect of the anti-rheumatic agent auranofin (AF) on RPE cell survival and studied the underlying signaling mechanisms in vitro. Our results showed that AF inhibited ARPE-19 cell survival in a dose and time-dependent manner. Application of AF induced several effects: a significant decrease in total epidermal growth factor receptor (EGFR) and an increase in phosphorylated EGFR and mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), c-Jun, mitogen activated protein kinase activated protein kinase 2(MAPKAPK2), and heat shock protein 27 (HSP27). AF also inhibited epidermal growth factor (EGF)-dependent cell proliferation and migration through affecting EGFR/MAPK signaling. The antioxidant N-acetylcysteine (NAC) blocked the AF-induced increase of reactive oxygen species (ROS) production, the reduction of total EGFR, and the phosphorylation of multiple nodes in EGFR/MAPK signaling pathway. P38MAPK inhibitor SB203580, but not inhibitors of EGFR (erlotinib), ERK (FR180204) and JNK (SP600125), suppressed AF-induced phosphorylation of EGFR/p38MAPK/MAPKAPK2/Hsp27. In conclusion, the ROS-dependent phosphorylation of EGFR/MAPK is an important signaling pathway for AF-induced inhibition of RPE cell survival, and AF may have the potential for treatment of abnormal survival of RPE cells in PVR. 10.1371/journal.pone.0166386
    Interaction of palmitate and LPS regulates cytokine expression and apoptosis through sphingolipids in human retinal microvascular endothelial cells. Lu Zhongyang,Li Yanchun,Ru Ji Hyun,Lopes-Virella Maria F,Lyons Timothy J,Huang Yan Experimental eye research Studies have implicated saturated fatty acid (SFA) and lipopolysaccharide (LPS) in diabetic retinopathy. Since type 2 diabetes is associated with increases in both SFA and LPS in circulation, we investigated how SFA interacts with LPS to regulate proinflammatory cytokine expression and apoptosis in human retinal microvascular endothelial cells (HRMVECs) and the underlying mechanisms. HRMVECs were challenged with palmitate, a major SFA, LPS or palmitate plus LPS and the expression of proinflammatory cytokines were quantified using real-time PCR and enzyme-linked immunosorbent assay. The interaction between palmitate and LPS on inflammatory signaling and sphingolipid metabolism was demonstrated by immunoblotting and lipidomic analysis, respectively. The effect of palmitate and LPS on apoptosis was also studied by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and histone-associated DNA fragment assays. Results showed that palmitate robustly stimulated the expression of proinflammatory cytokines including interleukin (IL)-6 and IL-1β, and the combination of palmitate and LPS further upregulated the proinflammatory cytokines by cooperatively stimulating inflammatory signaling pathways. Results also showed that while palmitate stimulated ceramide (CER) production via CER de novo synthesis and sphingomyelin (SM) hydrolysis, addition of LPS further increased CER de novo synthesis, but not SM hydrolysis. The involvement of sphingolipids in the cooperative stimulation by palmitate and LPS on cytokine expression was indicated by the findings that the inhibitor of CER de novo synthesis or SM hydrolysis attenuated the stimulation of IL-6 expression by palmitate and LPS. In addition, our study showed that fatty acid receptors GPR40 and CD36 were involved in the IL-6 upregulation by palmitate and LPS. Furthermore, palmitate induced apoptosis via CER production, but addition of LPS did not further increase apoptosis. Taken together, this study showed that palmitate interacted with LPS to upregulate cytokine expression via free fatty acid receptor-mediated inflammatory signaling and sphingolipid metabolism in HRMVECs. In contrast, the interaction between palmitate and LPS did not further increase apoptosis. 10.1016/j.exer.2018.09.016
    Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization. Kumar Raj,Singh Nikhlesh K,Rao Gadiparthi N Scientific reports Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo. 10.1038/srep26480
    Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization. Lin Tzu-Wei,Chien Yueh,Lin Yi-Ying,Wang Mong-Lien,Yarmishyn Aliaksandr A,Yang Yi-Ping,Hwang De-Kuang,Peng Chi-Hsien,Hsu Chih-Chien,Chen Shih-Jen,Chien Ke-Hung International journal of molecular sciences Age-related macular degeneration (AMD) is the eye disease with the highest epidemic incidence, and has great impact on the aged population. Wet-type AMD commonly has the feature of neovascularization, which destroys the normal retinal structure and visual function. So far, effective therapy options for rescuing visual function in advanced AMD patients are highly limited, especially in wet-type AMD, in which the retinal pigmented epithelium and Bruch's membrane structure (RPE-BM) are destroyed by abnormal angiogenesis. Anti-VEGF treatment is an effective remedy for the latter type of AMD; however, it is not a curative therapy. Therefore, reconstruction of the complex structure of RPE-BM and controlled release of angiogenesis inhibitors are strongly required for sustained therapy. The major purpose of this study was to develop a dual function biomimetic material, which could mimic the RPE-BM structure and ensure slow release of angiogenesis inhibitor as a novel therapeutic strategy for wet AMD. We herein utilized plasma-modified polydimethylsiloxane (PDMS) sheet to create a biomimetic scaffold mimicking subretinal BM. This dual-surface biomimetic scaffold was coated with laminin and dexamethasone-loaded liposomes. The top surface of PDMS was covalently grafted with laminin and used for cultivation of the retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSC-RPE). To reach the objective of inhibiting angiogenesis required for treatment of wet AMD, the bottom surface of modified PDMS membrane was further loaded with dexamethasone-containing liposomes via biotin-streptavidin linkage. We demonstrated that hiPSC-RPE cells could proliferate, express normal RPE-specific genes and maintain their phenotype on laminin-coated PDMS membrane, including phagocytosis ability, and secretion of anti-angiogenesis factor PEDF. By using in vitro HUVEC angiogenesis assay, we showed that application of our membrane could suppress oxidative stress-induced angiogenesis, which was manifested in decreased secretion of VEGF by RPE cells and suppression of vascularization. In conclusion, we propose modified biomimetic material for dual delivery of RPE cells and liposome-enveloped dexamethasone, which can be potentially applied for AMD therapy. 10.3390/ijms20020241
    Molecular Mechanisms Mediating Antiangiogenic Action of the Urokinase Receptor-Derived Peptide UPARANT in Human Retinal Endothelial Cells. Motta Carla,Lupo Gabriella,Rusciano Dario,Olivieri Melania,Lista Liliana,De Rosa Mario,Pavone Vincenzo,Anfuso Carmelina Daniela Investigative ophthalmology & visual science Purpose:To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. Methods:The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. Results:UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. Conclusions:These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A. 10.1167/iovs.16-19909
    Endothelial Robo4 suppresses breast cancer growth and metastasis through regulation of tumor angiogenesis. Zhao Helong,Ahirwar Dinesh K,Oghumu Steve,Wilkie Tasha,Powell Catherine A,Nasser Mohd W,Satoskar Abhay R,Li Dean Y,Ganju Ramesh K Molecular oncology Targeting tumor angiogenesis is a promising alternative strategy for improvement of breast cancer therapy. Robo4 (roundabout homolog 4) signaling has been shown to protect endothelial integrity during sepsis shock and arthritis, and inhibit Vascular Endothelial Growth Factor (VEGF) signaling during pathological angiogenesis of retinopathy, which indicates that Robo4 might be a potential target for angiogenesis in breast cancer. In this study, we used immune competent Robo4 knockout mouse model to show that endothelial Robo4 is important for suppressing breast cancer growth and metastasis. And this effect does not involve the function of Robo4 on hematopoietic stem cells. Robo4 inhibits breast cancer growth and metastasis by regulating tumor angiogenesis, endothelial leakage and tight junction protein zonula occludens protein-1 (ZO-1) downregulation. Treatment with SecinH3, a small molecule drug which deactivates ARF6 downstream of Robo4, can enhance Robo4 signaling and thus inhibit breast cancer growth and metastasis. SecinH3 mediated its effect by reducing tumor angiogenesis rather than directly affecting cancer cell proliferation. In conclusion, endothelial Robo4 signaling is important for suppressing breast cancer growth and metastasis, and it can be targeted (enhanced) by administrating a small molecular drug. 10.1016/j.molonc.2015.10.007
    Potential role of Peroxisome Proliferator Activated Receptor gamma analogues in regulation of endothelial progenitor cells in diabetes mellitus: An overview. Chhabra Manik,Sharma Saurabh Diabetes & metabolic syndrome Endothelial progenitor cells are recognized as the potential targets for the revascularization and angiogenesis because of their ability to get themselves transformed into mature endothelial cells. Underlying pathophysiology in diabetes mellitus leads to decrease in circulatory endothelial progenitor cells, resulting in diabetic macro-vascular and micro-vascular complications. Peroxisome Proliferator Activated Receptor (PPAR) gamma analogues serves as an effective therapy for controlling blood sugar levels and preventing its complications. Reports of clinical trials and meta-analysis of clinical trial suggests the beneficial aspects of PPAR gamma therapy in increasing the number and function of circulating endothelial progenitor cells. This review highlights the pleotropic effect of PPAR gamma analogs, apart from their antidiabetic action via reduction of oxidative stress, increasing expression of eNOS, reducing level of miR 22, miR 222 levels and positive modulation of rapamycin/Protein kinase B/phosphoinoside3-kinase pathways, preventing the early apoptosis, enhanced mobility proliferation and transformation into mature endothelial cells. PPAR gamma therapy in diabetes regulates endothelial progenitor cells, reduces complications of diabetes like retinopathy, nephropathy, neuropathy, cardiomyopathy, deep vein thrombosis, and maintains the healthy vasculature. 10.1016/j.dsx.2019.01.036
    Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation. Bruning Ulrike,Morales-Rodriguez Francisco,Kalucka Joanna,Goveia Jermaine,Taverna Federico,Queiroz Karla C S,Dubois Charlotte,Cantelmo Anna Rita,Chen Rongyuan,Loroch Stefan,Timmerman Evy,Caixeta Vanessa,Bloch Katarzyna,Conradi Lena-Christin,Treps Lucas,Staes An,Gevaert Kris,Tee Andrew,Dewerchin Mieke,Semenkovich Clay F,Impens Francis,Schilling Birgit,Verdin Eric,Swinnen Johannes V,Meier Jordan L,Kulkarni Rhushikesh A,Sickmann Albert,Ghesquière Bart,Schoonjans Luc,Li Xuri,Mazzone Massimiliano,Carmeliet Peter Cell metabolism The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade. 10.1016/j.cmet.2018.07.019
    Loss of Estrogen-Related Receptor Alpha Facilitates Angiogenesis in Endothelial Cells. Likhite Neah,Yadav Vikas,Milliman Eric J,Sopariwala Danesh H,Lorca Sabina,Narayana Nithya P,Sheth Megha,Reineke Erin L,Giguère Vincent,Narkar Vihang Molecular and cellular biology Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here, we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multifaceted role in vasculature. Of the three ERR subtypes, ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration, and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation but increased migration and tube formation. ERRα depletion increased basal as well as vascular endothelial growth factor A (VEGFA)- and ANG1/2-stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to that in wild-type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets, such as metabolism, mitochondrial biogenesis, and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory, and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, proangiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis and potentially fine-tunes growth factor-mediated angiogenesis. 10.1128/MCB.00411-18
    Joint Antiangiogenic Effect of ATN-161 and Anti-VEGF Antibody in a Rat Model of Early Wet Age-Related Macular Degeneration. Wang Wen-Qiu,Wang Feng-Hua,Qin Wen-Xin,Liu Hai-Yun,Lu Bing,Chung Christopher,Zhu Jie,Gu Qing,Shi William,Wen Cindy,Wu Frances,Zhang Kang,Sun Xiao-Dong Molecular pharmaceutics The wet form of age-related macular degeneration (AMD) is a leading cause of blindness among elderly Americans and is characterized by abnormal vessel growth, termed choroidal neovascularization (CNV). Integrin α5β1 is a transmembrane receptor that binds matrix macromolecules and proteinases to stimulate angiogenesis. We recently demonstrated that integrin α5β1 plays a critical role in the development of choroidal neovascularization. In this study, we determined the role and underlying mechanisms of integrin α5β1 in angiogenesis in human choroidal endothelial cells and evaluated the antiangiogenic effects of delivering a combination therapy of ATN-161, an integrin α5β1 inhibitor, and an anti-VEGF monoclonal antibody to rats with laser-induced CNV. Vascular endothelial growth factor (VEGF) is a signaling protein that stimulates vasculogenesis and angiogenesis through a pathway that is distinct from the integrin α5β1 signaling pathway. Our results indicate that fibronectin binds to integrin α5β1 and synergizes VEGF-induced angiogenesis via two independent signaling pathways, FN/integrin α5β1/FAK/ERK1/2 and FN/integrin α5β1/FAK/AKT. Integrin α5 knockdown by shRNA inhibits endothelial cell migration, tube formation, and proliferation, while ATN-161 only partially decreases integrin α5 function. Treatment with ATN-161 combined with anti-VEGF antibody showed joint effects in attenuating angiogenesis. In summary, our results provide the first evidence for the mechanisms by which integrin α5β1 is involved in ocular pathological neovascularization in vivo, suggesting that dual inhibition of integrin α5β1 and VEGF may be a promising novel therapeutic strategy for CNV in wet AMD. 10.1021/acs.molpharmaceut.6b00056
    Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration. Wang Haibo,Han Xiaokun,Kunz Eric,Hartnett M Elizabeth Investigative ophthalmology & visual science Purpose:This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV). Methods:Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA. Results:Thy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin-labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC-induced Rac1 and Src activation. Conclusions:Thy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted. 10.1167/iovs.16-19691
    MicroRNA-148a/b-3p regulates angiogenesis by targeting neuropilin-1 in endothelial cells. Kim Hyejeong,Ko Yeongrim,Park Hyojin,Zhang Haiying,Jeong Yoonjeong,Kim Yeomyeong,Noh Minyoung,Park Songyi,Kim Young-Myeong,Kwon Young-Guen Experimental & molecular medicine MicroRNAs (miRs) are crucial regulators of vascular endothelial cell (EC) functions, including migration, proliferation, and survival. However, the role of most miRs in ECs remains unknown. Using RNA sequencing analysis, we found that miR-148a/b-3p expression was significantly downregulated during the differentiation of umbilical cord blood mononuclear cells into outgrowing ECs and that decreased miR-148a/b-3p levels were closely related to EC behavior. Overexpression of miR-148a/b-3p in ECs significantly reduced migration, filamentous actin remodeling, and angiogenic sprouting. Intriguingly, the effects of decreased miR-148a/b-3p levels were augmented by treatment with vascular endothelial growth factor (VEGF). Importantly, we found that miR-148a/b-3p directly regulated neuropilin-1 (NRP1) expression by binding to its 3'-untranslated region. In addition, because NRP1 is the coreceptor for VEGF receptor 2 (VEGFR2), overexpression of miR-148a/b-3p inhibited VEGF-induced activation of VEGFR2 and inhibited its downstream pathways, as indicated by changes to phosphorylated focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase. Collectively, our results demonstrate that miR-148a/b-3p is a direct transcriptional regulator of NRP1 that mediates antiangiogenic pathways. These data suggest that miR-148a/b-3p is a therapeutic candidate for overcoming EC dysfunction and angiogenic disorders, including ischemia, retinopathy, and tumor vascularization. 10.1038/s12276-019-0344-x
    A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies. Asokan Priyadarsini,Mitra Rajendra N,Periasamy Ramesh,Han Zongchao,Borrás Teresa Investigative ophthalmology & visual science Purpose:Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Methods:Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. Results:The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. Conclusions:The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally. 10.1167/iovs.17-22992
    Secretogranin III as a novel target for the therapy of choroidal neovascularization. LeBlanc Michelle E,Wang Weiwen,Ji Yanli,Tian Hong,Liu Dachuan,Zhang Xuxiang,Li Wei Experimental eye research Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV. 10.1016/j.exer.2019.01.009
    Strand and Cell Type-specific Function of microRNA-126 in Angiogenesis. Zhou Qinbo,Anderson Chastain,Hanus Jakub,Zhao Fangkun,Ma Jing,Yoshimura Akihiko,Wang Shusheng Molecular therapy : the journal of the American Society of Gene Therapy microRNAs or miRs have been shown to be pivotal modulators of vascular development. The strand and cell type-specific function of miR-126 in angiogenesis, especially pathological angiogenesis, remains poorly defined. We characterized the retinal vascular phenotype of miR-126 mice, and tested the function of miR-126 strands (miR-126-3p and -5p) using in vitro angiogenesis models and a mouse model of neovascular age-related macular degeneration. We found that miR-126 is critical for retinal vascular development but has dual function in pathological angiogenesis. miR-126 mice showed defective postnatal retinal vascular development and remodeling, which is partially rescued by genetic knockout of its target gene Spred-1. Surprisingly, either silencing miR-126-3p by LNA-antimiR or overexpressing miR-126-3p by miRNA mimic repressed laser-induced choroidal neovascularization. To dissect the underlying mechanism, we found in endothelial cells, silencing of miR-126-3p repressed angiogenesis, while overexpression of miR-126-5p enhanced angiogenesis. However, in retinal pigment epithelial cells, miR-126-3p repressed vascular endothelial growth factor (VEGF-A) expression via a novel mechanism of regulating αB-Crystallin promoter activity and by directly targeting VEGF-A 3'-untranslated region. These findings provide first genetic evidence that miR-126 is required for the development of different retinal vascular layers, and also uncover a strand and cell type-specific function of miR-126 in ocular pathological angiogenesis. 10.1038/mt.2016.108
    Targeting eIF4E inhibits growth, survival and angiogenesis in retinoblastoma and enhances efficacy of chemotherapy. Wang Genguo,Li Zhi,Li Zhuojun,Huang Yi,Mao Xiaochun,Xu Chang,Cui Sha Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Although the eukaryotic translation initiation factor 4E (eIF4E) has been shown to be critically involved in the transformation and progression of various tumors, little is known about the role of eIF4E in retinoblastoma. In this work, we report that ribavirin, a pharmacologic inhibitor of eIF4E function, effectively targets retinoblastoma and angiogenesis. Ribavirin treatment dose-dependently blocked the growth and stimulated apoptosis in various retinoblastoma cell lines, with IC values that are within the clinically achievable range. Ribavirin also significantly inhibited angiogenesis via disrupting capillary network formation and suppressing VEGF-induced migration, proliferation and survival of human retinal endothelial cells. In addition, ribavirin significantly augments chemotherapy agent's inhibitory effects in retinoblastoma in vitro and in vivo. Mechanistically, ribavirin inhibited eIF4E function in retinoblastoma cells as shown by the decreased protein levels of Cyclin D1, c-Myc and VEGF without affecting their mRNA expression. Overexpression of the wildtype and phosphormimetic but not the nonphosphorylatable form of eIF4E significantly abolished the inhibitory effects of ribavirin, further demonstrating eIF4E as the target of ribavirin. Genetic knockdown of eIF4E using two independent siRNAs mirrored ribavirin's effects, confirming the role of eIF4E in retinoblastoma growth, survival and response to chemotherapy. Our findings provide a preclinical rationale to explore ribavirin as a strategy to treat retinoblastoma and highlight the therapeutic value of targeting eIF4E in retinoblastoma. 10.1016/j.biopha.2017.10.034
    High-glucose treatment regulates biological functions of human umbilical vein endothelial cells via Sirt1/FOXO3 pathway. Chen Yihui,Wang Yan,Jiang Yaping,Zhang Xiaoyan,Sheng Minjie Annals of translational medicine Background:Hyperglycaemia-induced angiogenesis plays an important role in diabetic retinopathy (DR). This study aimed to investigate the role of sirtuin1 (Sirt1)/forkhead box O3 (FOXO3) pathway in the effects of high-glucose on human umbilical vein endothelial cells (HUVECs). Methods:HUVECs were divided into normal control group (5 mM glucose), high glucose group (30 mM), 30 mM glucose + shsirt1 group, 30 mM glucose + Sirt1 over-expression group (30 mM + Sirt1), 30 mM glucose + Sirt1 agonist SRT group, 30 mM glucose + SRT + FOXO3 silencing group (30 mM + SRT + siFOXO3). Cell proliferation, migration, invasion and apoptosis were determined. Results:High glucose treatment reduced the expression of Sirt1 and FOXO3 in HUVECs. However, Sirt1 over-expression or SRT attenuated the high-glucose-induced inhibition of HUVEC proliferation and migration as well as reduced their apoptosis. In contrast, Sirt1 silencing deteriorated the high-glucose induced inhibition of HUVEC proliferation and migration and further increased HUVEC apoptosis. FOXO3 expression increased with the increase in Sirt1 expression, which was accompanied by enhanced cellular functions. These were abolished after FOXO3 silencing. In addition, Sirt1/FOXO3 regulated HUVEC activities via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Conclusions:Sirt1/FOXO3 pathway is essential for the survival of endothelial cells under high-glucose and plays an important role in the development of diabetes-induced retinal vascular endothelial injury. 10.21037/atm.2019.04.29
    [Uveal Melanoma Cell Under Oxidative Stress - Influence of VEGF and VEGF-Inhibitors]. Dithmer M,Kirsch A M,Gräfenstein L,Wang F,Schmidt H,Coupland S E,Fuchs S,Roider J,Klettner A K Klinische Monatsblatter fur Augenheilkunde BACKGROUND:The role of oxidative stress in cancer is complex. While the pathological alterations induced by oxidative stress may be involved in the induction of tumours, in the late stages of tumour development, it can facilitate the loss of tumour cells and might even prevent metastasis. Tumour cells show metabolic alterations, often inducing an increased production of reactive oxygen species, which makes these cells particularly vulnerable to additional oxidative stress. This is an important mode of action in the use of many chemotherapeutics and in the application of ionizing radiation. Uveal melanoma is the most frequent primary tumour in the adult eye. For metastasis of this tumour, which affects about 50 % of the patients, no appropriate treatment is currently available. However, the primary tumour can efficiently be treated with ionizing radiation. A frequent side effect of this treatment is radiation retinopathy, which is treated with vascular endothelial growth factor (VEGF) antagonists. A therapy of the primary tumour with VEGF antagonists is under discussion. So far, little data is available on this subject, however, a paradoxical worsening of the situation has been found in a mouse model of uveal melanoma treated with bevacizumab. METHODS:We have investigated the effect of VEGF and of the VEGF-antagonist bevacizumab on the survival of five different melanoma cell lines under oxidative stress treatment with hydrogen peroxide. In addition, we investigated the expression of relevant proteins and the effect of bevacizumab on the proliferation of the cells as well as its effect on the angiogenic behaviour of endothelial cells, co-cultured with uveal melanoma cells. RESULTS:Our study showed that not only VEGF but also, paradoxically, the VEGF-antagonist bevacizumab is able to protect uveal melanoma cells from oxidative stress-induced cell death. Bevacizumab did not influence the proliferation of the cells and showed only limited effectiveness to reduce angiogenic structures. CONCLUSION:Considering that oxidative stress is the mode of action for ionizing radiation to induce cell death, a protective effect of bevacizumab on uveal melanoma cells against oxidative stress is worrisome and argues against the use of VEGF in uveal melanoma. 10.1055/s-0043-103002
    Targeted Disruption of JCAD (Junctional Protein Associated With Coronary Artery Disease)/KIAA1462, a Coronary Artery Disease-Associated Gene Product, Inhibits Angiogenic Processes In Vitro and In Vivo. Hara Tetsuya,Monguchi Tomoko,Iwamoto Noriko,Akashi Masaya,Mori Kenta,Oshita Toshihiko,Okano Mitsumasa,Toh Ryuji,Irino Yasuhiro,Shinohara Masakazu,Yamashita Yui,Shioi Go,Furuse Mikio,Ishida Tatsuro,Hirata Ken-Ichi Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Recent genome-wide association studies newly identified the human KIAA1462 gene as a new locus for coronary artery disease. However, the function of the gene product, named JCAD (junctional protein associated with coronary artery disease), is unknown. Because JCAD is expressed at cell-cell junctions in endothelial cells, we hypothesized and tested whether JCAD regulates angiogenic processes in vitro and in vivo. APPROACH AND RESULTS:Cell culture experiments revealed impaired angiogenic ability (proliferation, migration, and cord formation) by the knockdown of JCAD with siRNA (<0.05 versus control siRNA). We have generated mice lacking JCAD (mKIAA1462) by gene-targeted deletion of JCAD to address in vivo angiogenic function. mKIAA1462 mice did not show morphological differences in development of retinal vasculature. Ex vivo aortic ring model demonstrated impaired neovascularization in aorta from mKIAA1462 mice than control wild-type mice (<0.05). Tumor growth was assessed by monitoring tumor volume after the subcutaneous injection of melanoma, LLC (Lewis lung carcinoma), and E0771 cells into the mice. mKIAA1462 mice exhibited significantly smaller tumor volume compared with wild-type mice (<0.001). Histological assessment of the tumor exhibited less smooth muscle actin-positive neovascularization determined by CD31-positive vascular structure in tumor of mKIAA1462 mice than wild-type mice, indicating that knockdown of JCAD inhibited the vascular maturation in pathological angiogenic process. CONCLUSIONS:These in vitro and in vivo studies suggest that JCAD has a redundant functional role in physiological angiogenesis but serves a pivotal role in pathological angiogenic process after birth. 10.1161/ATVBAHA.117.309721
    High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression. Farnoodian Mitra,Halbach Caroline,Slinger Cassidy,Pattnaik Bikash R,Sorenson Christine M,Sheibani Nader American journal of physiology. Cell physiology Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis. 10.1152/ajpcell.00001.2016
    Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis. Zahra Fatema Tuz,Sajib Md Sanaullah,Ichiyama Yusuke,Akwii Racheal Grace,Tullar Paul E,Cobos Christopher,Minchew Shelby A,Doçi Colleen L,Zheng Yi,Kubota Yoshiaki,Gutkind J Silvio,Mikelis Constantinos M Scientific reports Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting. 10.1038/s41598-019-48053-z
    Anti-VEGF Therapy for Neovascular AMD and Polypoidal Choroidal Vasculopathy. Cheung Gemmy Chui Ming,Lai Timothy Y Y,Gomi Fumi,Ruamviboonsuk Paisan,Koh Adrian,Lee Won Ki Asia-Pacific journal of ophthalmology (Philadelphia, Pa.) Anti-vascular endothelial growth factor (anti-VEGF) therapy has revolutionized the treatment of neovascular age-related macular degeneration (AMD). This review will summarize the current evidence of anti-VEGF therapy in neovascular AMD, including subtypes of retinal angiomatous proliferation and polypoidal choroidal vasculopathy (PCV). Importantly, 2 large multicenter randomized clinical trials evaluating the safety and efficacy of anti-VEGF monotherapy and combination with photodynamic therapy (PDT) have recently reported initial first-year outcomes. In this review, we summarize the latest updates in the efficacy and safety of anti-VEGF monotherapy and combination with PDT in common lesion subtypes. Remaining gaps in current understanding are highlighted where further research is needed. 10.22608/APO.2017260
    Low-Density Lipoprotein Receptor-Related Protein-1 Signaling in Angiogenesis. Mao Hua,Xie Liang,Pi Xinchun Frontiers in cardiovascular medicine Low-density lipoprotein receptor-related protein-1 (LRP1) plays multifunctional roles in lipid homeostasis, signaling transduction, and endocytosis. It has been recognized as an endocytic receptor for many ligands and is involved in the signaling pathways of many growth factors or cytokines. Dysregulation of LRP1-dependent signaling events contributes to the development of pathophysiologic processes such as Alzheimer's disease, atherosclerosis, inflammation, and coagulation. Interestingly, recent studies have linked LRP1 with endothelial function and angiogenesis, which has been underappreciated for a long time. During zebrafish embryonic development, LRP1 is required for the formation of vascular network, especially for the venous development. LRP1 depletion in the mouse embryo proper leads to angiogenic defects and disruption of endothelial integrity. Moreover, in a mouse oxygen-induced retinopathy model, specific depletion of LRP1 in endothelial cells results in abnormal development of neovessels. These loss-of-function studies suggest that LRP1 plays a pivotal role in angiogenesis. The review addresses the recent advances in the roles of LRP1-dependent signaling during angiogenesis. 10.3389/fcvm.2017.00034
    Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy. Park Hongryeol,Yamamoto Hiroyuki,Mohn Lucas,Ambühl Lea,Kanai Kenichi,Schmidt Inga,Kim Kee-Pyo,Fraccaroli Alessia,Feil Silke,Junge Harald J,Montanez Eloi,Berger Wolfgang,Adams Ralf H Nature communications Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR. 10.1038/s41467-019-13220-3
    Bradykinin alleviates DR retinal endothelial injury by regulating HMGB-1/NF-κB pathway. Zhu Y,Li X-Y,Wang J,Zhu Y-G European review for medical and pharmacological sciences OBJECTIVE:Diabetic retinopathy (DR) is one of the most important complications of diabetes (DM) and the leading cause of blindness in adults. Bradykinin (BK) is involved in several pathophysiological processes, such as inflammation, pain, cell proliferation, and tumors. It plays a crucial role in corneal epithelial cells, corneal stromal cells, and fibroblasts. However, the role of BK in DR retinal endothelial injury remains unclear. PATIENTS AND METHODS:Human retinal microvascular endothelial cells (hRECs) were cultured in vitro and randomly divided into 3 groups, control group in which hRECs were cultured in normal glucose concentration, high glucose group in which hRECs were cultured in the presence of high glucose, and BK group in which hRECs were cultured in the presence of high glucose with 1 μM BK. The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) was used to detect cell proliferation. Caspase-3 activity was adopted to detect Caspase-3 activity in hRECs. The colorimetric method was selected to determine lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and ROS content. Western blot was used to test HMGB-1/NF-κB and vascular endothelial growth factor (VEGF) expression changes. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion of inflammatory factor tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). RESULTS:In the presence of high glucose, hRECs cells proliferation was significantly reduced, Caspase-3 activity was enhanced, LDH and ROS levels were increased, SOD activity was declined with increased expression of HMGB-1, NF-κB, VEGF, as well as secretion of TNF-α and IL-1β compared with control group (p < 0.05). BK significantly inhibited the proliferation of hRECs cells, enhanced Caspase-3 activity, decreased the content of LDH and ROS, increased SOD activity, reduced the expressions of HMGB-1 and NF-κB protein, attenuated the expression of VEGF, and restrained the secretion of TNF-α and IL-1β compared with high glucose group (p < 0.05). CONCLUSIONS:BK can inhibit the growth and proliferation of retinal endothelial cells by regulating HMGB-1/NF-κB signaling pathway, attenuating oxidative stress and inflammation, thereby delaying DR development and progress. 10.26355/eurrev_201907_18286
    Effect of nanoencapsulation using poly (lactide-co-glycolide) (PLGA) on anti-angiogenic activity of bevacizumab for ocular angiogenesis therapy. Zhang Xiao-Pei,Sun Jian-Guo,Yao Jin,Shan Kun,Liu Bai-Hui,Yao Mu-Di,Ge Hui-Min,Jiang Qin,Zhao Chen,Yan Biao Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Antibody-based therapy is an effective strategy for treating ocular angiogenesis. However, short-acting efficacy and poor treatment compliance usually occurs in clinical practices. Thus, it is required to develop a drug delivery system to improve the bioavailability and decrease the toxicity of anti-angiogenic antibody. Bevacizumab is a recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGF). In this study, bevacizumab was encapsulated into poly (lactide-co-glycolide) (PLGA) nanoparticles. PLGA encapsulation could prolong the residency of bevacizumab in the vitreous and aqueous humor and produce long-lasting drug concentrations. Bevacizumab-encapsulated PLGA had no significant cytotoxicity and tissue toxicity effect in vitro and in vivo. In vitro studies showed that bevacizumab-encapsulated PLGA was more effective than bevacizumab in inhibiting VEGF-mediated endothelial cell proliferation, migration and tube formation. In vivo studies showed that bevacizumab-encapsulated PLGA enhanced the anti-angiogenic efficiency of bevacizumab for treating corneal neovascularization and retinal neovascularization. Thus, bevacizumab-encapsulated PLGA could increase the bioavailability and decrease the toxicity of bevacizumab during ocular angiogenesis therapy. 10.1016/j.biopha.2018.08.092
    Expression and effect of proline hydroxylase domain 2 in retina of diabetic rats. Li Zhen,Xing Yi-Qiao,Cui Wei,Lu Qiang International journal of ophthalmology AIM:To observe the expression of proline hydroxylase domain 2 (PHD2) in the retina of diabetic rats and investigate the relationship between PHD2 and relevant intraocular vascular proliferation factors. METHODS:Sixty male specific pathogen free (SPF) Sprague-Dawley (SD) rats were randomly divided into two groups: the diabetic group and the control group. The rats in the diabetic group were intraperitoneally injected with 60 mg/kg (0.60 mL/100g) of streptozotocin to induce a diabetic rat model. The rats in the control group were injected with an equal volume of sodium citrate buffer solution by the same method. Hematoxylin-eosin (HE) staining and immumofluorescence (IF) method were adopted to observe the pathological changes of retinal tissues and the expression of PHD2, glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF) by 8wk. RT-PCR method was applied to detect the expressions of mRNA of PHD2, VEGF and GFAP. The relationship between PHD2 and other vascular proliferation factors was analyzed. RESULTS:HE staining showed that there was the retinal tissue edema in the diabetic group, and the arrangement was in disorder, and proliferation could be seen. IF staining: in the retina of normal rats, PHD2 was not expressed, GFAP and VEGF were mainly expressed in astrocytes; while in the diabetic rats, PHD2, GFAP and VEGF staining showed strong positivity in all retinal layers, mainly in neurogliocytes. PHD2 was co-expressed with VEGF and GFAP. The mRNA expression levels of PHD2, GFAP and VEGF in the diabetic group were obviously higher than that in the control group,respectively 1.83 times, 1.75 times and 2.08 times. The difference had statistical significance (P<0.01). CONCLUSION:The high expression of PHD2 in the retina of early-stage diabetic rats might result from secretion of neurogliocytes induced by local high-concentration blood glucose, thus promoting the expression of VEGF and GFAP. PHD2 plays an important role during the occurrence of diabetic retinopathy. 10.18240/ijo.2016.03.05
    Gold Nanocrystals with Well-Defined Crystallographic {111} Facets Suppress Pathological Neovascularization. Jo Dong Hyun,Hong Jong Wook,Kim Jin Hyoung,Han Sang Woo,Kim Jeong Hun Journal of biomedical nanotechnology Vascular endothelial growth factor (VEGF) is a main factor in pathological neovascularization in various human diseases including age-related macular degeneration, cancer, and diabetic complications. Interestingly, gold nanospheres are known to bind to VEGF and to suppress VEGF-mediated angiogenesis. The anti-angiogenic effects are known to be governed by the size and surface charge of the nanoparticles. However, studies on the role of the shape in biological actions are limited. In this study, we investigate the anti-angiogenic properties of nanocrystals that have well-defined crystallographic {111} facets. Single-crystalline icosahedral and octahedral gold nanocrystals effectively scavenge VEGF just as nanospheres with similar diameter. In addition, they suppress the in vitro VEGF-induced activation of the VEGF receptor and the proliferation of endothelial cells. They also significantly inhibit in vivo VEGF-mediated retinal vascular permeability. These results thus suggest that gold nanocrystals with {111} facets can provide a useful platform for nanoparticle-based treatment of VEGF-driven pathological neovascularization beyond their current optical and catalytic applications.
    Nuclear FAK and its kinase activity regulate VEGFR2 transcription in angiogenesis of adult mice. Sun Shaogang,Wu Hsin-Jung,Guan Jun-Lin Scientific reports Focal adhesion kinase (FAK) is essential in embryonic angiogenesis by regulating endothelial cell (EC) survival and barrier functions through its kinase-independent and -dependent activities. Here, we generated EC-specific tamoxifen-inducible FAK knockout and FAK kinase-defective (KD) mutant knockin mice to investigate the role of FAK and its kinase activity in angiogenesis of adult animals. Unlike previous observations of their differential defects in embryonic vascular development, both FAK ablation and inactivation of its kinase activity resulted in deficient angiogenesis in wound-healing as well as retinal angiogenesis models. Consistent with these phenotypes, loss of FAK or its kinase activity decreased EC proliferation and migration to similar extents, suggesting FAK primarily acts as a kinase for the regulation of adult EC-mediated angiogenesis. Further mechanistic analyses were carried out using an established mouse EC line MS1 cells. Interestingly, we found that FAK regulated the expression of VEGFR2, a central mediator of various EC functions and angiogenesis, which requires both FAK kinase activity and its translocation into the nucleus. Moreover, nuclear FAK was detected in the RNA polymerase II complex associated with VEGFR2 promoter, suggesting its direct participation in the transcriptional regulation of VEGFR2. Together, our results provide significant insights into the signaling mechanisms of FAK in angiogenesis that may contribute to future design of more effective angiogenesis related therapy. 10.1038/s41598-018-20930-z
    Long noncoding RNA SNHG7 inhibits high glucose-induced human retinal endothelial cells angiogenesis by regulating miR-543/SIRT1 axis. Ke Ning,Pi Lian-Hong,Liu Qing,Chen Lin Biochemical and biophysical research communications Diabetic retinopathy (DR) is the serious complication of type 2 diabetes mellitus, which could lead to visual impairment. Growing evidence have revealed the involvement of long non-coding RNAs (lncRNAs) in the pathogenesis of DR. Thus, this study was performed to investigate the role of lncRNA SNHG7 (small nucleolar RNA host gene 7) in high glucose (HG)-induced proliferation, migration, and angiogenesis of human retinal endothelial cells (hRECs). We discovered that SNHG7 was decreased in hRECs under HG stimuli. Although SNHG7 had no influence on cell viability, migration and angiogenesis under condition, overexpression of SNHG7 inhibited the HG-induced cell proliferation, migration and angiogenesis, as well as vascular endothelial growth factor (VEGF) expression in HG condition. In terms of mechanism, we found that SNHG7 directly inhibited miR-543, which targeted the 3'-UTR of Silent information regulator T1 (SIRT1) mRNA and subsequently downregulated the VEGF expression in hRECs. Ultimately, upregulation of miR-543 or inhibition of SIRT1 both abrogated the effect of SNHG7 on HG-induced angiogenesis. Collectively, our results suggested that SNHG7 is a potential molecular target for attenuating HG-induced angiogenesis in the DR through regulation of the miR-543-mediated SIRT1/VEGF pathway. 10.1016/j.bbrc.2019.04.141
    Endothelial Cells Require CD98 for Efficient Angiogenesis-Brief Report. Liao Zhongji,Cantor Joseph M Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:CD98 regulates integrin signaling and is critical for tumor cell proliferation. It is also expressed on endothelial cells (EC), but its role in angiogenesis is unclear. APPROACH AND RESULTS:We used specific genetic targeting and antibody blockade approaches to examine the function of CD98 in EC proliferation, blood vessel growth, and tumor angiogenesis. It is upregulated on angiogenic ECs, and EC-specific deletion of CD98 in mice inhibited tumor growth, retinal angiogenesis, and EC proliferation. Reconstitution with CD98 mutants showed that integrin and CD98 interaction is necessary for EC survival and growth. Moreover, anti-CD98 treatment inhibited vessel formation and reversed EC-assisted tumor growth. CONCLUSIONS:Our findings demonstrate a requirement for CD98 in EC growth and suggest that CD98-specific reagents could have a dual anticancer effect: directly by inhibiting tumor cell proliferation and indirectly by preventing tumor angiogenesis. 10.1161/ATVBAHA.116.308335
    A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent. Gioelli Noemi,Maione Federica,Camillo Chiara,Ghitti Michela,Valdembri Donatella,Morello Noemi,Darche Marie,Zentilin Lorena,Cagnoni Gabriella,Qiu Yaqi,Giacca Mauro,Giustetto Maurizio,Paques Michel,Cascone Ilaria,Musco Giovanna,Tamagnone Luca,Giraudo Enrico,Serini Guido Science translational medicine Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis. 10.1126/scitranslmed.aah4807
    Regenerative Effects of Heme Oxygenase Metabolites on Neuroinflammatory Diseases. Lee Huiju,Choi Yoon Kyung International journal of molecular sciences Heme oxygenase (HO) catabolizes heme to produce HO metabolites, such as carbon monoxide (CO) and bilirubin (BR), which have gained recognition as biological signal transduction effectors. The neurovascular unit refers to a highly evolved network among endothelial cells, pericytes, astrocytes, microglia, neurons, and neural stem cells in the central nervous system (CNS). Proper communication and functional circuitry in these diverse cell types is essential for effective CNS homeostasis. Neuroinflammation is associated with the vascular pathogenesis of many CNS disorders. CNS injury elicits responses from activated glia (e.g., astrocytes, oligodendrocytes, and microglia) and from damaged perivascular cells (e.g., pericytes and endothelial cells). Most brain lesions cause extensive proliferation and growth of existing glial cells around the site of injury, leading to reactions causing glial scarring, which may act as a major barrier to neuronal regrowth in the CNS. In addition, damaged perivascular cells lead to the breakdown of the blood-neural barrier, and an increase in immune activation, activated glia, and neuroinflammation. The present review discusses the regenerative role of HO metabolites, such as CO and BR, in various vascular diseases of the CNS such as stroke, traumatic brain injury, diabetic retinopathy, and Alzheimer's disease, and the role of several other signaling molecules. 10.3390/ijms20010078
    Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class. Gille Hendrik,Hülsmeyer Martin,Trentmann Stefan,Matschiner Gabriele,Christian Hans Jürgen,Meyer Todd,Amirkhosravi Ali,Audoly Laurent P,Hohlbaum Andreas M,Skerra Arne Angiogenesis Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology. 10.1007/s10456-015-9490-5
    SiRNA silencing of VEGF, IGFs, and their receptors in human retinal microvascular endothelial cells. Nicolau Yona,Bany-Mohammed Fayez,Cai Charles L,Aranda Jacob V,Beharry Kay D American journal of translational research Vascular endothelial growth factor (VEGF) is a potent mitogen that regulates proliferation, migration, and tube formation of endothelial cells (EC). VEGF has recently become a target for severe retinopathy of prematurity (ROP) therapy. We tested the hypothesis that a specific VEGF isoform and/or receptor acts synergistically with insulin-like growth factor (IGF)-I to alter normal retinal microvascular EC angiogenesis and RNA interference can be used to reverse VEGF effects. We used small interfering RNA (SiRNA) transfection to target VEGF isoforms, IGFs, and their receptors in human retinal microvascular endothelial cells (HRECs). Media was collected at 24 and 48 hours post transfection for measurement of VEGF, sVEGFR-1 and IGF-1 levels; and HRECs were assessed for migration, tube formation, VEGF signaling genes, oxidative stress, and immune-reactivity. At 24 hours post transfection VEGF increased with VEGFR-2; sVEGFR-1 decreased with VEGF, VEGFR-2, and IGF-1R; and IGF-I increased with VEGF, VEGFR-1, IGF-2R, IGF+VEGF, and IGF+VEGF. IGF-I transfection with each VEGF isoform reduced sphere- forming and migration capacities with robust upregulation of caspase-9, COX-2, MAPK, PKC, and VEGF receptors. At 48 hours, the effects were reversed with a majority of genes downregulated, except with IGF-I and NP-1 transfection. Using RNA interference for targeted inhibition of VEGF isoforms in conjunction with IGF-I may be preferable for suppression of HREC overgrowth in vasoproliferative retinopathies such as ROP.
    PEDF expression affects the oxidative and inflammatory state of choroidal endothelial cells. Farnoodian Mitra,Sorenson Christine M,Sheibani Nader American journal of physiology. Cell physiology Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly population, and is associated with severe macular degeneration and choroidal neovascularization (CNV). Although the pathogenesis of AMD is associated with choroidal dysfunction and CNV, the detailed underlying mechanisms remain unresolved. Altered production of pigment epithelium-derived factor (PEDF), a neuroprotective and antiangiogenic factor, contributes to CNV. Furthermore, exogenous PEDF mitigates angiogenesis in preclinical CNV models. How PEDF expression affects choroidal endothelial cell (ChEC) function is unknown. Here we isolated ChECs from PEDF and PEDF-deficient (PEDF) mice and determined the impact of PEDF expression on the proangiogenic and pro-inflammatory properties of ChECs. We showed that PEDF expression significantly affects the proliferation, migration, adhesion, and oxidative and inflammatory state of ChECs. The PEDF ChECs were, however, more sensitive to HO challenge and exhibited increased rate of apoptosis and oxidative stress. We also observed a significant increase in production of cytokines with a primary role in inflammation and angiogenesis including vascular endothelial growth factor (VEGF) and osteopontin, and a reprograming of chemokines and cytokines expression profiles in PEDF ChECs. Collectively, our results indicate that PEDF expression has a significant impact on oxidative and inflammatory properties of ChECs, whose alteration could contribute to pathogenesis of chronic inflammatory diseases including exudative AMD. 10.1152/ajpcell.00259.2017
    Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo. Cui Kaixuan,Zhang Shanshan,Liu Xiaojuan,Yan Zhenzhen,Huang Lili,Yang Xiaowei,Zhu Rongrong,Sang Aimin Biochemical and biophysical research communications choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine-threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD. 10.1016/j.bbrc.2018.06.003
    Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization. Sheibani Nader,Wang Shoujian,Darjatmoko Soesiawati R,Fisk Debra L,Shahi Pawan K,Pattnaik Bikash R,Sorenson Christine M,Bhowmick Reshma,Volpert Olga V,Albert Daniel M,Melgar-Asensio Ignacio,Henkin Jack Experimental eye research Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 μg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 μM, PEDF 336 (100 μM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer. 10.1016/j.exer.2019.107798
    The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin ameliorates retinal endothelial cell dysfunction triggered by inflammation. Gonçalves Andreia,Almeida Luísa,Silva Ana Paula,Fontes-Ribeiro Carlos,Ambrósio António F,Cristóvão Armando,Fernandes Rosa Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Diabetic retinopathy is considered a low-grade chronic inflammatory disease and several inflammatory molecules, including tumor necrosis factor (TNF)-α, are known to play a major role in the degeneration of retinal capillaries. Previous studies have reported that sitagliptin, a DPP-4 inhibitor, prevents the increase in blood-retinal barrier (BRB) permeability and inhibits the tight junction disassembly induced by diabetes. AIM:Our goal was to investigate whether sitagliptin is able to prevent retinal endothelial cells (EC) dysfunction triggered by the pro-inflammatory cytokine TNF-α. MAIN METHODS:The effects of TNF-α and/or sitagliptin on primary cultures of bovine retinal EC were tested. The EC monolayer permeability was analyzed by using 70 kDa rhodamine isothiocyanate (RITC) dextran. The cellular distribution profile of claudin-5 was examined by immunofluorescence staining, and DPP-4 activity was evaluated by using a fluorogenic substrate. Cell viability was assessed by MTT assay, and cell proliferation by the BrdU incorporation assay. Retinal EC migration and angiogenesis were evaluated by a scratch assay and a capillary tube formation in matrigel assay, respectively. KEY FINDINGS:TNF-α increased the permeability of EC monolayer and induced the loss of claudin-5 immunostaining at the cell borders. This impairment was associated with decreased migration and capillary morphogenesis of retinal EC. Sitagliptin was unable to prevent the effect of TNF-α on EC permeability. However, it decreased DPP-4 activity in bovine retinal EC exposed to TNF-α, without affecting cell viability. Moreover, sitagliptin enhanced the migration and capillary morphogenesis in bovine retinal EC challenged with TNF-α. SIGNIFICANCE:These results suggest that sitagliptin is able to positively modulate vascular EC function under conditions of retinal inflammation. 10.1016/j.biopha.2018.03.144
    Antiangiogenic activity of PLGA-Lupeol implants for potential intravitreal applications. Soares Daniel Crístian Ferreira,de Paula Oliveira Diogo Coelho,Barcelos Luciola Silva,Barbosa Alan Sales,Vieira Lorena Carla,Townsend Danyelle M,Rubello Domenico,de Barros André Luis Branco,Duarte Lucienir Pains,Silva-Cunha Armando Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Uncontrolled angiogenesis is directly associated with ocular diseases such as macular degeneration and diabetic retinopathy. Implantable polymeric drug delivery systems have been proposed for intravitreal applications and in the present work, we evaluated the antiangiogenic potential of PLGA ocular implants loaded with the triterpene lupeol using in vitro and in vivo models. The drug/polymer physiochemical properties of the lupeol-loaded PLGA were validated as functionally similar using differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Interestingly, in an in vitro culture system, lupeol (100μg/mL and 250μg/mL) was capable to inhibited the proliferation as well as the migration of Human Umbilical Vein Endothelial Cells (HUVEC), without interfering in cell viability, promoting a significant reduction in the percentage of vessels (39.41% and 44.12%, respectively), compared with the control group. In vivo test, by using the chorioallantoic membrane (CAM) model, lupeol-loaded PLGA ocular implants showed antiangiogenic activity comparable to the FDA-approved anti-VEGF antibody Bevacizumab. Overall, our results suggest lupeol-loaded PLGA ocular implants were able to inhibit the angiogenic process by impairing both proliferation and migration of endothelial cells. 10.1016/j.biopha.2017.05.093
    MicroRNA-141-3p inhibits retinal neovascularization and retinal ganglion cell apoptosis in glaucoma mice through the inactivation of Docking protein 5-dependent mitogen-activated protein kinase signaling pathway. Zhang Li-Qiong,Cui Hao,Yu Yong-Bin,Shi Huan-Qi,Zhou Yuan,Liu Mei-Jiao Journal of cellular physiology Retinal neovascularization occurs in various ocular disorders including proliferative diabetic retinopathy and secondary neovascular glaucoma, resulting in blindness. This paper aims to investigate the effect of microRNA-141-3p (miR-141-3p) on retinal neovascularization and retinal ganglion cells (RGCs) in glaucoma mice through the Docking protein 5 (DOK5)-mediated mitogen-activated protein kinase (MAPK) signaling pathway. Chip retrieval and difference analysis were used for the potential mechanism of miR-141-3p on glaucoma. All modeled mice were transfected with different expression of mimic or inhibitor. The expressions of miR-141-3p, DOK5, and related genes and proteins of the MAPK signaling pathway were detected by Reverse transcription quantitative polymerase chain reaction and western blot analysis. Cell proliferation, lumen formation, and apoptosis in the retinal vascular epithelial cells and RGCs were detected using Matrigel angiogenesis and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling assays. Moreover, a total of 63 and 294 differentially expressed genes were obtained in GSE2378 and GSE9944 chips, and 4 genes were within the intersection of the chips. In addition, the results showed that miR-141-3p was found to inhibit the DOK5 gene and activate the MAPK pathway. The number of RGCs, the expression of p38, extracellular-signal-regulated kinases (ERK), Jun N-terminal kinase (JNK), IGF-1, VEGF, HIF1-α, Bax, caspase-3, and the extent of p38, ERK, and JNK phosphorylated were decreased with miR-141-3p upregulation. Lastly, the results obtained showed that miR-141-3p inhibited the proliferation of retinal vascular epithelial cells and inhibited angiogenesis, as well as promoted apoptosis of RGCs. The study suggests that miR-141-3p inhibits retinal neovascularization in glaucoma mice by impeding the activation of the DOK5-mediated MAPK signaling pathway. 10.1002/jcp.27549
    Long noncoding RNA-MEG3 is involved in diabetes mellitus-related microvascular dysfunction. Qiu Gui-Zhen,Tian Wei,Fu Hai-Tao,Li Chao-Peng,Liu Ban Biochemical and biophysical research communications Microvascular dysfunction is an important characteristic of diabetic retinopathy. Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. In this study, we investigated the role of lncRNA-MEG3 in diabetes-related microvascular dysfunction. We show that MEG3 expression level is significantly down-regulated in the retinas of STZ-induced diabetic mice, and endothelial cells upon high glucose and oxidative stress. MEG3 knockdown aggravates retinal vessel dysfunction in vivo, as shown by serious capillary degeneration, and increased microvascular leakage and inflammation. MEG3 knockdown also regulates retinal endothelial cell proliferation, migration, and tube formation in vitro. The role of MEG3 in endothelial cell function is mainly mediated by the activation of PI3k/Akt signaling. MEG3 up-regulation may serve as a therapeutic strategy for treating diabetes-related microvascular complications. 10.1016/j.bbrc.2016.01.164
    Effects of High Glucose on the Expression of LAMA1 and Biological Behavior of Choroid Retinal Endothelial Cells. Song Guangwei,Lin Da,Bao Licheng,Jiang Qi,Zhang Yinan,Zheng Haihua,Gao Qianying Journal of diabetes research Hyperglycemia is one of the main causes of proliferative diabetic retinopathy (PDR) characterized by thickening of the vascular basement membrane. Laminin alpha 1 (LAMA1) is a primary component of laminin, a major protein constituent of the basement membrane. In this study, we investigated the role of LAMA1 in the development of PDR. Retinal choroidal vascular endothelial cells (RF/6A line) were exposed to glucose at different concentrations (5 mM, 15 mM, 25 mM, and 35 mM) and analyzed for cell growth, migration, proliferation, and adhesion. LAMA1 expression was examined 24 and 48 h following glucose treatment using Western blotting, RT-PCR, and immunofluorescence. The results showed that the proliferation, migration, and adhesion of RF/6A cells were increased by high glucose, whereas LAMA1 expression was slightly higher at 15 mM but decreased at 25 mM and 35 mM glucose compared to control. Thus, the changes in the biological behavior of high glucose-exposed retinal vascular endothelial cells correspond to variations in LAMA1 expression, indicating a possibility for LAMA1 involvement in PDR development. Our findings suggest that LAMA1 may play a role in PDR and, thus, may serve as a potential target for DR diagnosis and/or treatment. 10.1155/2018/7504614
    A new role for cofilin in retinal neovascularization. Kumar Raj,Janjanam Jagadeesh,Singh Nikhlesh K,Rao Gadiparthi N Journal of cell science Pak1 plays an important role in several cellular processes, including cell migration, but its role in pathological angiogenesis is not known. Here, we have determined its role in pathological retinal angiogenesis using an oxygen-induced retinopathy (OIR) model. VEGFA induced phosphorylation of Pak1 and its effector cofilin in a manner that was dependent on time as well as p38MAPKβ (also known as MAPK11) in human retinal microvascular endothelial cells (HRMVECs). Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVEC F-actin stress fiber formation, migration, proliferation, sprouting and tube formation. In accordance with these observations, hypoxia induced Pak1 and cofilin phosphorylation with p38MAPKβ being downstream to Pak1 and upstream to cofilin in mouse retina. Furthermore, Pak1 deficiency abolished hypoxia-induced p38MAPKβ and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, small interfering RNA (siRNA)-mediated downregulation of p38MAPKβ or cofilin levels in the wild-type mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Taken together, these observations suggest that, although the p38MAPKβ-Pak1-cofilin axis is required for HRMVEC migration, proliferation, sprouting and tubulogenesis, Pak1-p38MAPKβ-cofilin signaling is also essential for hypoxia-induced mouse retinal endothelial cell proliferation, tip cell formation and neovascularization. 10.1242/jcs.179382
    Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models. Coroniti Roberta,Fario Rafal,Nuno Didier J,Otvos Laszlo,Scolaro Laura,Surmacz Eva Frontiers in molecular biosciences Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms. Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100-250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological vascularization with the efficacy similar to that of a standard treatment (anti-VEGF antibody, 1 μg/eye). Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inflammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases. 10.3389/fmolb.2016.00067
    Secretogranin III promotes angiogenesis through MEK/ERK signaling pathway. Tang Fen,Pacheco Mario Thiego F,Chen Ping,Liang Dan,Li Wei Biochemical and biophysical research communications Secretogranin III (Scg3) was recently discovered as the first highly diabetic retinopathy-associated angiogenic factor, and its neutralizing antibody alleviated the disease with high efficacy in diabetic mice. Investigation of its molecular mechanisms will facilitate the translation of this novel therapy. Scg3 was reported to induce the phosphorylation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Here we characterized the importance of MEK/ERK activation to Scg3 angiogenic activity. Our results showed that MEK inhibitor PD98059 blocked Scg3-induced proliferation of human umbilical vein endothelial cells (HUVECs). This finding was corroborated by PD98059 inhibition of HUVEC migration and tube formation. Furthermore, ERK inhibitor SCH772984 also suppressed Scg3-induced proliferation and migration of HUVECs. Taken together, these findings suggest that MEK-ERK pathway plays an important role in Scg3-induced angiogenesis. 10.1016/j.bbrc.2017.11.080
    Metformin suppresses retinal angiogenesis and inflammation in vitro and in vivo. Han Jing,Li Yue,Liu Xiuli,Zhou Tongrong,Sun Haijing,Edwards Paul,Gao Hua,Yu Fu-Shin,Qiao Xiaoxi PloS one The oral anti-diabetic drug metformin has been found to reduce cardiovascular complications independent of glycemic control in diabetic patients. However, its role in diabetic retinal microvascular complications is not clear. This study is to investigate the effects of metformin on retinal vascular endothelium and its possible mechanisms, regarding two major pathogenic features of diabetic retinopathy: angiogenesis and inflammation. In human retinal vascular endothelial cell culture, metformin inhibited various steps of angiogenesis including endothelial cell proliferation, migration, and tube formation in a dose-dependent manner. Its anti-angiogenic activity was confirmed in vivo that metformin significantly reduced spontaneous intraretinal neovascularization in a very-low-density lipoprotein receptor knockout mutant mouse (p<0.05). Several inflammatory molecules upregulated by tumor necrosis factor-α in human retinal vascular endothelial cells were markedly reduced by metformin, including nuclear factor kappa B p65 (NFκB p65), intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8). Further, metformin significantly decreased retinal leukocyte adhesion (p<0.05) in streptozotocin-induced diabetic mice. Activation of AMP-activated protein kinase was found to play a partial role in the suppression of ICAM-1 and MCP-1 by metformin, but not in those of NFκB p65 and IL-8. Our findings support the notion that metformin has considerable anti-angiogenic and anti-inflammatory effects on retinal vasculature. Metformin could be potentially used for the purpose of treating diabetic retinopathy in addition to blood glucose control in diabetic patients. 10.1371/journal.pone.0193031
    Down-regulation of microRNA-216a confers protection against yttrium aluminium garnet laser-induced retinal injury the GDNF-mediated GDNF/GFRα1/RET signalling pathway. Hu Xi-Bin,Fu Shu-Hua,Luo Q I,He Jian-Zhong,Qiu Yan-Fei,Lai Wei,Zhong Min Journal of biosciences Retinal injury plays a leading role in the onset of visual impairment. Current forms of treatment are not able to ameliorate scarring, cell death and tissue and axon regeneration. Recently, microRNA-216a (miR-216a) has been reported to regulate snx5, a novel notch signalling pathway component during retinal development. This study aims to elucidate the role of miR-216a in yttrium aluminium garnet (YAG) laser-induced retinal injury by targeting glial cell line-derived neurotrophic factor (GDNF) via GDNF/GDNF family neurotrophic factor receptor α1 (GFRα1)/rearranged during transfection (RET) signalling pathway. Wistar male rats were first randomly assigned into control and model groups. Immunohistochemistry was performed to detect the GDNF positive expression rate and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining for apoptotic index (AI) of retinal tissue. Retinal neurons were divided into normal, blank, negative control (NC), miR-216a mimic, miR-216a inhibitor, siRNA-GDNF and miR-216a inhibitor?siRNA-GDNF groups. Dual luciferase reporter assay was conducted in order to identify the targeting relationship between GDNF and miR-216a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot were used for the analysis of mRNA and protein levels of miR-216a and related genes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell proliferation and flow cytometry was used to observe cell cycle and apoptosis. Results show that the model group had an increased GDNF positive rate, AI of retinal tissue and mRNA and protein levels of cellular oncogene fos (c-fos), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), GDNF, GFRα1 and bcl-2-associated X protein (bax), declined miR-216a level and mRNA and protein levels of RET and bcl-2 compared with the control group. GDNF was verified as the target gene for miR-216a. Compared with the blank and NC groups, the miR-216a mimic and siRNA-GDNF groups had higher mRNA and protein levels of c-fos, VEGF and bax, cell number in the G1 phase and increased cell apoptosis but reduced BDNF, GDNF, GFRα1, RET and bcl-2 expression, cell proliferation and cell numbers in the S phase, while the opposite trend was observed in the miR-216a inhibitor group. Taken together, our findings demonstrate that elevated GDNF levels can reduce the retinal injury, whereby down-regulated miR-216a aggravates the YAG laser-induced retinal injury by targeting the GDNF level through the GDNF/ GFRα1/RET signalling pathway.
    Bioactive lipids and pathological retinal angiogenesis. Elmasry Khaled,Ibrahim Ahmed S,Abdulmoneim Samer,Al-Shabrawey Mohamed British journal of pharmacology Angiogenesis, disruption of the retinal barrier, leukocyte-adhesion and oedema are cardinal signs of proliferative retinopathies that are associated with vision loss. Therefore, identifying factors that regulate these vascular dysfunctions is critical to target pathological angiogenesis. Given the conflicting role of bioactive lipids reported in the current literature, the goal of this review is to provide the reader a clear road map of what has been accomplished so far in the field with specific focus on the role of polyunsaturated fatty acids (PUFAs)-derived metabolites in proliferative retinopathies. This necessarily entails a description of the different retina cells, blood retina barriers and the role of (PUFAs)-derived metabolites in diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration as the most common types of proliferative retinopathies. 10.1111/bph.14507
    Kaempferol targets estrogen-related receptor α and suppresses the angiogenesis of human retinal endothelial cells under high glucose conditions. Wu Yan,Zhang Qinmei,Zhang Rui Experimental and therapeutic medicine Diabetic retinopathy (DR) is the most common complication of diabetes and a major cause of new-onset blindness in the developed world. The present study aimed to examine the effect of kaempferol on high glucose-induced human retinal endothelial cells (HRECs) . The expression levels of various mRNAs and proteins were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The target of kaempferol was determined using a luciferase reporter assay. In addition, HREC proliferation, migration and cell sprouting were determined using Cell Counting kit-8, wound scratch and tube formation assays, respectively. RT-qPCR and western blotting results showed that treatment with 30 mM glucose for 12, 24 and 48 h increased the expression level of estrogen-related receptor α (ERRα) mRNA and protein. The luciferase reporter assay demonstrated that kaempferol inhibited ERRα activity in HRECs. Compared with 5 mM normal glucose treatment, high (30 mM) glucose significantly promoted the proliferation, migration and tube formation of HRECs, which was antagonized by 10 and 30 µM kaempferol in a dose-dependent manner. Treatment with 30 mM glucose also increased the expression of vascular endothelial growth factor (VEGF) mRNA and protein, and the expression levels of VEGF mRNA and protein were suppressed by kaempferol (10 and 30 µM). Kaempferol (30 µM) treatment also increased the expression levels of thrombospondin 1 (TSP-1) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS-1) mRNA; however, TSP-1 and ADAMTS-1 levels did not differ between high glucose and normal (5 mM) glucose conditions. The results of this study suggest that kaempferol targets ERRα and suppresses the angiogenesis of HRECs under high glucose conditions. Kaempferol may be a potential drug for use in controlling the progression of DR; however, studies are required to evaluate its efficacy and safety. 10.3892/etm.2017.5261
    Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis. Xiong Si-Qi,Jiang Hai-Bo,Li Yan-Xiu,Li Hai-Bo,Xu Hui-Zhuo,Wu Zhen-Kai,Zheng Wei,Xia Xiao-Bo Molecular vision OBJECTIVE:To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. METHODS:siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor-reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. RESULTS:The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. CONCLUSIONS:Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo Endogenous Islet-1 regulates angiogenesis via VEGF.
    RNCR3: A regulator of diabetes mellitus-related retinal microvascular dysfunction. Shan Kun,Li Chao-Peng,Liu Chang,Liu Xin,Yan Biao Biochemical and biophysical research communications Retinal microvascular abnormality is an important pathological feature of diabetic retinopathy. Herein, we report the role of lncRNA-RNCR3 in diabetes mellitus-induced retinal microvascular abnormalities. We show that RNCR3 is significantly up-regulated upon high glucose stress in vivo and in vitro. RNCR3 knockdown alleviates retinal vascular dysfunction in vivo, as shown by decreased acellular capillaries, decreased vascular leakage, and reduced inflammatory response. RNCR3 knockdown decreases retinal endothelial cell proliferation, and reduces cell migration and tube formation in vitro. RNCR3 regulates endothelial cell function through RNCR3/KLF2/miR-185-5p regulatory network. RNCR3 inhibition may be a treatment option for the prevention of diabetes mellitus-induced retinal microvascular abnormalities. 10.1016/j.bbrc.2016.11.110
    Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization. Montassar Fadoua,Darche Marie,Blaizot Amandine,Augustin Sébastien,Conart Jean-Baptiste,Millet Aurélie,Elayeb Mohamed,Sahel José-Alain,Réaux-Le Goazigo Annabelle,Sennlaub Florian,Marrakchi Naziha,Messadi Erij,Guillonneau Xavier FASEB journal : official publication of the Federation of American Societies for Experimental Biology Angiogenesis is a cause of visual impairment and blindness in the wet form of age-related macular degeneration and in ischemic retinopathies. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization (CNV) and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF and possible adverse effects of long-term VEGF inhibition in the retina and choroid highlight a need for additional alternative therapies. Integrins αvβ3 and αvβ5, which regulate endothelial cell proliferation and stabilization, have been implicated in ocular angiogenesis. Lebecetin (LCT) is a 30-kDa heterodimeric C-type lectin that is isolated from venom and interacts with α5β1- and αv-containing integrins. We previously showed that LCT inhibits human brain microvascular endothelial cell adhesion, migration, proliferation, and tubulogenesis. To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on CNV in the mouse CNV model and on retinal neovascularization in the oxygen-induced retinopathy model. Our data demonstrate that a single injection of LCT efficiently reduced CNV and retinal neovascularization in these models.-Montassar, F., Darche, M., Blaizot, A., Augustin, S., Conart, J.-B., Millet, A., Elayeb, M., Sahel, J.-A., Réaux-Le Goazigo, A., Sennlaub, F., Marrakchi, N., Messadi, E., Guillonneau, X. Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization. 10.1096/fj.201600351R
    Pharmacology study of a chimeric decoy receptor trap fusion protein on retina neovascularization by dual blockage of VEGF and FGF-2. Jiang Jing,Xu Ke,Wang Ling,Xin Wei,Zhao Guorui,Huang Min,Li Shenjun,Luan Xuejing,Fang Jianmin European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences BACKGROUND:Clinical anti-vascular epithelial growth factor (VEGF) therapy trials faced a major challenge due to upregulated expression of other pro-angiogenic factors, such as fibroblast growth factor-2 (FGF-2). RC28, a novel recombinant dual decoy receptor IgG1 Fc-fusion protein, can block VEGFA and FGF-2 simultaneously. It is designed for the treatment of neovascular age-related macular degeneration and other pathological ocular neovascularization. The present study investigated the prevention efficacy of RC28 on choroidal neovascularization (CNV) in a monkey model and compared to the other mono VEGF antagonists; biodistribution and pharmacokinetics performance were also investigated. METHODS:ELISA and endothelial cell proliferation, migration, and tubule formation assay evaluated the bioactivity of RC28 in vitro, and an initial comparison was made among the mono target antagonists, Bevacizumab (Avastin), Ranibizumab (Lucentis), Aflibercept (EYLEA), Conbercept (KH902), and Ranibizumab (Lucentis). Laser-induced CNV in monkeys, and both VEGF and FGF-2 serum levels were detected in animals before and after the CNV model were induced. RC28 prevention efficacy was compared to other VEGF antagonists on CNV with respect to the incidence of CNV and several ophthalmic examinations. Ocular and systemic levels of RC28 were analyzed by Zr-labeled RC28 after single intravitreal administration for the biodistribution and pharmacokinetic profiles. RESULTS:RC28 is a unique fusion protein with high affinity to both VEGF and FGF-2, and beneficial to in vitro and in vivo bioactivity. The in vivo pharmacological studies demonstrated that the incidence of CNV formation was largely reduced in RC28 treatment groups with a low dosage as compared to other VEGF antagonist control groups. Furthermore, traces of RC28 were detected as dispersing from eyeballs to the liver after 20 days, and a prolonged half-time pharmacokinetic profile was exhibited. 10.1016/j.ejps.2018.04.043
    Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration. Kim Sung Hwan,Kim Hyunjin,Ku Hyeong Jun,Park Jung Hyun,Cha Hanvit,Lee Seoyoon,Lee Jin Hyup,Park Jeen-Woo Redox biology Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF), secreted by the retinal pigment epithelium (RPE), in pathological angiogenesis and the development of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA) is a competitive inhibitor of NADP-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS). Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development. 10.1016/j.redox.2016.10.008
    Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina. Niimi Kenta,Adachi Yumi,Ishikawa Hiroko,Yamaguchi Wataru,Kubota Yoshiaki,Inagaki Shinobu,Furuyama Tatsuo Biochemical and biophysical research communications Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1-3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFβ activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3 arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant. The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology. 10.1016/j.bbrc.2019.10.040
    [Effect of MiR-200b on retinal endothelial cell function in high-glucose condition and the mechanism]. Jiang Qun,Zhu Xiao-Hua,Liu Xin-Min,Liu Jian-Ming Nan fang yi ke da xue xue bao = Journal of Southern Medical University OBJECTIVE:To investigate the effect of MiR-200b on human retinal endothelial cells (hRECs) cultured in high glucose and explore the mechanism. METHODS:hRECs cultured in high glucose or in normal media were examined for MiR-200b mRNA expression using real-time PCR. The effect of MiR-200b transfection on hREC proliferation in high-glucose culture was evaluated with MTT assay, and real-time PCR and Western blotting were performed to determine vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) expression in the transfected cells. RESULTS:The cells in high-glucose culture showed significantly decreased MiR-200b expression and active proliferation. Compared with those in normal control cells, VEGF and TGFβ1 mRNA and protein expressions increased markedly in cells cultured in high glucose (P<0.05). MiR-200b transfection of the cells caused significantly increased cellular expression of MiR-200b but decreased expression levels of VEGF and TGFβ1 mRNA and protein, and suppressed hREC proliferation in high glucose culture (P<0.05). CONCLUSION:MiR-200b can regulate REC growth and proliferation by changing VEGF and TGFβ1 expressions and thus play a role in the pathogenesis and progression of diabetic retinopathy.
    Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin Signaling Complex to Inhibit VEGF-Induced Retinal Angiogenesis. Toutounchian Jordan J,Pagadala Jayaprakash,Miller Duane D,Baudry Jerome,Park Frank,Chaum Edward,Morales-Tirado Vanessa,Yates Charles R Molecular pharmacology Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor-induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye. 10.1124/mol.116.105031
    Combined VEGF and PDGF inhibition for neovascular AMD: anti-angiogenic properties of axitinib on human endothelial cells and pericytes in vitro. Siedlecki Jakob,Wertheimer Christian,Wolf Armin,Liegl Raffael,Priglinger Claudia,Priglinger Siegfried,Eibl-Lindner Kirsten Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie PURPOSE:Drugs currently approved for neovascular age-related macular degeneration (nAMD) offer anti-VEGF monotherapy only. Platelet-derived growth factor (PDGF) signaling is pivotal to pericyte-induced stabilization of choroidal neovascularizations (CNV), and causes partial anti-VEGF resistance. No combination therapy for VEGF and PDGF has been approved yet. Axitinib is a tyrosine kinase inhibitor interfering with VEGF and PDGF signaling, and has been approved for the treatment of renal cell carcinoma. This study evaluates anti-angiogenic properties of axitinib in an in-vitro model of choroidal neovascularizations in nAMD. METHODS:Human endothelial cells (HUVEC) and human pericytes (hPC-PL) were treated with axitinib doses ranging from 1.0 ng/ml to 10 μg/ml. Cellular viability and proliferation were assessed with a modified MTT assay. VEGF- and PDGF-stimulated migration was observed in modified Boyden chambers. Formation of capillary structures was evaluated on Cultrex basement membrane. RESULTS:Proliferation was significantly inhibited in both cell lines in a dose-dependent manner. VEGF and PDGF significantly induced, whereas simultaneous axitinib normalized cellular migration in HUVEC and pericytes. On growth-factor-reduced Cultrex, VEGF induced the formation of capillary structures, while axitinib significantly reverted this effect. Axitinib reduced the amount of vessel associated tissue on full growth factor Cultrex. All effects on both cell lines were observed in non-toxic concentrations of axitinib. CONCLUSIONS:Axitinib inhibits angiogenesis in endothelial cells and pericytes via VEGFR and PDGFR modulation in vitro. Further studies are needed to elucidate whether axitinib may also improve therapy of CNV in AMD in vivo by interference with pericyte stabilization of pathological vessels. 10.1007/s00417-017-3595-z
    Oxidative stress-induced angiogenesis is mediated by miR-205-5p. Oltra Maria,Vidal-Gil Lorena,Maisto Rosa,Sancho-Pelluz Javier,Barcia Jorge M Journal of cellular and molecular medicine miR-205-5p is known to be involved in VEGF-related angiogenesis and seems to regulate associated cell signalling pathways, such as cell migration, proliferation and apoptosis. Therefore, several studies have focused on the potential role of miR-205-5p as an anti-angiogenic factor. Vascular proliferation is observed in diabetic retinopathy and the 'wet' form of age-related macular degeneration. Today, the most common treatments against these eye-related diseases are anti-VEGF therapies. In addition, both AMD and DR are typically associated with oxidative stress; hence, the use of antioxidant agents is accepted as a co-adjuvant therapy for these patients. According to previous data, ARPE-19 cells release pro-angiogenic factors when exposed to oxidative insult, leading to angiogenesis. Matching these data, results reported here, indicate that miR-205-5p is modulated by oxidative stress and regulates VEGFA-angiogenesis. Hence, miR-205-5p is proposed as a candidate against eye-related proliferative diseases. 10.1111/jcmm.14822
    The regulatory role of hepatoma-derived growth factor as an angiogenic factor in the eye. LeBlanc Michelle E,Wang Weiwen,Chen Xiuping,Ji Yanli,Shakya Akhalesh,Shen Chen,Zhang Chenming,Gonzalez Vivianne,Brewer Megan,Ma Jian-Xing,Wen Rong,Zhang Fangliang,Li Wei Molecular vision PURPOSE:Hepatoma-derived growth factor (HDGF) is a mitogen that promotes endothelial proliferation and neuronal survival. Using a unique technology of ligandomics, we recently identified HDGF as a retinal endothelial binding protein. The purpose of this study is to examine the role of HDGF in regulating ocular vasculature and the expression of HDGF in the retina. METHODS:HDGF expression in the retinal was analyzed with western blot and immunohistochemistry. Angiogenic activity was investigated in human retinal microvascular endothelial cells (HRMVECs) with in vitro endothelial proliferation, migration, and permeability assays. In vivo angiogenic activity was quantified with a corneal pocket assay. The Evans blue assay and western blot using anti-mouse albumin were performed to detect the capacity of HDGF to induce retinal vascular leakage. RESULTS:Immunohistochemistry revealed that HDGF is expressed in the retina with a distinct pattern. HDGF was detected in retinal ganglion cells and the inner nuclear layer but not in the inner plexiform layer, suggesting that HDGF is expressed in the nucleus, but not in the cytoplasm, of retinal neurons. In contrast to family member HDGF-related protein 3 (HRP-3) that has no expression in photoreceptors, HDGF is also present in the outer nuclear layer and the inner and outer segments of photoreceptors. This suggests that HDGF is expressed in the nucleus as well as the cytoplasm of photoreceptors. In vitro functional assays showed that HDGF induced the proliferation, migration, and permeability of HRMVECs. Corneal pocket assay indicated that HDGF directly stimulated angiogenesis in vivo. Intravitreal injection of HDGF significantly induced retinal vascular leakage. CONCLUSIONS:These results suggest that HDGF is an angiogenic factor that regulates retinal vasculature in physiologic and pathological conditions. Identification of HDGF by ligandomics and its independent characterization in this study also support the validity of this new technology for systematic identification of cellular ligands, including angiogenic factors.
    Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis. Genet Gael,Boyé Kevin,Mathivet Thomas,Ola Roxana,Zhang Feng,Dubrac Alexandre,Li Jinyu,Genet Nafiisha,Henrique Geraldo Luiz,Benedetti Lorena,Künzel Steffen,Pibouin-Fragner Laurence,Thomas Jean-Leon,Eichmann Anne Nature communications Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions. 10.1038/s41467-019-10359-x
    Müller cells in pathological retinal angiogenesis. Li Xiaorui,Liu Jing,Hoh Josephine,Liu Ju Translational research : the journal of laboratory and clinical medicine Müller cells are the major glial cells spanning the entire layer of the retina and maintaining retinal structure. Under pathological conditions, Müller cells are involved in retinal angiogenesis, a process of growing new blood vessels from pre-existing capillaries. In response to hypoxia, high glucose, and inflammation conditions, multiple signaling pathways are activated in Müller cells, followed by the increased production of proangiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, matrix metalloproteinases, Netrin-4, and angiopoietin-like 4. Expression of antiangiogenic factors is also downregulated in Müller cells. Besides, proliferation and dedifferentiation of Müller cells facilitates retinal angiogenesis. In this review, we summarized molecular mechanisms of Müller cells-related retinal angiogenesis. The potential of Müller cells as a therapeutic target for retinal angiogenesis was also discussed. 10.1016/j.trsl.2018.12.006
    Chebulagic acid Chebulinic acid and Gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation. Shanmuganathan Sivasankar,Angayarkanni Narayanasamy Vascular pharmacology Tumor necrosis factor-α (TNFα) a pleiotropic cytokine induces pro-inflammatory and pro-angiogenic changes in conditions such as diabetic retinopathy (DR) and neovascular age related macular degeneration (NV-AMD). Hence, inhibition of TNFα mediated changes can benefit the management of DR and NV-AMD. Triphala, an ayurvedic herbal preparation is known to have immunomodulatry functions. In this study we evaluated the alcoholic extract of triphala (AlE) and its compounds Chebulagic acid (CA), Chebulinic acid (CI) and Gallic acid (GA) for their anti-TNFα activity. TNFα induced pro-inflammatory and pro-angiogenic changes in the retinal-choroid microvascular endothelial cells (RF/6A). Treatment with CA/CI/GA and the whole Triphala extract showed characteristic inhibition of MMP-9, cell proliferation/migration and tube formation as well the expression of IL-6, IL-8 and MCP-1 without affecting cell viability. This was mediated by inhibition of p38, ERK and NFκB phosphorylation. Ex vivo angiogenesis assay using chick chorioallantoic membrane (CAM) model also showed that TNFα-induced angiogenesis and it was inhibited by AlE and its active principles. Further, in silico studies revealed that CA, CI and GA are capable of binding the TNFα-receptor-1 to mediate anti-TNFα activity. This study explains the immunomodulatory function of Triphala, evaluated in the context of retinal and choroid vasculopathies in vitro and ex vivo; which showed that CA, CI and GA can be a potential pharmacological agents in the management of DR and NV-AMD. 10.1016/j.vph.2018.04.005
    Extract and Its Constituent Ishophloroglucin A Attenuated In Vitro and In Vivo High Glucose-Induced Angiogenesis. Fernando K H N,Yang Hye-Won,Jiang Yunfei,Jeon You-Jin,Ryu BoMi International journal of molecular sciences Diabetes is associated with vascular complications, such as impaired wound healing and accelerated vascular growth. The different clinical manifestations, such as retinopathy and nephropathy, reveal the severity of enhanced vascular growth known as angiogenesis. This study was performed to evaluate the effects of an extract of (IO) and its constituent, Ishophloroglucin A (IPA) on high glucose-induced angiogenesis. A transgenic zebrafish (:EGFP) embryo model was used to evaluate vessel growth. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), gap closure, transwell, and Matrigel assays were used to analyze the proliferation, migration, and capillary formation of EA.hy926 cells. Moreover, protein expression were determined using western blotting. IO extract and IPA suppressed vessel formation in the transgenic zebrafish (:EGFP) embryo. IPA attenuated cell proliferation, cell migration, and capillary-like structure formation in high glucose-treated human vascular endothelial cells. Further, IPA down regulated the expression of high glucose-induced vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream signaling molecule cascade. Overall, the IO extract and IPA exhibited anti-angiogenic effects against high glucose-induced angiogenesis, suggesting their potential for use as therapeutic agents in diabetes-related angiogenesis. 10.3390/ijms20225542
    Curcumolide, a unique sesquiterpenoid from Curcuma wenyujin displays anti-angiogenic activity and attenuates ischemia-induced retinal neovascularization. Lin Weiwei,Tu Hongfeng,Zhu Yao,Guan Yijian,Liu Hui,Ling Wei,Yan Pengcheng,Dong Jianyong Phytomedicine : international journal of phytotherapy and phytopharmacology BACKGROUND:Targeting vascular endothelial growth factor is a common treatment strategy for neovascular eye disease, a leading cause of visual impairment and blindness. However, these approaches are limited or carry various complications. Therefore, there is an urgent need for the development of unique therapeutic approaches. PURPOSE:To investigate the anti-angiogenic effects of curcumolide and its mechanism of action. METHODS /STUDY DESIGNS:In this study, we examine the effects of curcumolide on the process of vasculature formation, including cell proliferation, migration, tube formation and apoptosis in vitro using human umbilical vascular endothelial cells (HUVECs). We also assess the anti-angiogenic effects of curcumolide in vivo using a mouse model of oxygen induced retinopathy (OIR). The mechanism of anti-angiogenic effects was investigated by measuring the expression level of various signaling proteins and the molecular docking simulations. RESULTS:Intravitreal injection of curcumolide reduced the formation of retinal neovascular tufts and VEGFR2 phosphorylation in the murine OIR model at concentrations administered without definite cellular and retinal toxicities. Curcumolide suppressed VEGF-induced HRMECs proliferation, migration and tube formation in a dose-dependent manner. Meanwhile, it promoted caspase-dependent apoptosis. Curcumolide also inhibited VEGF-induced phosphorylation of VEGFR-2 tyrosine kinase, and suppressed downstream protein kinases of VEGFR2, including Src, FAK, ERK, AKT, and mTOR in HRMECs. In silico study revealed that curcumolide bound with ATP-binding sites of the VEGFR2 kinase unit by the formation of a hydrogen bond and hydrophobic interactions. CONCLUSION:Curcumolide has anti-angiogenic activity in HUVECs and in a murine OIR model of ischemia-induced retinal neovascularization, and it might be a potential drug candidate for the treatment of proliferative diabetic retinopathy. 10.1016/j.phymed.2019.152923
    [Comparative Clinical and Ultrastructural Analysis of the Results from Ranibizumab and Aflibercept in Patients with PDR]. Vidinova Christina Nicolaeva,Gouguchkova Pravoslava Tz,Dimitrov Tzvetomir,Vidinov Kalin Nicolaev,Nocheva Hristina Klinische Monatsblatter fur Augenheilkunde PURPOSE:Diabetic retinopathy is one of the worst complications of diabetes and can threaten sight. The aim of our study is to compare the clinical outcomes and ultrastructural changes in diabetic retinopathy patients after intravitreal admission of either ranibizumab (Lucentis) or aflibercept (Eylea). METHODS:In our prospective study, 27 patients with PDR were enrolled. They were divided into two groups - 14 PDR patients treated with ranibizumab and 13 PDR patients treated with aflibercept. In both groups, a complete ophthalmological examination was performed, including OCT. In 12 patients (6 from each group), pars plana vitrectomy was performed with excision of epiretinal membranes. Transmission and scanning electron microscopy of the membranes was performed. RESULTS:Clinical findings - in both groups there was a decrease in the neovascular proliferation and macular oedema. CRT was reduced with approximately 100 - 120мk. In the Eylea group, there was general shrinking of neovascular proliferation with lower risk of bleeding. The ultrastructural analysis showed more significant reduction in endothelial fenestration after Eylea application, with clustering of platelets and erythrocytes in the capillary lumen. Increased numbers of macrophages were found in the membranes of both groups. CONCLUSION:Our results show that aflibercept is very effective in treating PDR. It causes more significant reduction of endothelial fenestration and more evident thrombotic microangiopathy than other anti-VEGF drugs, and thus diminishes macular oedema and prevents neovascular tissue from bleeding. Eylea inhibits both VEGF and placental growth factor and thus modifies the whole pathophysiology of DR. It is therefore more effective than other treatment medications. 10.1055/a-0767-6951
    Occludin S490 Phosphorylation Regulates Vascular Endothelial Growth Factor-Induced Retinal Neovascularization. Liu Xuwen,Dreffs Alyssa,Díaz-Coránguez Monica,Runkle E Aaron,Gardner Thomas W,Chiodo Vince A,Hauswirth William W,Antonetti David A The American journal of pathology Occludin is a transmembrane tight junction protein that contributes to diverse cellular functions, including control of barrier properties, cell migration, and proliferation. Vascular endothelial growth factor (VEGF) induces phosphorylation of occludin at S490, which is required for VEGF-induced endothelial permeability. Herein, we demonstrate that occludin S490 phosphorylation also regulates VEGF-induced retinal endothelial cell proliferation and neovascularization. Using a specific antibody, phospho-occludin was located in centrosomes in endothelial cell cultures, animal models, and human surgical samples of retinal neovessels. Occludin S490 phosphorylation was found to increase with endothelial tube formation in vitro and in vivo during retinal neovascularization after induction of VEGF expression. More important, expression of occludin mutated at S490 to Ala, completely inhibited angiogenesis in cell culture models and in vivo. Collectively, these data suggest a novel role for occludin in regulation of endothelial proliferation and angiogenesis in a phosphorylation-dependent manner. These findings may lead to methods of regulating pathological neovascularization by specifically targeting endothelial cell proliferation. 10.1016/j.ajpath.2016.04.018
    Nap Interferes with Hypoxia-Inducible Factors and VEGF Expression in Retina of Diabetic Rats. D'Amico Agata Grazia,Maugeri Grazia,Bucolo Claudio,Saccone Salvatore,Federico Concetta,Cavallaro Sebastiano,D'Agata Velia Journal of molecular neuroscience : MN The retinal microvascular damage is a complication of diabetic retinopathy (DR). Hyperglycemia and hypoxia are responsible of aberrant vessel's proliferation. The cellular response to hypoxia is mediated through activation of hypoxia-inducible factors (HIFs). Among these, HIF-1α modulates expression of its target gene, VEGF, whose upregulation controls the angiogenic event during DR development. In a previous study, we have demonstrated that a small peptide, NAP, is able to protect retina from hyperglycemic insult. Here, we have demonstrated that its intraocular administration in a rat model of diabetic retinopathy has reduced expression of HIF-1α, HIF-2α, and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization study by confocal microscopy. Although these evidences need to be further deepened to understand the molecular mechanism involved in the protective NAP action, the present data suggest that this small peptide may be effective to prevent the development of this ocular pathology. 10.1007/s12031-016-0869-6
    LncRNAs in ocular neovascularizations. Cissé Yacouba,Bai Lang,Chen Min-Ting International journal of ophthalmology The prevalence of eye diseases worldwide is dramatically increasing and represents a major concern in underdeveloped and developed regions. Ocular diseases, previously associated with a higher depression risk, also impose a substantial economic burden on affected families, thus early detection and/or accurate treatment in order to avoid and prevent blindness should be emphasized. Ocular neovascularization (NV), the leading cause of blindness in a variety of eye diseases, is a pathologic process characterized by the formation, proliferation and infiltration of anomalous, tiny and leaky fragile blood vessels within the eye. Genetics have been suspected to play an important role in the occurrence of eye diseases, with the detection of a numbers of specific gene mutations. Long non-coding RNA (lncRNAs) are novel class of regulatory molecules previously associated with various biological processes and diseases, however the nature of the relation and pathways by which they might contribute to the development of corneal, choroidal and retinal NV have not yet been completely elucidated. In this review, we focus on the regulation and characteristics of lncRNAs, summarize results from ocular NV-related studies and discuss the implication of lncRNAs in ocular NV development. 10.18240/ijo.2019.12.19
    Cyclin-dependent kinase 1 disruption inhibits angiogenesis by inducing cell cycle arrest and apoptosis. Gao Xin,Zhang Yuan,Zhang Rui,Zhao Zichan,Zhang Haorui,Wu Jinhui,Shen Wei,Zhong Ming Experimental and therapeutic medicine Angiogenesis is a complex process, which involves the sprouting of new blood vessels from pre-existing vasculature. Pathological retinal angiogenesis can lead to vision loss and even blindness. Cyclin-dependent kinase 1 (CDK1) is involved in regulation of the cell cycle and is reported to contribute to tumor progression. However, the role of CDK1 in retinal angiogenesis is largely unknown. The purpose of the present study was to investigate the role of CDK1 in retinal angiogenesis. Western blotting, reverse transcription-quantitative PCR (RT-qPCR) analysis, immunofluorescence and immunohistochemistry were used to evaluate the expression of CDK1 in pathological angiogenesis using an oxygen-induced retinopathy (OIR) mouse model. Small interfering (si)RNA sequences against CDK1 were synthesized and incubated with retinal cells. The efficiency of knockdown was confirmed by western blot and RT-qPCR assays. The effect of CDK1 siRNAs on angiogenesis was investigated using EdU cell proliferation, cell migration and tube formation assays. Subsequently, flow cytometry was used to assess the effects of CDK1 siRNAs on cell cycle distribution and on the induction of apoptosis. The expression levels of cell cycle- and apoptosis-related genes were detected using western blotting. CDK1 was overexpressed in pathological retinal angiogenesis. CDK1 siRNAs inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. The possible mechanisms involved the induction of cell cycle arrest at the G2/M phase and the induction of apoptosis via an increase in the expression levels of p21 and p53. In conclusion, the data indicated that CDK1 was overexpressed in the OIR model and that silencing of CDK1 inhibited angiogenesis . CDK1 may be a novel therapeutic target for pathological retinal angiogenesis. 10.3892/etm.2019.7883
    Apolipoprotein E2 and E3, but Not E4, Promote Retinal Pathologic Neovascularization. Masuda Tomomi,Shimazawa Masamitsu,Hashimoto Yuhei,Kojima Atsushi,Nakamura Shinsuke,Suemori Shinsuke,Mochizuki Kiyofumi,Kawakami Hideaki,Kawase Kazuhide,Hara Hideaki Investigative ophthalmology & visual science Purpose:To determine the relationship between the different isoforms of apolipoprotein E (ApoE) and retinal neovascularization. Methods:The concentrations of ApoE and VEGF in vitreous humor samples with either a macular hole (MH), or diabetic macular edema (DME), or proliferative diabetic retinopathy (PDR) with or without intravitreal injection of bevacizumab (IVB) were measured by ELISA. The effects of each isoform of ApoE on human retinal microvascular endothelial cells (HRMECs) in culture or on the retina of oxygen-induced retinopathy (OIR) mice were investigated. Results:The concentrations of ApoE and VEGF were significantly higher in the vitreous humor of patients with PDR and DME than in patients with an MH. There was a significant positive correlation between the concentrations of ApoE and VEGF in vitreous humor of patients. In vitro assays showed that ApoE2 and ApoE3, but not ApoE4, promoted the VEGF-induced cell proliferation and migration. In vivo assays showed that intravitreal injections of ApoE2 and ApoE3 increased the number and area of nodes in the retina of OIR mice. Moreover, ApoE was expressed in the vascular endothelial cell in both normal and OIR retinas, but their expression levels were different at postnatal day (P) 12 and P17. Conclusions:These results demonstrate that ApoE2 and ApoE3, but not ApoE4, have proangiogenic effects, and the increased expression of ApoE in the vitreous humor of patients with PDR and DME indicates that ApoE2 and ApoE3 are involved in the development of retinal neovascularization in eyes. 10.1167/iovs.16-20539
    RKIP negatively regulates the glucose induced angiogenesis and endothelial-mesenchymal transition in retinal endothelial cells. Feng Le,Zhang Conghui,Liu Guodong,Wang Fang Experimental eye research Diabetic retinopathy (DR), a common microvascular complication of diabetes, is reported to be the leading cause of blindness worldwide. In our previous study, we found that the Raf kinase inhibitor protein (RKIP) is significantly decreased in vitreous humor of proliferative diabetic retinopathy (PDR) patients, which indicated that RKIP might play a role in the development of PDR. To investigate the role of RKIP in PDR, stable overexpression and knockdown of RKIP in Human retinal capillary endothelial cells (HRCECs) were generated by using lentivirus constructs. Then, the glucose-induced cell viability, migration, angiogenesis, and (endothelial to mesenchymal transition) EndMT were determined in the RKIP-wide type (WT), -knocking down (KD) and -overexpression (OE) HRCECs. The results showed that, compared with the RKIP-WT groups, the glucose-induced cell viabilities, migration and angiogenesis were significantly increased in the RKIP-KD groups, while significantly decreased in the RKIP-OE groups. Besides, compared with the control groups, CD31 and vWF were upregulated, while α-SMA was downregulated in the RKIP-KD groups, while CD31 and vWF were downregulated, while α-SMA was upregulated in the RKIP-OE groups induced by glucose. In conclusion, our results showed that RKIP negatively regulates glucose-induced cell viability, migration, angiogenesis, and EndMT in HRCECs, suggesting that the downregulation of RKIP in the vitreous humor of PDR patients might contribute to the development of DR. 10.1016/j.exer.2019.107851
    BET Bromodomain Suppression Inhibits VEGF-induced Angiogenesis and Vascular Permeability by Blocking VEGFR2-mediated Activation of PAK1 and eNOS. Huang Mingcheng,Qiu Qian,Xiao Youjun,Zeng Shan,Zhan Mingying,Shi Maohua,Zou Yaoyao,Ye Yujin,Liang Liuqin,Yang Xiuyan,Xu Hanshi Scientific reports The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a critical modulator of angiogenesis. Increasing evidence indicate the important role of bromodomain and extra-terminal domain (BET) of chromatin adaptors in regulating tumor growth and inflammatory response. However, whether BET proteins have a role in angiogenesis and endothelial permeability is unclear. In this study, we observed that treatment with JQ1, a specific BET inhibitor, suppressed in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis in a Matrigel plug and oxygen-induced retinopathy neovascularization. JQ1 attenuated the VEGF-induced decrease in TEER in HUVECs and prevented Evans blue dye leakage in the VEGF-induced Miles assay in athymic Balb/c nude mice. BET inhibition with JQ1 or shRNA for Brd2 or Brd4 suppressed VEGF-induced migration, proliferation, and stress fiber formation of HUVECs. Furthermore, BET inhibition suppressed phosphorylation of VEGFR2 and PAK1, as well as eNOS activation in VEGF-stimulated HUVECs. Inhibition with VEGFR2 and PAK1 also reduced migration and proliferation, and attenuated the VEGF-induced decrease in TEER. Thus, our observations suggest the important role of BET bromodomain in regulating VEGF-induced angiogenesis. Strategies that target the BET bromodomain may provide a new therapeutic approach for angiogenesis-related diseases. 10.1038/srep23770
    The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2. Terlizzi Vincenzo,Kolibabka Matthias,Burgess Janette Kay,Hammes Hans Peter,Harmsen Martin Conrad Stem cells (Dayton, Ohio) Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to functionally replace retinal pericytes and normalize retinal microvasculature during disease progression. We hypothesized that Notch signaling in ASC underlies regulation and stabilization of dysfunctional retinal microvascular networks such as in DR. ASC prominently and constitutively expressed NOTCH2. Genetic knockdown of NOTCH2 in ASC (SH-NOTCH2) disturbed the formation of vascular networks of human umbilical cord vein endothelial cells both on monolayers of ASC and in organotypical three-dimensional cocultures with ASC. On ASC SH-NOTCH2, cell surface platelet-derived growth factor receptor beta was downregulated which disrupted their migration toward the chemoattractant platelet-derived growth factor beta subunits (PDGF-BB) as well as to conditioned media from EC and bovine retinal EC. This chemoattractant is secreted by pro-angiogenic EC in newly formed microvascular networks to attract pericytes. Intravitreal injected ASC SH-NOTCH2 in oxygen-induced retinopathy mouse eyes did not engraft in the preexisting retinal microvasculature. However, the in vivo pro-angiogenic capacity of ASC SH-NOTCH2 did not differ from controls. In this respect, multifocal electroretinography displayed similar b-wave amplitudes in the avascular zones when either wild type ASC or SH-NOTCH2 ASC were injected. In conclusion, our results indicate that NOTCH2 is essential to support in vitro vasculogenesis via juxtacrine interactions. In contrast, ongoing in vivo angiogenesis is influenced by paracrine signaling of ASC, irrespective of Notch signaling. Stem Cells 2018;36:240-251. 10.1002/stem.2726
    Kaempferol inhibited VEGF and PGF expression and in vitro angiogenesis of HRECs under diabetic-like environment. Xu X H,Zhao C,Peng Q,Xie P,Liu Q H Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR. 10.1590/1414-431X20165396
    Somatostatin protects human retinal pericytes from inflammation mediated by microglia. Mazzeo Aurora,Arroba Ana I,Beltramo Elena,Valverde Angela M,Porta Massimo Experimental eye research Diabetic retinopathy (DR) is usually considered a microvascular disease. However, involvement of the neuroretina in the early stages of DR has recently gained major credit. Inflammatory processes, leading to glial activation and neuronal apoptosis, develop early in the retina of diabetic subjects. Pericytes constitute a link between the vascular and the neural retina, play a central role in blood-retinal barrier maintenance, and may influence neuroinflammation. Somatostatin (SST) is a potent neuroprotective factor, which is down-regulated during early DR. In this paper, we have investigated the effects of the inflammatory signals triggered by the activation of microglia on inflammation and apoptosis/survival pathways in pericytes. Microglia cells (Bv-2) were stimulated with lipopolysaccharide (LPS) and/or SST. Human retinal pericytes (HRP) were exposed to conditioned media (CM) collected from Bv-2 cells in physiological conditions and in the settings described above. A panel of inflammation, apoptosis and survival mediators was analyzed. HRP treated with LPS-CM showed a significant increase of pro-inflammatory (iNos and TNFα) and pro-apoptotic mediators (FasL, active caspase-8, tBid and Bax), and a concomitant decrease in pro-survival factors (BclxL and pAkt). SST added to LPS was able to counteract these effects in all conditions. In conclusion, SST is able to modulate apoptosis/survival pathways in HRP during microglia-mediated inflammation. These results demonstrate a crosstalk between microglia and retinal pericytes, evidencing a possible defensive role of microglia in the early phases of DR. 10.1016/j.exer.2017.07.011
    LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell Proliferation. Mao Hua,Lockyer Pamela,Townley-Tilson W H Davin,Xie Liang,Pi Xinchun Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:We recently demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis. APPROACH AND RESULTS:Depletion of LRP1 in endothelial cells results in increased retinal neovascularization in a mouse model of oxygen-induced retinopathy. Specifically, retinas in mice lacking endothelial LRP1 have more branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Increased endothelial proliferation as detected by Ki67 staining was observed in LRP1-deleted retinal endothelium in response to hypoxia. Using an array of biochemical and cell biology approaches, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1) directly interacts with LRP1 in human retinal microvascular endothelial cells. This interaction between LRP1 and PARP-1 decreases under hypoxic condition. Moreover, LRP1 knockdown results in increased PARP-1 activity and subsequent phosphorylation of both retinoblastoma protein and cyclin-dependent kinase 2, which function to promote cell cycle progression and angiogenesis. CONCLUSIONS:Together, these data reveal a pivotal role for LRP1 in endothelial cell proliferation and retinal neovascularization induced by hypoxia. In addition, we demonstrate for the first time the interaction between LRP1 and PARP-1 and the LRP1-dependent regulation of PARP-1-signaling pathways. These data bring forth the possibility of novel therapeutic approaches for pathological angiogenesis. 10.1161/ATVBAHA.115.306713
    A Protective Effect of PPARα in Endothelial Progenitor Cells Through Regulating Metabolism. Shao Yan,Chen Jianglei,Dong Li-Jie,He Xuemin,Cheng Rui,Zhou Kelu,Liu Juping,Qiu Fangfang,Li Xiao-Rong,Ma Jian-Xing Diabetes Deficiency of endothelial progenitor cells, including endothelial colony-forming cells (ECFCs) and circulating angiogenic cells (CACs), plays an important role in retinal vascular degeneration in diabetic retinopathy (DR). Fenofibrate, an agonist of peroxisome proliferator-activated receptor α (PPARα), has shown therapeutic effects on DR in both patients and diabetic animal models. However, the function of PPARα in ECFC/CACs has not been defined. In this study, we determined the regulation of ECFC/CAC by PPARα. As shown by flow cytometry and Seahorse analysis, ECFC/CAC numbers and mitochondrial function were decreased in the bone marrow, circulation, and retina of mice, correlating with PPARα downregulation. Activation of PPARα by fenofibrate normalized ECFC/CAC numbers and mitochondrial function in diabetes. In contrast, knockout exacerbated ECFC/CAC number decreases and mitochondrial dysfunction in diabetic mice. Primary ECFCs from mice displayed impaired proliferation, migration, and tube formation. Furthermore, ECFCs showed reduced mitochondrial oxidation and glycolysis compared with wild type, correlating with decreases of Akt phosphorylation and expression of its downstream genes regulating ECFC fate and metabolism. These findings suggest that PPARα is an endogenous regulator of ECFC/CAC metabolism and cell fate. Diabetes-induced downregulation of PPARα contributes to ECFC/CAC deficiency and retinal vascular degeneration in DR. 10.2337/db18-1278
    Efficacy of Lenvatinib, a multitargeted tyrosine kinase inhibitor, on laser-induced CNV mouse model of neovascular AMD. Wei Xian,Zhang Ting,Yao Yuqin,Zeng Shaoxue,Li Min,Xiang Haotian,Zhao Chengjian,Cao Guiqun,Li Minhui,Wan Ran,Yang Ping,Yang Jinliang Experimental eye research Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ± 0.2939 vs 5.202 ± 0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ± 0.4334 vs 3.122 ± 0.3466 (P = .0064) at day 14th, 1.401 ± 0.6577 vs 2.781 ± 0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T in retina was 3.81 ± 0.77 h, the T was 4.60 ± 0.73 h, the AUC was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD. 10.1016/j.exer.2017.12.009
    Downregulation of CKS1B restrains the proliferation, migration, invasion and angiogenesis of retinoblastoma cells through the MEK/ERK signaling pathway. Zeng Zhou,Gao Zhao-Lin,Zhang Zhi-Pei,Jiang Hai-Bo,Yang Chang-Quan,Yang Jie,Xia Xiao-Bo International journal of molecular medicine Retinoblastoma (RB) is a common neoplasm that is exhibited in individuals globally. Increasing evidence demonstrated that cyclin‑dependent kinase regulatory subunit 1B (CKS1B) may be involved in the pathogenesis of various tumor types, including multiple myeloma and breast cancer. In the present study, the hypothesis that CKS1B downregulation would effectively inhibit the proliferation, invasion and angiogenesis of RB cells through the mitogen‑activated protein kinase kinase (MEK)/extracellular signal‑regulated kinase (ERK) signaling pathway was examined. Initial investigation of the expression profile of CKS1B in RB and adjacent retina tissues was performed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. A total of three RB cell lines, SO‑RB50, Y79 and HXO‑RB44, were examined for selection of the cell line with the highest expression of CKS1B, and human normal retinal vascular endothelial cells (ACBRI‑181) were also evaluated. CKS1B short hairpin RNA (shRNA) sequences (shRNA CKS1B‑1, shRNA CKS1B‑2 and shRNA CKS1B‑3) and negative control shRNA sequences were constructed and transfected into cells at the third generation to evaluate the role of shCKS1B and the MEK/ERK signaling pathway in RB. Furthermore, the effect of shCKS1B on cell proliferation, migration, invasion, apoptosis and angiogenesis was investigated. CKS1B was determined to be highly expressed in RB tissue, compared with adjacent retina tissue. SO‑RB50 and HXO‑RB44 cells treated with shRNA CKS1B‑1 and shRNA CKS1B‑2 were selected for the present experiments. Activation of the MEK/ERK signaling pathway increases the expression of MEK, ERK, B‑cell lymphoma 2, proliferating cell nuclear antigen, cyclin D1, vascular endothelia growth factor and basic fibroblast growth factor, enhances cell proliferation, migration, invasion and lumen formation, and decreases apoptosis. Following silencing CKS1B, the aforementioned conditions were reversed. The key observations of the present study demonstrated that shCKS1B can inhibit the proliferation, invasion and angiogenesis of RB cells by suppressing the MEK/ERK signaling pathway. Thus, CKS1B represents a potential research target in the development of therapeutics for RB. 10.3892/ijmm.2019.4183
    Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis. Sun Jian,Huang Wei,Yang Shuo-Fei,Zhang Xiao-Pei,Yu Qing,Zhang Zhi-Qing,Yao Jin,Li Ke-Ran,Jiang Qin,Cao Cong Theranostics VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis. Using genetic strategies, we tested the requirement of α subunits of heterotrimeric G proteins (Gαi1/3) in the process. Gαi1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. Gαi1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that Gαi1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. , Gαi1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of Gαi1/3 shRNA lentivirus. Moreover, angiogenesis by alkali burn and OIR was significantly attenuated in Gαi1/3 double knockout mice. Significantly, Gαi1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients. These results provide mechanistic insights into the critical role played by Gαi1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis. 10.7150/thno.26203
    Simultaneous interference of SP1 and HIF1α retarding the proliferation, migration, and invasion of human microvascular endothelial cells (HMEC-1) under hypoxia. Ai Liqianyu,Lin Sen,Huang Chanjuan,Gao Ling,Zhou Jiaxing,Chen Chunlin,Ye Jian Journal of cellular biochemistry OBJECTIVE:To investigate the regulation of special protein 1 (SP1) and hypoxia-inducible factor-1α (HIF1α) on human microvascular endothelial cells (HMEC-1) under hypoxic conditions. METHODS:The expression of SP1 and HIF1α under normoxia and hypoxic conditions were assessed by Western blot. SP1 and HIF1α were knocked down by small interfering RNA (siRNA) under hypoxic conditions. The proliferation, migration, and invasion of HMEC-1 were measured by cell counting kit 8, 5-ethynyl-2'-deoxyuridine and Transwell coculture system. Western blot analysis and Immunofluorescence were carried out to study the mechanisms of simultaneously inhibiting the adenosine triphosphatase (CD39), 5'-nucleotidase (CD73), adenosine, and vascular endothelial growth factor (VEGF). We compared the inhibitory effects between groups concurrently interfering SP1, HIF-1α, and ranibizumab under hypoxic conditions. RESULTS:Under hypoxic conditions, the protein expression of SP1 and HIF1α was increased in HMEC-1, contrarily, SP1 siRNA and HIF1α siRNA downregulated the expression. Simultaneous inhibition of SP1 and HIF1α demonstrated a much greater restraint of proliferation, migration, and invasion characteristics on HMEC-1 than respectively knocking down SP1 or HIF1α and anti-VEGF drugs (0.5 mg/mL ranibizumab) (siRNA and the VEGF inhibitor were applied separately in different groups). Meanwhile, simultaneous inhibition of SP1 and HIF1α effectively reduced the expression of CD39, CD73, adenosine, and VEGF on HMEC-1 under hypoxic conditions. CONCLUSIONS:Our study demonstrated that both SP1 and HIF1α played important roles in HMEC-1 under hypoxia condition. Simultaneous inhibition of SP1 and HIF1α effectively decreased the activity of HMEC-1 under hypoxic conditions through the CD39-CD73-adenosine and VEGF angiogenesis pathways. Our study may provide a new approach to the treatment of retinal neovascular diseases. 10.1002/jcb.29059
    Activation of ERK pathway is required for 15-HETE-induced angiogenesis in human umbilical vascular endothelial cells. Wang Shuang,Cao Weiwei,Xing Hao,Chen Ying Li,Li Qian,Shen Tingting,Jiang Chun,Zhu Daling Journal of receptor and signal transduction research Angiogenesis plays a critical role in the progression of cardiovascular disease, retinal ischemia, or tumorigenesis. The imbalance of endothelial cell proliferation and apoptosis disturbs the establishment of the vasculogenesis, which is affected by several arachidonic acid metabolites. 15-Hydroxyeicosatetraenoic acid (15-HETE) is one of the metabolites. However, the underlying mechanisms of angiogenesis induced by 15-HETE in human umbilical vascular endothelial cells (HUVECs) are still poorly understood. Since extracellular signal-regulated kinase (ERK) is a critical regulator of cell proliferation, there may be a crosstalk between 15-HETE-regulating angiogenic process and ERK-proliferative effect in HUVECs. To test this hypothesis, we study the effect of 15-HETE on cell proliferation, angiogenesis, and apoptosis using cell viability measurement, cell cycle analysis, western blot, scratch-wound, tube formation assay, and nuclear morphology determination. We found that 15-HETE promoted HUVEC angiogenesis, which were mediated by ERK. Moreover, 15-HETE-induced proliferation and cell cycle transition from the G(0)/G(1) phase to the G(2)/M + S phase. All these effects were reversed after blocking ERK with PD98059 (an ERK inhibitor). In addition, HUVEC apoptosis was relieved by 15-HETE through the ERK pathway. Thus, ERK is necessary for the effects of 15-HETE in the regulation of HUVEC angiogenesis, which may be a novel potential target for the treatment of angiogenesis-related diseases. 10.3109/10799893.2015.1077865
    Subfoveal Choroidal Thickness in Eyes with Neovascular Age-Related Macular Degeneration Treated with Anti-Vascular Endothelial Growth Factor Agents. Kanadani Tereza Cristina Moreira,Veloso Carlos Eduardo,Nehemy Márcio B Ophthalmologica. Journal international d'ophtalmologie. International journal of ophthalmology. Zeitschrift fur Augenheilkunde PURPOSE:We aimed to assess the subfoveal choroidal thickness (SFChT) and the effect of treatment with anti-vascular endothelial growth factor (anti-VEGF) agents on the SFChT in age-related macular degeneration (AMD) subtypes. METHODS:We enrolled 128 eyes of 107 patients with neovascular AMD (60 women; 47 men; mean age, 73.6 ± 8.9 years), and prospectively evaluated the best-corrected visual acuity (BCVA) and SFChT at baseline and at 3, 6, and 12 months after treatment with anti-VEGF agents. Patients were assigned to the typical AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP) subgroups. RESULTS:In total, 85 (66.4%), 31 (24.2%), and 12 (9.4%) eyes were assigned to the typical AMD, PCV, and RAP subgroups, respectively. The baseline mean BCVA was 0.75 ± 0.26, 0.72 ± 0.21, and 0.77 ± 0.24 logMAR in the typical AMD, PCV, and RAP subgroups, respectively (p = 0.774). The mean baseline SFChT was 203.20 ± 35.80, 271.80 ± 24.50, and 182.93 ± 31.31 µm, respectively (p < 0.001). Mean SFChT significantly decreased from baseline to 3, 6, and 12 months after treatment. The RAP subtype presented a significantly higher decrease in SFChT compared to the other subtypes (p = 0.01). The percentage reduction in SFChT was not significantly correlated with the number of injections (r = -0.02; p = 0.823). No association was observed between baseline SFChT and final visual acuity at 12 months (r = 0.0; p = 0.586). CONCLUSIONS:SFChT was greatest in eyes with PCV and least in eyes with RAP. The reduction in SFChT after treatment was greater in the RAP cases. The decrease in SFChT after 12 months of anti-VEGF treatment was not associated with the number of injections and there was no correlation between the baseline SFChT and visual acuity in all AMD subtypes. 10.1159/000488254
    Protein kinase C-mediated insulin receptor phosphorylation in diabetic rat retina. Kida Teruyo,Oku Hidehiro,Horie Taeko,Osuka Sho,Fukumoto Masanori,Ikeda Tsunehiko Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie PURPOSE:Diabetic retinopathy (DR) involves a proliferation of vascular endothelial cells and loss of pericytes. There is a link among the action of protein kinase C (PKC) and insulin signaling. Thus, we investigated the differences between these cells in insulin receptor (IR) phosphorylation in DR. METHODS:Retinas were removed from streptozotocin-induced diabetic or healthy rats, and IR expression levels were compared by immunoblot and immunohistochemistry. In vitro assays also were performed in order to determine the expressions of phosphorylated IR in both cells cultured under 5.5 or 25 mM glucose by immunoblot. Cell viability was determined in both cells cultured under different concentrations of phorbol myristate acetate (PMA), a PKC activator. To determine the involvement of the PI3 kinase pathway of IR, PMA with or without wortmannin-induced changes in Akt was also analyzed. RESULTS:Immunoreactivity to the IR was decreased in diabetic retina. High glucose (25 mM) increased phosphorylated IR levels in endothelial cells but not in pericytes. PMA (1 nM or higher) induced death of pericytes, while endothelial cells were increased. PMA increased phosphorylated Akt in endothelial cells and decreased in pericytes. Wortmannin suppressed the PMA-induced phosphorylation of Akt in endothelial cells. CONCLUSIONS:The different responses to 25 mM glucose and PMA were observed between retinal endothelial cells and pericytes. Thus, IR phosphorylation is likely important for retinal vascular cells to survive in diabetic retina. 10.1007/s00417-019-04324-z
    Short-term efficacy of intravitreal Aflibercept injections for retinal angiomatous proliferation. Chou Hung-Da,Wu Wei-Chi,Wang Nan-Kai,Chuang Lan-Hsin,Chen Kuan-Jen,Lai Chi-Chun BMC ophthalmology BACKGROUND:To evaluate the short-term efficacy of intravitreal injections of aflibercept (IVA) to treat retinal angiomatous proliferation (RAP) and identify factors related to functional outcomes. METHODS:This retrospective case series consisted of 19 eyes in 19 patients with RAP. All 19 eyes received 3 monthly consecutive IVA. The primary outcome measures were best-corrected visual acuity (BCVA) and central retinal thickness (CRT) after the last IVA. RESULTS:Of the 19 treated eyes, 8 (42%) were pre-treated with 1 dose of bevacizumab one month prior to the initiation of treatment with aflibercept. BCVA was significantly improved and CRT was significantly reduced after 3 consecutive IVAs (P = 0.014 and P = 0.0002, respectively). Stabilization or improvement in BCVA was observed in 17 eyes (90%) treated with IVA. Eyes with baseline fibrovascular pigment epithelial detachment (PED) showed no significant gain in BCVA, and fibrovascular PED was negatively correlated with final BCVA (Spearman's correlation coefficient = - 0.481, P = 0.037). The mean follow-up was 3.5 ± 0.5 months. CONCLUSIONS:In this short-term study, three consecutive IVAs showed efficacy for improving vision and reducing retinal edema in RAP patients. Eyes with fibrovascular PED showed poorer responses, and the presence of fibrovascular PED at baseline was negatively correlated with visual outcomes. 10.1186/s12886-017-0497-0
    The Impact of Hyperglycemia on VEGF Secretion in Retinal Endothelial Cells. Caprnda Martin,Kubatka Peter,Saxena Sandeep,Valaskova Jela,Stefanickova Jana,Kobyliak Nazarii,Zulli Anthony,Kruzliak Peter Folia medica BACKGROUND:Diabetic retinopathy is a serious sight-threatening complication which is manifested by excessive angiogenesis in diabetic patients. AIM:We hypothesize that cultured Rhesus monkey retinal endothelial cells (RhRECs) respond to high glucose with a change in cell proliferation and vascular endothelial growth factor (VEGF) secretion. MATERIALS AND METHODS:In our study, 20 000 cells per well were treated without glucose or with 5.5 mM, 18.5 mM and 30 mM glucose for 24 hours. Viable cells were counted using trypan blue dye exclusion method. VEGF concentrations were measured in cell media by ELISA method. RESULTS:The number of viable cells incubated with 5.5 mM glucose increased significantly by 53.7% after 24 hours. In comparison, the number of viable cells decreased by 2.8% at 18.5 mM of glucose and by 20.4% at 30 mM of glucose after 24 hours of incubation. In contrast to this effect of glucose on the number of viable cells, a significant increase in VEGF levels (pg/mL) in the cell media with a glucose concentration of 0 mM compared to 5.5 mM of glucose was found. VEGF secretion in cell medium with 18.5 and 30 mM of glucose increased non-significantly in comparison with euglycemic levels. CONCLUSION:Our results show that viability of retinal endothelial cells and VEGF release are highly responsive to changes in glucose concentration. Such glucose-induced changes in retinal endothelial cells may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and microaneursym. 10.1515/folmed-2017-0029
    Low expression of microRNA-15b promotes the proliferation of retinal capillary endothelial cells and pericytes by up-regulating VEGFA in diabetic rats. Xu Y,Xie S-C,Ma Y-C European review for medical and pharmacological sciences OBJECTIVE:To investigate the role of microRNA-15b in diabetic retinopathy and its underlying mechanism. MATERIALS AND METHODS:Diabetes rat model was established by streptozotocin injection. The mRNA expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor A (VEGFA) were detected by qRT-PCR and Western blot, respectively. MicroRNA-15b mimics or inhibitor were transfected into retinal capillary endothelial cells and pericytes of diabetic rats, respectively. The mRNA expressions of microRNA-15b and VEGFA were detected by qRT-PCR. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation of capillary endothelial cells and pericytes. Dual-Luciferase reporter gene assay was conducted to verify the binding condition of microRNA-15b and VEGFA. RNA immunoprecipitation (RIP) assay was performed to determine whether microRNA-15b could bind to AGO2. Rescue experiments were finally carried out by detecting the proliferation of retinal capillary endothelial cells and pericytes after downregulation or overexpression of microRNA-15b and VEGFA. RESULTS:The expression of microRNA-15b decreased, whereas VEGFA expression increased in retinal capillary endothelial cells and pericytes of diabetic rats. High expression of microRNA-15b in retinal capillary endothelial cells and pericytes resulted in VEGFA down-regulation and decreased proliferation. RIP assay results indicated that microRNA-15b could interact with AGO2. Additionally, Dual-Luciferase reporter gene assay showed that VEGFA is a direct target gene of microRNA-15b. VEGFA overexpression could reverse the inhibited proliferation of retinal capillary endothelial cells and pericytes induced by microRNA-15b overexpression. Similarly, VEGFA knockdown could reverse the effect of the low expression of microRNA-15b on the proliferation of retinal capillary endothelial cells and pericytes. CONCLUSIONS:Low expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats promotes the development of diabetic retinopathy by up-regulating VEGFA. 10.26355/eurrev_201907_18413
    Knockdown of Fibromodulin Inhibits Proliferation and Migration of RPE Cell via the VEGFR2-AKT Pathway. Hu He,Li Shanshan,Li Jianqiao,Huang Chao,Zhou Fang,Zhao Li,Yu Wenzhen,Qin Xiao Journal of ophthalmology Purpose:Recent research has provided novel insight into the function of fibromodulin (FMOD) in wound healing and angiogenesis. The role of FMOD in initiation of proliferative vitreoretinopathy (PVR) has not been studied. This study investigated the effect of FMOD on human retinal pigment epithelial (RPE) cell, which plays an essential role in the progression of PVR, and the possible mechanisms. Methods:Small interfering (si) RNA-based gene transfer technology was used to decrease FMOD expression and to study its effects on RPEs . Cell Counting Kit-8 assays, transwells, and flow cytometry analysis were used to measure cell proliferation, migration, cell cycle, and apoptosis. Western blot was used to measure expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), extracellular signal-related kinase 1/2 (ERK1/2), and phosphoinositide 3 kinase (PI3K/AKT). Results:After transfection of RPEs with a FMOD-specific siRNA, cell proliferation and migration were inhibited to the percentage of 65% ± 5% and 39% ± 10%, respectively, compared to the control group. Depletion of FMOD induced cell cycle arrest and apoptosis in RPE cells. Downregulation of VEGF, VEGFR2, and phosphorylated AKT (p-AKT) were detected in transfected RPEs. Conclusion:Depletion of FMOD selectively downregulated the expression of VEGF and VEGFR2 and inhibited the signaling pathway of AKT phosphorylation, which consequently inhibited the proliferation and migration of RPE Cell. 10.1155/2018/5708537
    Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis. Lam Jonathan D,Oh Daniel J,Wong Lindsay L,Amarnani Dhanesh,Park-Windhol Cindy,Sanchez Angie V,Cardona-Velez Jonathan,McGuone Declan,Stemmer-Rachamimov Anat O,Eliott Dean,Bielenberg Diane R,van Zyl Tave,Shen Lishuang,Gai Xiaowu,D'Amore Patricia A,Kim Leo A,Arboleda-Velasquez Joseph F Diabetes Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31 vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis. 10.2337/db16-1035
    Protective effect of miR-200b/c by inhibiting vasohibin-2 in human retinal microvascular endothelial cells. Ding Yuzhi,Hu Zizhong,Luan Jie,Lv Xuehua,Yuan Dongqing,Xie Ping,Yuan Songtao,Liu Qinghuai Life sciences AIMS:Proliferative diabetic retinopathy (PDR), characterized by angiogenesis, can cause serve vision loss and even blindness. Recent studies have suggested a pivotal role of vasohibin-2 (VASH2) in the promotion of angiogenesis in tumor tissues. Here we further investigated the role of VASH2 in the proliferation and migration of retinal endothelial cells. MAIN METHODS:The expression of VASH2 in vascular endothelial cells of epiretinal fibrovascular membranes (FVMs) from PDR patients were detected by immunofluorescence. VASH2 gene interfering lentiviral vectors (VASH2-shRNA) and miR-200b/c were constructed for the evaluation of the VASH2 effect on high glucose induced human retinal microvascular endothelial cell line (HRMECs). Cell proliferation, cell cycle and cell migration were carried out subsequently. The relationship between VASH2 and miR-200b/c was determined by luciferase reporter gene assays. KEY FINDINGS:A positive expression of VASH2 was identified in vascular endothelial cells of FVMs from PDR patients. In HRMECs, cells transfected with shRNA or miR-200b/c mimics showed a significantly reduced VASH2 expression compared with negative control group by real time-polymerase chain reaction and western-blot analysis. Inhibition of VASH2 was demonstrated to suppress cell proliferation and migration from Day 2 to Day 4. The luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200b/c in HRMECs. SIGNIFICANCE:The present study suggests a protective effect of miR-200b/c on high glucose induced HRMECs dysfunction by inhibiting VASH2. It could be a potential therapeutic strategy to inhibit angiogenesis for the treatment of retinal vascular disease. 10.1016/j.lfs.2017.09.001
    Olfactomedin 2 Regulates Smooth Muscle Phenotypic Modulation and Vascular Remodeling Through Mediating Runt-Related Transcription Factor 2 Binding to Serum Response Factor. Shi Ning,Li Chen-Xiao,Cui Xiao-Bing,Tomarev Stanislav I,Chen Shi-You Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. APPROACH AND RESULTS:Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB-induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury-induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury-induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo. CONCLUSIONS:Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases. 10.1161/ATVBAHA.116.308606
    Newly Identified Peptide, Peptide Lv, Promotes Pathological Angiogenesis. Shi Liheng,Zhao Min,Abbey Colette A,Tsai Shu-Huai,Xie Wankun,Pham Dylan,Chapman Samantha,Bayless Kayla J,Hein Travis W,Rosa Robert H,Ko Michael L,Kuo Lih,Ko Gladys Y-P Journal of the American Heart Association Background We recently discovered a small endogenous peptide, peptide Lv, with the ability to activate vascular endothelial growth factor receptor 2 and its downstream signaling. As vascular endothelial growth factor through vascular endothelial growth factor receptor 2 contributes to normal development, vasodilation, angiogenesis, and pathogenesis of various diseases, we investigated the role of peptide Lv in vasodilation and developmental and pathological angiogenesis in this study. Methods and Results The endothelial cell proliferation, migration, and 3-dimensional sprouting assays were used to test the abilities of peptide Lv in angiogenesis in vitro. The chick chorioallantoic membranes and early postnatal mice were used to examine its impact on developmental angiogenesis. The oxygen-induced retinopathy and laser-induced choroidal neovascularization mouse models were used for in vivo pathological angiogenesis. The isolated porcine retinal and coronary arterioles were used for vasodilation assays. Peptide Lv elicited angiogenesis in vitro and in vivo. Peptide Lv and vascular endothelial growth factor acted synergistically in promoting endothelial cell proliferation. Peptide Lv-elicited vasodilation was not completely dependent on nitric oxide, indicating that peptide Lv had vascular endothelial growth factor receptor 2/nitric oxide-independent targets. An antibody against peptide Lv, anti-Lv, dampened vascular endothelial growth factor-elicited endothelial proliferation and laser-induced vascular leakage and choroidal neovascularization. While the pathological angiogenesis in mouse eyes with oxygen-induced retinopathy was enhanced by exogenous peptide Lv, anti-Lv dampened this process. Furthermore, deletion of peptide Lv in mice significantly decreased pathological neovascularization compared with their wild-type littermates. Conclusions These results demonstrate that peptide Lv plays a significant role in pathological angiogenesis but may be less critical during development. Peptide Lv is involved in pathological angiogenesis through vascular endothelial growth factor receptor 2-dependent and -independent pathways. As anti-Lv dampened the pathological angiogenesis in the eye, anti-Lv may have a therapeutic potential to treat pathological angiogenesis. 10.1161/JAHA.119.013673
    Circular RNAs: Novel Promising Biomarkers in Ocular Diseases. Guo Nan,Liu Xiu-Fen,Pant Om Prakash,Zhou Dan-Dan,Hao Ji-Long,Lu Cheng-Wei International journal of medical sciences Circular RNAs (circRNAs) are a novel class of endogenous non-coding RNAs produced by back-splicing. They are found to be expressed in eukaryotic cells and play certain roles in various cellular functions, including fibrosis, cell proliferation, differentiation, apoptosis and angiogenesis. Dysregulated circRNAs are found in several human disorders including, malignancy, vascular, inflammatory as well as nervous diseases. Although, increasing evidence suggests that circRNAs may also contribute in different ocular diseases, the outline of circRNAs in ocular diseases remains obscure. In this review we consider the current state of knowledge regarding the potential role and underlying mechanism of circRNAs in ocular diseases including pterygium, age-related cataract, glaucoma, diabetic retinopathy, retinoblastoma, retinal vascular dysfunction and hyperhomocysteinemia induced ocular diseases, emphasizing that circRNAs could be promising biomarkers for the diagnosis and prognosis evaluation. Future circRNAs-targeted intervention may become a novel therapeutic tool for the treatment of ocular diseases. 10.7150/ijms.29750
    Application of recombinant peroxisome proliferator-activated receptor-γ coactivator-1α mediates neovascularization in the retina. Jiang Jian,Zhang Lixin,Zhang Lu,Xia Xiaobo Molecular medicine reports Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is able to induce the expression of vascular endothelial growth factor (VEGF), promoting the formation of new blood vessels in skeletal muscle. The aim of the current study was to determine whether PGC-1α is able to regulate angiogenesis in human retinal vascular endothelial cells (hRVECs) in vitro and in retinas in vivo. hRVECs treated with recombinant PGC-1α were incubated for 24 h and then placed into a normoxic (20% O2) or hypoxic (1% O2) environment for a further 16 h. Following this, VEGF mRNA and protein levels were significantly increased. Cellular proliferation was enhanced by treatment with recombinant PGC-1α in normoxic and hypoxic conditions. At 24 h following recombinant PGC-1α treatment, hRVECs were plated into Matrigel-coated plates and cultured under normoxic (20% O2) or hypoxic (1% O2) conditions for a further 24 h. Recombinant PGC-1α-treated cells were observed to form significantly greater numbers of tubes. In a C57BL/6J mouse model of ischemic retinopathy, mice received an intravitreal injection of recombinant PGC-1α, resulting in a significant increase in VEGF mRNA and protein levels in the retina. Retinal neovascular tufts and neovascular nuclei were investigated by angiographic and cross-sectional analysis and were observed to be significantly increased in the PGC-1α group compared with the control group. These results indicate that PGC-1α is able to induce angiogenesis in hRVECs and retinas, and suggests that PGC-1α is a potential anti-angiogenic target in retinal neovascularization. 10.3892/mmr.2015.4678
    Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2. Guo Ting,Song Hongyuan,Zhao Zichang,Qi Zhongtian,Zhao Shihong Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology BACKGROUND/AIMS:Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. METHODS:We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. RESULTS:Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2. CONCLUSIONS:Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future. 10.1159/000489209
    Anti-Human VEGF Repebody Effectively Suppresses Choroidal Neovascularization and Vascular Leakage. Hwang Da-Eun,Ryou Jeong-Hyun,Oh Jong Rok,Han Jung Woo,Park Tae Kwann,Kim Hak-Sung PloS one Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers. 10.1371/journal.pone.0152522
    Discovery of 4-Chromen-4-one Derivatives as a New Class of Selective Rho Kinase (ROCK) Inhibitors, which Showed Potent Activity in ex Vivo Diabetic Retinopathy Models. Zhao Lanying,Li Yueshan,Wang Yujiao,Qiao Zeen,Miao Zhuang,Yang Jiao,Huang Luyi,Tian Chenyu,Li Linli,Chen Danian,Yang Shengyong Journal of medicinal chemistry Diabetic retinopathy (DR) is a major cause of blindness, and there is a lack of effective treatment at present. Rho-associated coiled-coil containing serine/threonine protein kinases (ROCKs) have recently been suggested as potential targets for the DR treatment. We herein report the discovery of 4-chromen-4-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship analyses led to the identification of the most active compound, 4-(dimethylamino)--(3-{2-[(4-oxo-4-chromen-7-yl)oxy]acetamido}phenyl) (). This compound showed excellent kinase selectivity for ROCK I and ROCK II against 387 other kinases. In retinal explants, compound protected retinal neurons from high glucose-induced oxidative stress and apoptosis-mediated cell death. Furthermore, administration suppressed the improper proliferation of Müller cells and promoted the regression of vascular vessels in retinal explants cultured in a high glucose microenvironment. Collectively, our data suggest that could be a potential lead compound for the treatment of DR, hence deserving further in-depth studies. 10.1021/acs.jmedchem.9b01143
    MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways. Wu Jin-Hui,Wang Yi-Han,Wang Wei,Shen Wei,Sang Yan-Zhi,Liu Lin,Chen Cui-Min The international journal of biochemistry & cell biology MicroRNAs (miRNAs) are important for the proliferation of endothelial cells and have been shown to be involved in diabetic retinopathy (DR). In previous study, we found that miRNAs might play a critical role in hyperglycemia-induced endothelial cell proliferation based on miRNA expression profiling. Here, the roles of microRNA-18b (miR-18b) in the proliferation of human retinal endothelial cells (HRECs) were investigated in an in vitro model of HRECs grown in high glucose. We identified that levels of miR-18b were decreased in high-glucose-induced HRECs, compared with those in cells incubated in normal glucose. However, the reduction of miR-18b up-regulated vascular endothelial growth factor (VEGF) secretion and promoted effects on in vitro proliferation of HRECs. Mechanistically, insulin growth factor-1 (IGF-1) was identified as a target of miR-18b. IGF-1 simulation could antagonize the effect induced by miR-18b up-regulation, promoting cell proliferation and increasing VEGF production. In contrast, the opposite results were observed with silencing IGF-1, which was consistent with the effects of miR-18b overexpression. MiR-18b exerted its function on VEGF synthesis and cell proliferation by suppressing the IGF-1/insulin growth factor-1 receptor (IGF1R) pathway, consequently inhibiting the downstream phosphorylation of Akt, MEK, and ERK. Hence, this may provide a new insight into understanding the mechanism of DR pathogenesis, as well as a potential therapeutic target for proliferative DR. 10.1016/j.biocel.2016.02.002
    Caspase-8 modulates physiological and pathological angiogenesis during retina development. Tisch Nathalie,Freire-Valls Aida,Yerbes Rosario,Paredes Isidora,La Porta Silvia,Wang Xiaohong,Martín-Pérez Rosa,Castro Laura,Wong Wendy Wei-Lynn,Coultas Leigh,Strilic Boris,Gröne Hermann-Josef,Hielscher Thomas,Mogler Carolin,Adams Ralf H,Heiduschka Peter,Claesson-Welsh Lena,Mazzone Massimiliano,López-Rivas Abelardo,Schmidt Thomas,Augustin Hellmut G,Ruiz de Almodovar Carmen The Journal of clinical investigation During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-8ECKO) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECKO pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP. 10.1172/JCI122767
    Prognostic factors of revitrectomy for complications in eyes with proliferative diabetic retinopathy: a retrospective multicentre study. Takayama Kei,Someya Hideaki,Yokoyama Hiroshi,Kimura Takeshi,Takamura Yoshihiro,Morioka Masakazu,Sameshima Seiji,Ueda Tetsuo,Ogata Nahoko,Kitano Shigehiko,Tashiro Maki,Sugimoto Masahiko,Kondo Mineo,Sakamoto Taiji,Takeuchi Masaru Acta ophthalmologica PURPOSE:To identify prognostic factors for revitrectomy in patients who underwent vitrectomy for complications with proliferative diabetic retinopathy (PDR) in multicentre study. METHODS:Consecutive 452 eyes of 452 patients with PDR undergoing 25-gauge microincision vitrectomy system (MIVS) in seven centres were retrospectivity reviewed. Preoperative ocular factors (baseline visual acuity [VA], vitreous haemorrhage [VH], tractional retinal detachment [TRD] and retinal photocoagulation), general factors (sex, age, diabetes duration, HbA1c level, hypertension, anti-coagulant medication and estimated glomerular filtration rate), surgical procedures (preoperative anti-vascular endothelial growth factor injection, internal limiting membrane peeling, combined cataract surgery, retinal break, and tamponade), postoperative complications for revitrectomy and postoperative VA at 6 months were evaluated. RESULTS:In the follow-up period of 6 months, revitrectomy was performed in 56 eyes (26.3%), and postoperative complications for revitrectomy were VH in 31 eyes (15%), TRD in 13 eyes (6.2%) and membrane proliferation in 12 eyes (5.2%). The mean LogMAR improvement from baseline to 6 months in revitrectomy group (0.39) was significantly worse than in single vitrectomy group (0.74). Diabetic duration, low baseline VA, less simple VH, TRD and air tamponade were statistical risk factors of revitrectomy, and logistic regression analysis identified low baseline VA and air tamponade also as prognostic factors of revitrectomy. CONCLUSION:Our results indicated that prognosis of VA was worse in PDR patients with revitrectomy and low baseline VA and air as the tamponade material were the potential prognostic factors of revitrectomy. 10.1111/aos.14292
    Endothelial Protein C-Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells. Arta Anthoula,Eriksen Anne Z,Melander Fredrik,Kempen Paul,Larsen Michael,Andresen Thomas L,Urquhart Andrew J Investigative ophthalmology & visual science Purpose:To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid-loaded functionalized liposomes that exhibit both enhanced uptake by HRECs and superior biologic activity compared to nontargeting liposomes and free drug. Methods:EPCR expression of HRECs was investigated through flow cytometry and Western blot assays. EPCR-targeting liposomes were developed by functionalizing EPCR-specific antibodies onto liposomes, and the uptake of liposomes was assessed with flow cytometry and confocal laser scanning microscopy. The therapeutic potential of EPCR-targeting liposomes was determined by loading them with prednisolone either through bilayer insertion and/or by remote loading into the aqueous core. The carrier efficacy was assessed in two ways through its ability to inhibit secretion of interleukins in cells stimulated with high glucose and angiogenesis in vitro by using an endothelial cell tube formation assay. Results:HRECs express EPCR at a similar level in both human aortic and umbilic vein endothelial cells. The EPCR-targeting liposomes displayed at least a 3-fold higher uptake compared to nontargeting liposomes. This enhanced uptake was translated into superior anti-inflammatory efficacy, as the corticosteroid-loaded EPCR-targeting liposomes significantly reduced the secretion of IL-8 and IL-6 and inhibited the development of cell tube formations in contrast to nontargeting liposomes. Conclusions:We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised. 10.1167/iovs.18-23800
    Apelin-13 Is an Early Promoter of Cytoskeleton and Tight Junction in Diabetic Macular Edema via PI-3K/Akt and MAPK/Erk Signaling Pathways. Li Yang,Bai Yu-Jing,Jiang Yan-Rong,Yu Wen-Zhen,Shi Xuan,Chen Li,Feng Jing,Sun Gui-Bo BioMed research international Diabetic macular edema is major cause of vision loss associated with diabetic retinopathy. Breakdown of blood-retinal barrier, especially inner BRB, is an early event in pathogenesis of DR. Apelin, an endogenous ligand of APJ, mediates angiogenesis and is involved in the development of DR. The present study aimed to investigate effects and mechanism of apelin-13 in vascular permeability during DME. We verified apelin-13 was upregulated in DME patients' vitreous. High glucose incubation led to a progressive increase of apelin-13, APJ, cytoskeleton, and tight junction proteins, including VE-Cadherin, FAK, Src, ZO-1, and occludin. Apelin-13 promoted HRMEC proliferation and migration and phosphorylation of both cytoskeleton and tight junction under both normal and high glucose conditions. Besides, apelin-13 activated PI-3K/Akt and MAPK/Erk signaling pathways, including PLC1, p38, Akt, and Erk both in HRMEC and in C57BL/6 mice. Meanwhile, F13A performed opposite effects compared with apelin-13. In study, apelin-13 was also upregulated in retina of mice. Taken together, apelin-13 increased biologic activity of HRMEC, as well as expression of both cytoskeleton and tight junction in DME via PI-3K/Akt and MAPK/Erk signaling pathways. Apelin-13 as an early promoter of vascular permeability may offer a new perspective strategy in early treatment of DR. 10.1155/2018/3242574
    Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats. Teng Fukang,Yin Ying,Cui Yajun,Deng Yanping,Li Defang,Cho Kenka,Zhang Ge,Lu Aiping,Wu Wanying,Yang Min,Liu Xuan,Guo De-an,Yin Jun,Jiang Baohong Life sciences AIMS:Despite the numerous pharmacological agents available for hypertension therapy, hypertension-related microvascular remodeling is not resolved, eventually leading to end-organ damage. The aim of the present study was to investigate the protection of salvianolic acid A (SalA) against microvascular remodeling in vitro and in vivo. MAIN METHODS:Spontaneously hypertensive rats (SHRs) were administered 2.5, 5 or 10 mg/kg SalA via intraperitoneal injection once a day for 4 weeks. The tail-cuff method was applied to monitor blood pressure; the microvascular structure of the retina was detected by hematoxylin-eosin and immunohistochemical staining; the function of mesenteric arteries was measured by DMT wire myography; endothelial cell proliferation was estimated using the Cell Counting Kit-8; endothelial cell migration was evaluated by wound healing and transwell assay; and endothelial cell integrity was detected by transendothelial electrical resistance and permeability assays. KEY FINDINGS:Although no antihypertensive effects of SalA were observed, SalA attenuated the microvascular inward remodeling of the retina and improved microvascular function in the mesenteries in vivo. Further cell experiments confirmed the beneficial effects of SalA on the integrity of the endothelial monolayer in vitro. SIGNIFICANCE:Salvianolic acid A inhibited endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats. Therefore, salvianolic acid A could be a potential drug therapy to prevent further targeted organ damage induced by vascular remodeling. 10.1016/j.lfs.2015.06.010
    ATN-161 as an Integrin α5β1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis. Sui Ailing,Zhong Yisheng,Demetriades Anna M,Shen Jikui,Su Ting,Yao Yiyun,Gao Yushuo,Zhu Yanji,Shen Xi,Xie Bing Medical science monitor : international medical journal of experimental and clinical research BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5β1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5β1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5β1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV. 10.12659/MSM.907446
    Chebulagic acid and Chebulinic acid inhibit TGF-β1 induced fibrotic changes in the chorio-retinal endothelial cells by inhibiting ERK phosphorylation. Shanmuganathan Sivasankar,Angayarkanni Narayanasamy Microvascular research PURPOSE:Diabetic retinopathy (DR) is characterized by pro-inflammatory, pro-angiogenic and pro-fibrotic environment during the various stages of the disease progression. Basement membrane changes in the retina and formation of fibrovascular membrane are characteristically seen in DR. In the present study the effect of Alcoholic (AlE) extracts of Triphala an ayurvedic herbal formulation and its chief compounds, Chebulagic (CA), Chebulinic (CI) and Gallic acid (GA) were evaluated for TGFβ1-induced anti-fibrotic activity in choroid-retinal endothelial cells (RF/6A). METHOD:RF/6A cells were treated with TGFβ1 alone or co-treated with AlE, CA, CI or GA. The mRNA and protein expression of fibrotic markers (αSMA, CTGF) were assessed by qPCR and western blot/ELISA. Functional changes were assessed using proliferation assay and migration assay. To deduce the mechanism of action, downstream signaling was assessed by western blot analysis along with in silico docking studies. RESULT:AlE (50 μg/ml) CA and CI at 10 μM reduced the expression of pro-fibrotic genes (αSMA and CTGF) induced by TGFβ1, by inhibiting ERK phosphorylation. GA did not inhibit TGFβ1 mediated changes in RF/6A cells. In silico experiments shows that CA and CI has favourable binding energy to bind with TGFβ receptor and inhibit the downstream signaling, while GA did not. CONCLUSION:Hence this study identifies Triphala and its chief compounds CA and CI as potential adjuvants in the management of DR. 10.1016/j.mvr.2018.09.001
    Tanshinone IIA inhibits high glucose‑induced proliferation, migration and vascularization of human retinal endothelial cells. Fan Keshun,Li Suyan,Liu Gaoqin,Yuan Honghua,Ma Li,Lu Peirong Molecular medicine reports Diabetic retinopathy is the most universal and severe complication of diabetes mellitus. The primary aim of the present study was to determine whether tanshinone IIA (TSA) has an inhibitory effect on the proliferation, migration and vascularization of human retinal endothelial cells (HREC) under high glucose (HG) conditions and the associated underlying mechanism. It was demonstrated that TSA exhibited a significant inhibitory effect on the proliferation, migration and vascularization of HRECs in a dose‑dependent manner, under conditions of high glucose (25 mM) medium. However, there was no distinct inhibitory effect on HREC proliferation, migration and vascularization under normal glucose (NG, 5.5 mM glucose) conditions. Reverse transcription‑quantitative polymerase chain reaction, western blotting and immunofluorescence experiments were conducted to evaluate the effects of TSA on the expression levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)‑1. It was demonstrated that TSA significantly downregulated the expression levels of VEGF and ICAM‑1 in a dose‑dependent manner under HG conditions. Overall, the results of the present study indicate that TSA‑mediated inhibition of proliferation, migration and vascularization in HRECs may be associated with its ability to affect the expression levels of VEGF and ICAM‑1. 10.3892/mmr.2017.7743
    MicroRNA-423 may regulate diabetic vasculopathy. Blum Arnon,Meerson Ari,Rohana Hanan,Jabaly Hanin,Nahul Nahul,Celesh Dorina,Romanenko Olga,Tamir Snait Clinical and experimental medicine To test the hypothesis that microRNAs may play a role in diabetic retinopathy, we measured the levels of different markers [microRNAs, vascular endothelial growth factor (VEGF), nitric oxide (NO), and total antioxidant capacity (TAO)] in patients with type 2 diabetes mellitus (T2DM) and microvascular complications. Sixty-nine patients were recruited: 22 healthy subjects, ten T2DM patients without retinopathy, 22 with nonproliferative diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). Serum levels of NO, VEGF, TAO and 16 candidate microRNAs were measured. Additionally, the mRNA levels of endothelial nitric oxide synthase (eNOS), induced NOS (iNOS), C reactive protein (CRP), VEGF, tumor necrosis factor α (TNFα), PON2, p22, and SOD2 were measured in human vascular endothelial cells cultured in the presence of pooled sera from the subject groups. Plasma miR-423 levels showed a significant ~ twofold decrease in patients with PDR compared to controls. P lasma NO levels were significantly higher in retinopathy, VEGF levels were significantly lower, and TAO was significantly decreased. eNOS mRNA levels were lower in the cells of T2DM patients without retinopathy, but higher in PDR. PON2, p22, and SOD2 mRNA levels were all significantly lower in PDR. CRP, TNFα, iNOS, and VEGF mRNA levels showed no significant association with disease status. Lowered miR-423 levels in diabetic patients showed a correlation with VEGF and an inverse correlation between NO and eNOS expression. Our findings suggest a cross talk between miR-423 and VEGF signaling, affecting eNOS function. miR-423 may be involved in the regulation of diabetic vascular retinal proliferation. 10.1007/s10238-019-00573-8
    Dexamethasone reverses the effects of high glucose on human retinal endothelial cell permeability and proliferation in vitro. Stewart E A,Saker S,Amoaku W M Experimental eye research Diabetic macular oedema (DMO), a leading cause of preventable visual loss in the working population, is caused by an increase in microvascular endothelial cell permeability, and its prevalence is on the increase in parallel with the rising worldwide prevalence of diabetes. It is known that retinal vascular leakage in DMO is contributed to by VEGF upregulation as well as non-VEGF dependent inflammatory pathways, and the potential use of anti-inflammatory agents such as the glucocorticoids, including dexamethasone are being extensively studied. However, the mechanisms of action of dexamethasone in DMO reduction are not fully understood. Using human primary retinal endothelial cells (REC) the in vitro effect of dexamethasone in modulating the proliferation, permeability and gene expression of key tight and adheren junction components, and the expression of angiopoietins (Ang) 1 and 2 in high (25 mM) glucose conditions were investigated. High glucose decreased REC proliferation, an effect that was reversed by dexamethasone. High glucose conditions significantly increased REC permeability and decreased claudin-5, occludin and JAM-A gene expression; dexamethasone was effective in partially reversing these changes, restoring EC permeability to the normal or near normal state. High glucose levels resulted in reduction of Ang1 secretion, although Ang2 levels were consistently high. DEX increased Ang1 and decreased Ang2, indicating that the balance of Ang1/Ang2 may be important in determining functional changes in REC under high glucose conditions. 10.1016/j.exer.2016.08.005
    Recombinant human maspin inhibits high glucose-induced oxidative stress and angiogenesis of human retinal microvascular endothelial cells via PI3K/AKT pathway. Qiu Feng,Tong Huijuan,Wang Yawen,Tao Jun,Wang Hailin,Chen Lei Molecular and cellular biochemistry Maspin is known as a tumor suppressor and a potent angiogenesis inhibitor, however, its effects on proliferative diabetic retinopathy (PDR) have not been fully elucidated. This study aimed at evaluating the effects of maspin on high glucose-induced oxidative stress and angiogenesis in human retinal microvascular endothelial cells (HRMECs). Herein, HRMECs were treated with 0.25, 0.5, or 1 µM recombinant human maspin in the presence of 30 mM glucose, and their proliferation, tube formation, and oxidative stress responses were further detected. Our results revealed that maspin inhibited the high glucose-induced proliferation, migration, and tube formation of HRMECs. Maspin also decreased reactive oxygen species, nitric oxide level, and increased glutathione S-transferase activity in HRMECs. Meanwhile, maspin reduced the mRNA and protein levels of hypoxia-inducible factor-1α and vascular endothelial growth factor in high glucose-stimulated cells in a dose-dependent manner. Additionally, the high glucose-induced elevation of phosphorylated phosphoinositide-3-kinase (p-PI3K) and phosphorylated AKT was also suppressed by maspin. In summary, our data suggest that maspin inhibits high glucose-induced proliferation, oxidative stress, and angiogenesis of HRMECs at least by modulating the PI3K/AKT pathway. Maspin may be a potential therapeutic agent for the prevention and treatment of PDR. 10.1007/s11010-018-3280-5
    IL-10 inhibits retinal pigment epithelium cell proliferation and migration through regulation of VEGF in rhegmatogenous retinal detachment. Zhao Quiqing,Ji Mingli,Wang Xuemei Molecular medicine reports Rhegmatogenous retinal detachment (RRD) is a disorder of the eye that affects physical and mental health. Retinal pigment epithelium (RPE) is closely associated with RRD, and it is hypothesized that RPE-secreted inflammatory cytokines may induce early pathological changes of RRD and may participate in RPE proliferation and migration. The present study determined a role for interleukin (IL)‑10 as an RPE‑secreted immune regulatory factor that contributes to RRD. A rat RRD model was established and RPE cells were isolated and cultivated. RPE cells were randomly divided into four groups, three treated with different concentrations of IL‑10 (100, 50, and 20 mM) and one untreated. RPE cell proliferation was evaluated by MTT assay and the activity of caspase‑3 was also measured. RPE cell invasion was determined by Transwell assay. Vascular endothelial growth factor A (VEGF) expression was examined by reverse transcription‑quantitative polymerase chain reaction and western blotting; IL‑1 and IL‑6 levels were measured by ELISA. IL‑10 treatment suppressed RPE cell proliferation and migration, promoted caspase‑3 activity, inhibited VEGF mRNA and protein expression, and downregulated the secretion of inflammatory cytokines IL‑1 and IL‑6 in RRD group compared with the untreated Model group. The aforementioned effects of IL‑10 became more evident with increasing IL‑10 concentration. IL‑10 suppressed inflammation, facilitated RPE cell apoptosis and inhibited cell proliferation and migration through the regulation of VEGF expression. 10.3892/mmr.2018.8787
    Long non-coding RNA HEIH contributes to diabetic retinopathy by regulating miR-939/VEGF axis. Zhao Chengyuan,Fei Xiaoqiang,Xu Bangkui,Lu Yu,Zhang Qingqing International journal of clinical and experimental pathology Diabetes is one of the most prevalent metabolic diseases in the world. This study explored the role of long non-coding RNA HEIH in regulating the development of diabetic retinopathy (DR). The expression of HEIH gene was detected in the serum of patients with DR. Subsequently, high concentrations of D-glucose (HG) were used to stimulate ARPE-19 cells to construct a cell model of DR. HEIH was overexpressed and suppressed to further investigate the effects of HEIH on HG-induced ARPE-19 cell injury. Moreover, the regulatory relationship between HEIH and miR-939 was investigated, and a target relationship between miR-939 and VEGF in ARPE-19 cells was explored. We elucidated an association between HEIH/miR-939/VEGF axis and the PI3K/AKT pathway. HEIH was highly expressed in the serum of patients with DR. Moreover, HG-induced ARPE-19 cell injury and expression of HEIH. The overexpression of HEIE aggravated HG-induced ARPE-19 cell injury by significantly inhibiting cell viability, inducing apoptosis, promoting cytochrome C release from mitochondria to cytoplasm, and enhancing the caspase-3 activity, whereas suppression of HEIE had the opposite effects. In addition, the effects of the suppression of HEIH on HG-induced ARPE-19 cell injury were markedly reversed by inhibiting miR-939. miR-939 regulated HG-induced ARPE-19 cell injury by targeting VEGF. The suppression of HEIH reversed HG-induced activation of the PI3K/AKT signaling pathway. Our findings revealed that HEIH may contribute to DR by sponging miR-939 to target VEGF expression and by regulating the activation of the PI3K/AKT pathway. Inhibition of epidermal growth factor receptor and PI3K/Akt signaling suppresses cell proliferation and survival through regulation of Stat3 activation in human cutaneous squamous cell carcinoma. HEIH/miR-939/VEGF axis may provide a novel perspective for DR therapy.
    TLR2/4 deficiency prevents oxygen-induced vascular degeneration and promotes revascularization by downregulating IL-17 in the retina. He Chang,Lai Peilong,Wang Jing,Zhou Tian,Huang Zijing,Zhou Lingli,Liu Xialin Scientific reports Vascular degeneration is a critical pathological process in many human degenerative diseases, which need efficient ways to revascularization. However, little is known about cellular and molecular mechanisms that are used during vascular degeneration and revascularization. Here, we show that Toll-like receptor 2 and 4 (TLR2/4) double deficiency suppressed hyperoxia induced retinal vessel regression in an oxygen-induced retinopathy (OIR) model. Notably, the TLR2/4-/- mice experienced more revascularization after reduced vessel regression compared with wild-type mice, accompanied with less activation of glial cells. Mechanistically, TLR2/4 activation can tip the balance between Th17 cells and regulatory T cells towards Th17 cells, a critical source of the IL-17A. Less migration and infiltration of IL-17A-expressing proinflammatory cells but elevated regulatory T cells were observed in OIR-retinae from TLR2/4-/- mice. Coincidentally, TLR2/4 deficiency suppressed IL-17A production and increased expressions of anti-inflammatory genes. Furthermore, IL-17A promoted activation of glial cells. IL-17A blockade using a neutralizing antibody alleviated retinal cell apoptosis and glial activation in C57/B6-OIR mice, demonstrating the important role of IL-17A pathway in glial function during revascularization. Thus TLR2/4-mediated IL-17A inflammatory signaling is involved in vessel degeneration and revascularization, indicating that modulation of the TLR2/4-IL-17A pathway may be a novel therapeutic strategy for degenerative diseases. 10.1038/srep27739
    Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization. Ali Zaheer,Mukwaya Anthony,Biesemeier Antje,Ntzouni Maria,Ramsköld Daniel,Giatrellis Sarantis,Mammadzada Parviz,Cao Renhai,Lennikov Anton,Marass Michele,Gerri Claudia,Hildesjö Camilla,Taylor Michael,Deng Qiaolin,Peebo Beatrice,Del Peso Luis,Kvanta Anders,Sandberg Rickard,Schraermeyer Ulrich,Andre Helder,Steffensen John F,Lagali Neil,Cao Yihai,Kele Julianna,Jensen Lasse Dahl Arteriosclerosis, thrombosis, and vascular biology Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article. 10.1161/ATVBAHA.118.312190
    Imbalance between pro-apoptotic and pro-survival factors in human retinal pericytes in diabetic-like conditions. Beltramo Elena,Arroba Ana I,Mazzeo Aurora,Valverde Angela M,Porta Massimo Acta ophthalmologica PURPOSE:Loss of pericytes is one the key events in the pathogenesis of diabetic retinopathy. We have previously demonstrated that human retinal pericytes (HRP) are more vulnerable to intermittent than stable high glucose concentrations, with an increase in apoptosis. Our aim was to explore the expression of molecules involved in pro-apoptotic and survival pathways in pericytes cultured in stable/intermittent high glucose and/or hypoxia, to clarify the mechanisms of action of these diabetic-like stressing stimuli. METHODS:Human retinal pericytes (HRP) were exposed intermittently at 48-hr intervals to high/physiological glucose for 8 days (intHG) and/or hypoxia over the last 48 hr. Control cells were kept in stable physiological and high glucose. Cell proliferation and apoptosis were assessed. The expression of pro-apoptotic and pro-survival molecules was evaluated by Western blotting. Caspase-8 translocation from the cytoplasm into the nucleus was checked by Western blotting of nuclear versus cytoplasmic fractions and immunofluorescence. RESULTS:Hypoxia, alone and combined with intHG, increased HRP apoptosis and decreased proliferation. Pro-apoptotic molecules increased in HRP cultured in these conditions, while some survival markers decreased. Conversely, in stable HG, pro-apoptotic molecules were stable or even decreased, and survival factors increased. Translocation of caspase-8 from cytoplasm into nucleus indicates a primary role for this molecule in inducing apoptosis. CONCLUSION:Diabetic-like conditions are able to stimulate pericyte apoptosis through activation of pro-apoptotic molecules, leading to an imbalance between pro-apoptotic and survival signalling pathways, with caspase-8 playing a pivotal role. Our identification of such intermediates could help finding new therapeutic approaches for the prevention of diabetic retinopathy. 10.1111/aos.13377
    YAP1 is required for the angiogenesis in retinal microvascular endothelial cells via the inhibition of MALAT1-mediated miR-200b-3p in high glucose-induced diabetic retinopathy. Han Ning,Tian Wen,Yu Na,Yu Li Journal of cellular physiology Diabetic retinopathy (DR) is one of the severest complications in the development of diabetes with a characteristic of intraretinal new vessel formation. Our present study attempts to probe into the involvement of Yes-associated protein 1 (YAP1) in retinal microvascular endothelial cells (RMECs) and the underlying mechanism. The DR mouse model was induced by streptozotocin injection and a high-glucose/high-fat diet. After that, with the utilization of the Pearson's correlation analysis, the correlation between YAP1 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was analyzed. The relationship between microRNA-200b-3p (miR-200b-3p) and MALAT1 or vascular endothelial growth factor A (VEGFA) was then validated. Finally, the cellular processes including the abilities of proliferation, migration, as well as tube formation, were evaluated after the alteration of YAP1, MALAT1, and miR-200b-3p expression. YAP1 was observed to be highly expressed in retinas from DR mice, which promoted the cellular processes of RMECs through upregulating the MALAT1 expression. It was further confirmed that MALAT1 could sponge miR-200b-3p, and miR-200b-3p directly targeted VEGFA. When YAP1 was silenced, the RMEC proliferation, migration, and angiogenesis in the retina of DR mice were reduced. From these data, we conclude that YAP1 may exert some promotive effects on the development of DR through its regulation of the MALAT1/miR-200b-3p/VEGFA axis, highlighting that YAP1 silencing may be instrumental for the therapeutic targeting of DR. 10.1002/jcp.29047
    miR-132 promotes retinal neovascularization under anoxia and reoxygenation conditions through up-regulating Egr1, ERK2, MMP2, VEGFA and VEGFC expression. Zhang Lixin,Tao Lijuan International journal of clinical and experimental pathology Retinal neovascularization (RNV) is a prominent pathological angiogenesis, which causes detrimental outcomes in visual functions. Previous literature represents that miR-132 induces angiogenesis in tumor development and ischemic diseases. Considering the important role in angiogenesis, we hypothesized that miR-132 might be involved in RNV. In this study, human retinal microvascular endothelial cells were maintained in hypoxia for indicated time, followed by further incubation in normoxic conditions to establish hypoxia/reoxygenation (H/R) models . mRNA microarray analysis was undertaken to detect alterations in gene profiles in the cells. qRT-PCR and Western blotting were performed to evaluate expression of genes that are closely associated to neovascularization. Results showed that miR-132 expression was increased under hypoxic conditions. Reoxygenation for a limited time (6 h) failed to restore miR-132 expression to basal level. Interference of miR-132 expression via its inhibitor suppressed the cell proliferation under H/R conditions, increasing the apoptosis rate. mRNA microarray analysis revealed that miR-132 is involved in the regulation of vasculature development, blood vessel morphogenesis, and proliferation and migration of microvascular endothelial cells through regulating genes such as early growth response gene 1 (Egr1), extracellular signal-regulated kinase (ERK), metal matrix proteinase (MMP2), vascular endothelial growth factor (VEGF)-A and VEGF-C. qRT-PCR and Western blotting further demonstrated that miR-132 up-regulated their gene and protein expression under H/R conditions. In summary, miR-132 was involved in the development of RNV under H/R conditions, at least partly, through up-regulating Egr1, ERK2, MMP2, VEGFA and VEGFC expression. This finding facilitates the understanding of pathogenic mechanisms of RNV.
    Interleukin-17A neutralization alleviated ocular neovascularization by promoting M2 and mitigating M1 macrophage polarization. Zhu Yanji,Tan Wei,Demetriades Anna M,Cai Yujuan,Gao Yushuo,Sui Ailing,Lu Qing,Shen Xi,Jiang Chunhui,Xie Bing,Sun Xinghuai Immunology Neovascularization (NV), as a cardinal complication of several ocular diseases, has been intensively studied, and research has shown its close association with inflammation and immune cells. In the present study, the role of interleukin-17A (IL-17A) in angiogenesis in the process of ocular NV both in vivo and in vitro was investigated. Also, a paracrine role of IL-17A was demonstrated in the crosstalk between endothelial cells and macrophages in angiogenesis. In the retinas of mice with retinopathy of prematurity, the IL-17A expression increased significantly at postnatal day 15 (P15) and P18 during retinal NV. Mice given IL-17A neutralizing antibody (NAb) developed significantly reduced choroidal NV and retinal NV. Studies on vascular endothelial growth factor (VEGF) over-expressing mice suggested that IL-17A modulated NV through the VEGF pathway. Furthermore, IL-17A deficiency shifted macrophage polarization toward an M2 phenotype during retinal NV with significantly reduced M1 cytokine expression compared with wild-type controls. In vitro assays revealed that IL-17A treated macrophage supernatant gave rise to elevated human umbilical vascular endothelial cell proliferation, tube formation and VEGF receptor 1 and receptor 2 expression. Therefore, IL-17A could potentially serve as a novel target for treating ocular NV diseases. The limitation of this study involved the potential mechanisms, such as which transcription accounted for macrophage polarization and how the subsequent cytokines were modulated when macrophages were polarized. Further studies need to be undertaken to definitively determine the extent to which IL-17A neutralizing anti-angiogenic activity depends on macrophage modulation compared with anti-VEGF treatment. 10.1111/imm.12571
    Involvement of IL-37 in the Pathogenesis of Proliferative Diabetic Retinopathy. Zhao Mengmeng,Hu Yongguang,Yu Ying,Lin Qing,Yang Jianhua,Su Shao Bo,Xu Guo-Tong,Yang Tianshu Investigative ophthalmology & visual science PURPOSE:Interleukin-37 is suggested as a novel proangiogenic factor in our previous study. In this study, the role of IL-37 was investigated in proliferative diabetic retinopathy (PDR). METHODS:Vitreous fluids from 10 patients with PDR and 8 controls were collected. The levels of IL-37 were determined by ELISA and the relationship between IL-37 and VEGF-A/Ang-2 was analyzed. The effects of IL-37 on chorioretinal endothelial cell (RF/6A) proliferation, migration, and tube formation were determined by BrdU incorporation assay, Boyden chamber assay, scratch-wound assay and tube formation assay. RESULTS:The concentration of IL-37 in the PDR group was 95.09 ± 5.22 pg/mL and 34.91 ± 5.61 pg/mL in control group (P = 0.001). The level of IL-37 was highly related to the level of Ang-2 (P = 0.009, r = 0.772) and VEGF-A (P = 0.003, r = 0.827) in the PDR group, and VEGF expression in RF/6A cell was upregulated by IL-37 at low concentration. Interleukin-37 remarkably promoted RF/6A cell proliferation and migration. Interleukin-37 (1 ng/mL) remarkably stimulated tube formation with an increase of 85.3% for total tubule length and 74.1% for branching points compared with PBS control. CONCLUSIONS:The level of IL-37 is elevated in vitreous fluids of patients with PDR and correlates with the level of VEGF-A and Ang-2. Interleukin-37 stimulates proangiogenic response of retinal endothelial cells in vitro, suggesting the involvement of IL-37 in the pathogenesis of PDR. 10.1167/iovs.15-18505
    Endocan Blockade Suppresses Experimental Ocular Neovascularization in Mice. Su Ting,Zhong Yisheng,Demetriades Anna M,Shen Jikui,Sui Ailing,Yao Yiyun,Gao Yushuo,Zhu Yanji,Shen Xi,Xie Bing Investigative ophthalmology & visual science Purpose:Ocular neovascularization (NV) is a pathologic process characterized by the proliferation and infiltration of various types of cells such as RPE, glial, and endothelial cells, which interact with proangiogenic factors and inflammatory cytokines. Endocan is known to be enriched in retinal endothelial tip cells under hypoxia, but the effect of endocan on ocular NV progression is largely unknown. In this study, we investigated the role of endocan in the ocular NV pathologic process and the possible mechanisms involved. Methods:In the eyes of mice with oxygen-induced retinopathy (OIR); choroidal NV (CNV); and rhodopsin promoter (rho)/VEGF transgenic mice, endocan expression was assessed by quantitative real-time PCR (RT-PCR) and Western blot. In vivo, a specific functional antibody was used to neutralize endocan and ocular NV levels were evaluated by RT-PCR, Western blot and immunostaining of flat-mounts. In vitro, the effect of endocan on human retinal microvascular endothelial cell (HREC) tube formation was observed using a routine method. Results:Endocan was significantly elevated in these three experimental mice models. Endocan blockade with the neutralizer intravitreal injection not only suppressed the area of retinal, choroidal and subretinal NV, but also resulted in a decrease in several angiogenesis-associated molecules. Recombinant endocan protein (rhEndocan) was found to induce tube formation on HRECs directly. Conclusions:The current data suggest that endocan is a potential therapeutic or an additional target for retinal and subretinal NV diseases. 10.1167/iovs.17-22945
    Anti-angiogenic effect of adiponectin in human primary microvascular and macrovascular endothelial cells. Palanisamy Karthikka,Nareshkumar Ragavachetty Nagaraj,Sivagurunathan Suganya,Raman Rajiv,Sulochana Konerirajapuram Natarajan,Chidambaram Subbulakshmi Microvascular research Neovascularization in retina and choroid involves interplay of many cytokines and growth factors. Vascular endothelial growth factor (VEGF) being a pro-angiogenic molecule has been found to be high in aqueous and vitreous humour of patients with proliferative diabetic retinopathy (PDR). VEGF is also found in the fibroblast and retinal pigment epithelial cells (RPE) of choroidal neovascular (CNV) membranes isolated from patients. Though anti-VEGF agents cause regression of clinically visible new vessels, there is evidence that they increase the occurrence of retinal tractional detachment and other adverse effects in PDR and CNV treatments. Adiponectin (APN) is a cytokine, found to be involved in the pathobiology of PDR. It is unclear whether APN plays a reparative or pathological role in the disease condition. In this study, we explored the effect of APN on tube formation in the primary culture of human umbilical vein macrovascular endothelial cells (HUVEC), human retinal microvascular endothelial cells (hREC) and human choroidal endothelial cells (hCEC). Anti-VEGF agent, bevacizumab (avastin) was used as a control. Full-length pAc-APN transfected in HUVEC, hRECs and hCECs inhibited basal tube formation and migration comparable to bevacizumab (Avastin™). In hRECs, full length pAc-APN reduced VEGF or PDR vitreous mediated migration. In a similar way, rAPN significantly disrupted VEGF and PDR vitreous induced tube formation in HUVEC and hREC. Moreover, rAPN significantly reduced VEGF influenced proliferation and phosphorylation of ERK1/2 in hREC. Altogether, our study suggests that APN may be effective in the treatment of retinal neovascularization. 10.1016/j.mvr.2018.08.002
    APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2. Jiao Chunhua,Eliott Dean,Spee Christine,He Shikun,Wang Kai,Mullins Robert F,Hinton David R,Sohn Elliott H Retina (Philadelphia, Pa.) PURPOSE:We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy. METHODS:Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed. RESULTS:High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF. CONCLUSION:Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes. 10.1097/IAE.0000000000001952
    Bilateral proliferative retinopathy and ischemic optic neuropathy in a patient with atypical hemolytic-uremic syndrome: A case report. Lin I-Hung,Chen Ying-Jen,Chang Ping-Ying,Hsiao Po-Wei,Weng Tzu-Heng,Chang Yun-Hsiang Medicine RATIONALE:To report a rare case of severe atypical hemolytic-uremic syndrome (HUS) in a patient who presented with vitreous hemorrhage and tractional retinal detachment (TRD) in both eyes. To our knowledge, this is the first reported case of atypical HUS complicated with bilateral TRD in the literature. PATIENT CONCERNS:A 20-year-old man with atypical HUS demonstrated bilateral visual acuity of hand motion at 30 cm. DIAGNOSES:Dilated fundus examination revealed diffuse intraretinal hemorrhage with vascular engorgement, neovascularization of the disc, and neovascularization elsewhere bilaterally. Fluorescein angiography revealed bilateral proliferative retinopathy, retinal hemorrhage, and a large nonperfusion area with extensive neovascularization. Intravitreal antivascular endothelial growth factor (ranibizumab) injection was administered in both eyes, but his ophthalmic condition did not improve, and TRD developed bilaterally. Therefore atypical HUS complicated with bilateral TRD was diagnosed. INTERVENTIONS:Pars plana vitrectomy was performed with panretinal photocoagulation and silicone oil tamponade in the right eye. OUTCOMES:After the pars plana vitrectomy of right eye, the retina was well-attached after surgery, but visual acuity remained poor. Visual evoked potential examination showed poor waveforms bilaterally, which suggested ischemic optic neuropathy. LESSONS:Atypical HUS can cause systemic thrombotic microangiopathy, resulting in ischemic retinal changes. These ischemic retinal changes can then cause hypoxia, which triggers production of angiogenic factors and subsequently causes retinal vascular hyperpermeability, retinal and vitreous neovascularization, fibrovascular proliferation, vitreous hemorrhage, and TRD, in a manner similar to that of other ischemia-induced proliferative retinopathies. Despite successful surgery in the right eye, our patient's visual acuity did not improve, possibly because of severe and generalized ischemia of intraocular tissue, which resulted in ischemic optic neuropathy. 10.1097/MD.0000000000017232
    Efficacy of aflibercept (EYLEA) on inhibition of human VEGF in vitro. Schicht M,Hesse K,Schröder H,Naschberger E,Lamprecht W,Garreis F,Paulsen F P,Bräuer L Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft INTRODUCTION:Pathological formation of blood vessels plays a key role in the growth and metastasis of tumors and also in several serious ophthalmological diseases such as wet age-related macular degeneration (AMD) or diabetic retinopathy. In AMD treatment, aflibercept (tradename EYLEA) is used to deactivate the underlying pathological neovascularisation. Aflibercept is a recombinant fusion protein which binds to vascular endothelial growth factor (VEGF) receptors, thereby inhibiting VEGF pathway activation. VEGF is one of the most important angiogenesis factors. OBJECTIVE:This analysis investigates lasting efficacy of aflibercept in vitro for later application as therapeutic agent against macular degeneration (AMD). MATERIAL AND METHODS:VEGF-ELISA assays were performed to investigate binding affinities at different aflibercept concentrations. The impact of VEGF on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated using proliferation assays. Moreover, time-dependent kinetic studies were performed to analyze different aflibercept storage durations with regard to its inhibitory capabilities on human VEGF. RESULTS AND CONCLUSION:Our results reveal that aflibercept significantly lowers the amount of unbound VEGF as well as the proliferation rate of HUVEC. Moreover, in contrast to specifications given by the manufacturer, aflibercept retains its full inhibitory effect up to at least 120h after transference from the original vial into the injection syringe. 10.1016/j.aanat.2017.02.005
    Anti-angiogenic effect of rapamycin in mouse oxygen-induced retinopathy is mediated through suppression of HIF-1alpha/VEGF pathway. Sun Yi Zhou,Liu Lei,Cai Na,Liu Ning Ning International journal of clinical and experimental pathology Hypoxia-inducible factor-1alpha (HIF-1α)-vascular endothelial growth factor (VEGF) pathway, a downstream of mammalian target of rapamycin (mTOR), plays a major role in the formation of pathological retinal angiogenesis. Rapamycin (RAPA), a highly specific inhibitor of mTOR, is widely used in cancer studies for its antiangiogenic activity. However, the inhibitory effects of RAPA on the HIF-1α-VEGF pathway in retinal tissues were rarely researched. The study aimed to investigate the efficacy and potential mechanisms of RAPA in inhibiting retinal neovascularization. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia and in the presence of different concentrations of RAPA. RAPA was injected intraperitoneally in oxygen-induced retinopathy (OIR) C57BL/6 mice from postnatal day 12 (P12) to P17. The proliferation of HUVECs, the protein and mRNA expressions of HIF-1α and VEGF were evaluated in HUVECs or OIR mice using MTT assay, ELISA, immunohistochemistry, Western-blot and real-time PCR. Histological methods were used to count blood vessel profiles in the inner retina. RAPA inhibited HUVECs proliferation and retinal neovascularization by reducing protein and mRNA expressions of HIF-1α and VEGF. RAPA suppresses hypoxia-induced HUVECs cell proliferation and pathological ocular angiogenesis through a mechanism linked to the targeting of HIF-1α/VEGF signaling.
    LncRNA-MALAT1 promotes neovascularization in diabetic retinopathy through regulating miR-125b/VE-cadherin axis. Liu Ping,Jia Song-Bai,Shi Jing-Ming,Li Wen-Jie,Tang Luo-Sheng,Zhu Xia-Hua,Tong Ping Bioscience reports Diabetic retinopathy (DR) is currently the leading cause of blindness and visual disability in adults with diabetes mellitus (DM). Neovascularization has been identified as an important clinical property in DR, however, the exact mechanisms in DR neovascularization are still unclear and need further elucidation. Quantitative real-time PCR (qRT-PCR) was conducted to detect the expression level of long non-coding RNA (lncRNA)-metastasis associated lung adenocarcinoma transcript 1 (MALAT1), miR-125b and vascular endothelial-cadherin (VE-cadherin) in human retina microvascular endothelial cells (hRMECs) treated with high glucose (HG). Luciferase assay was used to detect interaction of MALAT1 with miR-125b and miR-125b with VE-cadherin. MTT assay, transwell assay, tube formation assay and vascular permeability assay were conducted to detect the cell viability, migration tube formation ability and permeability of hRMECs, respectively. ELISA was used to examine the release of VE-cadherin and vascular endothelial growth factor (VEGF). Western blotting was used to access the protein expression of VE-cadherin, VEGF, β-catenin, matrix metalloproteinase (MMP) 2 (MMP2) and MMP9. MALAT1 and VE-cadherin were up-regulated while miR-125b was down-regulated in hRMECs treated with HG. MALAT1 could competitively bind to miR-125b against VE-cadherin at the site of 3'-untranslated region (3'-UTR), leading to the up-regulation of VE-cadherin. Knockdown of MALAT1 inhibited the proliferation, migration, tube formation and vascular permeability of hRMECs induced by HG through up-regulating miR-125b. Furthermore, we found the deletion of MALAT1 suppressed the VE-cadherin/β-catenin complex and neovascularization related proteins expression, which was up-regulated by HG. Knockdown of MALAT1 inhibited cell proliferation, migration and angiogenesis of hRMECs via suppressing the VE-cadherin/β-catenin complex through targeting miR-125b. Inhibition of MALAT1 may serve as a potential target for anti-angiogenic therapy for DR. 10.1042/BSR20181469
    OCT angiography demonstrates retinal angiomatous proliferation and chorioretinal anastomosis of type 3 neovascularization. Bansal Reema,Hemanth Varshitha,Mulkutkar Samyak,Singh Ramandeep,Gupta Vishali,Dogra Mangat R,Gupta Amod International ophthalmology PURPOSE:To report the OCT angiography findings of type 3 neovascularization. METHODS:Two cases (one each with retinal angiomatous proliferation and chorioretinal anastomosis) are reported, in which the conventional fundus fluorescein angiography and spectral-domain optical coherence tomography findings were inconclusive. RESULTS:OCT angiography demonstrated flow within the abnormal anastomotic network and delineated the extent of the lesion with respect to the retinal layers. CONCLUSION:OCT angiography enables a quick diagnosis of type 3 neovascularization by demonstrating a supranormal flow within the lesion and delineates the lesion within different retinal layers. 10.1007/s10792-017-0664-6
    Apatinib, an Inhibitor of Vascular Endothelial Growth Factor Receptor 2, Suppresses Pathologic Ocular Neovascularization in Mice. Kim Koung Li,Suh Wonhee Investigative ophthalmology & visual science Purpose:Vascular endothelial growth factor (VEGF) signaling via VEGF receptor 2 (VEGFR2) plays a crucial role in pathologic ocular neovascularization. In this study, we investigated the antiangiogenic effect of apatinib, a pharmacologic inhibitor of VEGFR2 tyrosine kinase, against oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) in mice. Methods:Western blotting and in vitro angiogenesis assays were performed using human retinal microvascular endothelial cells (HRMECs). OIR was induced in neonatal mice by exposure to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17. Experimental CNV was induced in mice using laser photocoagulation. Apatinib was intravitreally and orally administered to mice. Neovascularization and phosphorylation of VEGFR2 were evaluated by immunofluorescence staining. Results:Apatinib inhibited VEGF-mediated activation of VEGFR2 signaling and substantially reduced VEGF-induced proliferation, migration, and cord formation in HRMECs. A single intravitreal injection of apatinib significantly attenuated retinal or choroidal neovascularization in mice with OIR or laser injury-induced CNV, respectively. Retinal or choroidal tissues of the eyes treated with apatinib exhibited substantially lower phosphorylation of VEGFR2 than those of controls injected with vehicle. Intravitreal injection of apatinib did not cause noticeable ocular toxicity. Moreover, oral administration of apatinib significantly reduced laser-induced CNV in mice. Conclusions:Our study demonstrates that apatinib inhibits pathologic ocular neovascularization in mice with OIR or laser-induced CNV. Apatinib may, therefore, be a promising drug for the prevention and treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration. 10.1167/iovs.17-21416
    Angiogenic Factors produced by Hypoxic Cells are a leading driver of Anastomoses in Sprouting Angiogenesis-a computational study. Moreira-Soares Maurício,Coimbra Rita,Rebelo Luís,Carvalho João,D M Travasso Rui Scientific reports Angiogenesis - the growth of new blood vessels from a pre-existing vasculature - is key in both physiological processes and on several pathological scenarios such as cancer progression or diabetic retinopathy. For the new vascular networks to be functional, it is required that the growing sprouts merge either with an existing functional mature vessel or with another growing sprout. This process is called anastomosis. We present a systematic 2D and 3D computational study of vessel growth in a tissue to address the capability of angiogenic factor gradients to drive anastomosis formation. We consider that these growth factors are produced only by tissue cells in hypoxia, i.e. until nearby vessels merge and become capable of carrying blood and irrigating their vicinity. We demonstrate that this increased production of angiogenic factors by hypoxic cells is able to promote vessel anastomoses events in both 2D and 3D. The simulations also verify that the morphology of these networks has an increased resilience toward variations in the endothelial cell's proliferation and chemotactic response. The distribution of tissue cells and the concentration of the growth factors they produce are the major factors in determining the final morphology of the network. 10.1038/s41598-018-27034-8
    Activation of the sweet taste receptor T1R3 by sucralose attenuates VEGF-induced vasculogenesis in a cell model of the retinal microvascular endothelium. Lizunkova Polina,Enuwosa Emmanuella,Chichger Havovi Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie BACKGROUND:One of the most prevalent microvascular complications for patients with diabetes is diabetic retinopathy (DR) associated with increased retinal endothelial blood vessel formation. Treatments to reduce vascularisation in the retinal endothelium are linked to improved sight in patients with DR. Recently, we have demonstrated the novel protective role of the artificial sweetener, sucralose, and the sweet taste receptor, T1R3, in the pulmonary endothelium to reduce vascular leak. In the present study, we examined the role of sucralose and sweet taste receptors on vasculogenic processes (proliferation, migration, adhesion and tube formation) in a cell model of the retinal endothelium. METHODS:We exposed human retinal microvascular endothelial cells (RMVEC) to VEGF as an in vitro model of DR in the presence and absence of T1R3 agonist sucralose. RESULTS:In RMVEC, we observed increased VEGF-induced cell proliferation, migration, adhesion and tube formation, which was significantly attenuated by exposure to the artificial sweetener sucralose. Following siRNA knockdown of the sweet taste receptor, T1R3, but not T1R2, the protective effect of sucralose on VEGF-induced RMVEC vasculogenic processes was blocked. We further demonstrate that sucralose attenuates VEGF-induced Akt phosphorylation to protect the retinal microvasculature. CONCLUSION:These studies are the first to demonstrate a protective effect of an artificial sweetener, through the sweet taste receptor T1R3, on VEGF-induced vasculogenesis in a retinal microvascular endothelial cell line. 10.1007/s00417-018-4157-8
    Protection of tauroursodeoxycholic acid on high glucose-induced human retinal microvascular endothelial cells dysfunction and streptozotocin-induced diabetic retinopathy rats. Wang Chun-Fei,Yuan Jia-Rui,Qin Dong,Gu Jun-Fei,Zhao Bing-Jie,Zhang Li,Zhao Di,Chen Juan,Hou Xue-Feng,Yang Nan,Bu Wei-Quan,Wang Jing,Li Chao,Tian Gang,Dong Zi-Bo,Feng Liang,Jia Xiao-Bin Journal of ethnopharmacology ETHNOPHARMACOLOGICAL RELEVANCE:Tauroursodeoxycholic acid (TUDCA), one of the main ingredients from bear gall which hold "Clearing heat and detoxification, Removing liver fire for improving eyesight" functions, is formed by the conjugation of ursodeoxycholic acid (UDCA) with taurine. However, the limited information of TUDCA on protecting diabetic retinopathy (DR) has been known. The present study was conducted to evaluate the protection of TUDCA on high glucose-induced human retinal microvascular endothelial cells (HRMECs) dysfunction and streptozotocin (STZ)-induced diabetic retinopathy (DR) rats and the possible mechanism underlying was also explored. MATERIALS AND METHODS:The proliferation of high glucose-induced HRMECs was determined by MTT assay. DR rats' model was established by an administration of high-glucose-fat diet and an intraperitoneal injection of STZ (30mg/kg). The cell supernatant and rats' serum were collected for the assays of NO content by ELISA kits. Retinas were stained with hematoxylin and eosin (HE) to observe pathological changes. Immunohistochemical assay was applied to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in rat retinas. Furthermore, western blot analysis was carried out to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in high glucose-induced HRMECs. RESULTS:After treating with TUDCA, high glucose-induced HRMECs proliferation could be significantly inhibited. TUDCA (5.0μM, 25.0μM and 125.0μM) could decrease NO content in high glucose-induced HRMECs. Furthermore, TUDCA (500mg/kg/d and 250mg/kg/d) also decrease NO content in serum of DR rats. Additionally, both immunocytochemistry analysis and western blot analysis showed that the over-expression of ICAM-1, NOS, NF-κB p65 and VEGF were significantly decreased by TUDCA. CONCLUSION:The data indicated that TUDCA could ameliorate DR by decreasing NO content and down-regulating the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF. Thus, our experimental results suggested that TUDCA might be a potential drug for the prevention and treatment of DR. 10.1016/j.jep.2016.03.026
    Apelin induces the proliferation, migration and expression of cytoskeleton and tight junction proteins in human RPE cells via PI-3K/Akt and MAPK/Erk signaling pathways. Li Yang,Bai Yu-Jing,Jiang Yan-Rong International journal of clinical and experimental pathology Diabetic retinopathy is major cause of vision loss during working age. Breakdown of blood-retinal barrier is an early event in pathogenesis of DR. RPE is the major part of outer BRB. Apelin, an endogenous ligand of APJ, mediates angiogenesis. Our previous study showed that apelin induced proliferation, migration, and collagen I mRNA expression in human RPE cells via PI-3K/Akt and MAPK/Erk signaling pathways. Now we investigate the connection between apelin and RPE in vascular permeability of diabetic retinopathy and its working mechanism. Our study showed that apelin promotes the proliferation, migration and expression of cytoskeleton and tight junction proteins in human RPE cells using MTS and transwell chamber assay. Apelin also activated the expression of PI-3K/Akt and MAPK/Erk signaling pathways proteins, such as PLCγ1, p38, Akt and Erk phosphorylation in RPE cells using laser scanning confocal detection, PCR and western blot. Pretreatment with the inhibitor of apelin receptor APJ, F13A, abolished the apelin-induced activations of the proliferation, migration and expression of cytoskeleton, tight junction and PI-3K/Akt and MAPK/Erk signaling pathways proteins in human RPE cells. It suggested that apelin as a promoter in retinal vascular permeability during early stage of DR, provides further evidence for neurovascular crosstalk in pathogenesis of DR, which may offer a new target in early prevention and treatment of DR.
    Intravitreal Anti-Vascular Endothelial Growth Factor Drugs for Retinal Angiomatous Proliferation in Real-Life Practice. Parodi Maurizio Battaglia,Donati Simone,Semeraro Francesco,Danzi Paola,Introini Ugo,Viola Francesco,Bottoni Ferdinando,Pucci Vincenzo,Musig Andrea,Pece Alfredo,Azzolini Claudio Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics PURPOSE:To describe the outcomes of intravitreal anti-vascular endothelial growth factor (VEGF) in the treatment of retinal angiomatous proliferation (RAP) in real-life practice in 7 Italian centers under the Progetto Luce initiative. METHODS:Clinical data of 95 eyes of 95 patients affected by RAP, regularly followed up and treated with either intravitreal ranibizumab or bevacizumab over 12 months, were examined. After a loading phase of 3 consecutive injections, retreatments were administered following a pro-re-nata regimen on the basis of the persistence or the recurrence of subretinal/intraretinal fluid on optical coherence tomography, or leakage on fluorescein angiography. RESULTS:Overall, the mean best corrected visual acuity changed from 0.66 to 0.53 LogMAR (P: 0.0003); 36.8% of eyes gained at least 3 ETDRS lines, whereas 13.7% lost >3 lines at the end of the follow-up. Mean central retinal thickness improved from 384 μm at baseline to 262 μm at the 12-month examination (P < 0.001). A serous pigment epithelium detachment (PED) was identified in 68.4% of eyes at baseline, and it was still detectable in 30.5% at the end of follow-up. The mean number of injections was 4.4 over the follow-up. A significantly greater proportion of eyes showed PED resolution in the subgroup treated with ranibizumab (P < 0.001). CONCLUSIONS:Intravitreal anti-VEGF treatment in routine clinical practice allows a significant improvement in visual function in patients affected by RAP. A limited number of anti-VEGF injections are generally required in most cases. 10.1089/jop.2016.0091
    Fufang Xueshuantong protects retinal vascular endothelial cells from high glucose by targeting YAP. Xing Wei,Song Yongli,Li Hongli,Wang Zhenglin,Wu Yan,Li Chun,Wang Yong,Liu Yonggang,Wang Wei,Han Jing Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Fufang Xueshuantong (FXST), a Chinese patent medicine, is composed of Panax notoginseng, Salviae miltiorrhizae, Astragali Radix and Radix Scrophulariae and has been found to prevent diabetic retinopathy. Yes-associated protein (YAP) participates in the pathophysiology of retinal disease and promotes endothelial cell proliferation and angiogenesis. Although it is known that YAP activity is altered by FXST, the role of YAP in mediating the effect of FXST remains unclear. In high glucose-treated retinal vascular endothelial cells (RVECs), FXST significantly reduced cell viability, the number of migrating cells and tube length in the present study. Moreover, FXST decreased the levels of YAP mRNA and protein and inhibited the expression of vascular endothelial growth factor (VEGF). Transfection of sh-YAP into the cells decreased the ability of FXST to modulate cell migration and tube formation. The effect of FXST on VEGF expression was also decreased. Similar results were obtained when the cells were stimulated with a YAP inhibitor in combination with FXST. Thus, FXST is shown to protect high glucose-injured RVECs via YAP-mediated effects. 10.1016/j.biopha.2019.109470
    Inhibition of Aberrant IGF-I Signaling in Diabetic Male Rat Retina Prevents and Reverses Changes of Diabetic Retinopathy. Xi Gang,Wai Christine,Clemmons David Journal of diabetes research Hyperglycemia results in inhibition of cleavage of integrin-associated protein (IAP) thereby allowing it to bind to SHPS-1 which results in pathophysiologic changes in endothelial function. This study determined if an anti-rat IAP antibody directed against the SHPS-1 binding site which disrupts IAP/SHPS-1 association could inhibit these pathophysiologic changes. The anti-IAP antibody inhibited IGF-I-stimulated SHPS-1, p52Shc, MAP kinase phosphorylation, and proliferation in endothelial cells. To determine if it could reverse established pathophysiologic changes in vivo, this antibody or normal rat IgG F(ab)2 was injected intraperitoneally for 6 weeks into rats that had diabetes for 4 weeks. Optical coherence tomography (OCT) showed that retinal thickness increased at 4 weeks and this increase was maintained in rats treated with the control antibody for an additional 6 weeks. The increase was reversed by anti-IAP antibody treatment (84.6 ± 2.0 compared to 92.3 ± 2.5 m, < 0.01). This value was similar to nondiabetic animals (82.2 ± 1.6 m, , NS). The anti-IAP antibody also decreased retinal vascular permeability (0.62 ± 0.12 vs. 0.96 ± 0.25%/g/h, < 0.001). To determine if it was effective after local injection, this antibody or control was administered via intravitreal injection. After 3 weeks, retinal thickness increased to 6.4 ± 2.8% in diabetic rats, and IAP antibody treatment prevented this increase (0.8 ± 2.5%, < 0.01). It also prevented the increase of retinal vascular permeability (0.92 ± 0.62 vs. 1.63 ± 0.99%/g/h, < 0.001). Biochemical analyses of retinal extracts showed that the anti-IAP antibody inhibited IAP/SHPS-1 association and SHPS-1 phosphorylation. This resulted in inhibition of AKT activation and VEGF synthesis in the retina: changes associated with increased vascular permeability. We conclude the anti-rat IAP antibody disrupts IAP/SHPS-1 association and attenuates aberrant IGF-I signaling thereby preventing or reversing the progression of retinal pathophysiological changes. 10.1155/2019/6456032
    β-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation. Yun Jang-Hyuk,Jeong Han-Seok,Kim Kyung-Jin,Han Man Hyup,Lee Eun Hui,Lee Kihwang,Cho Chung-Hyun FASEB journal : official publication of the Federation of American Societies for Experimental Biology Pericytes (PCs) are crucial in maintaining the quiescence of endothelial cells (ECs) and the integrity of EC tight junctions. Especially in diabetic retinopathy (DR), PC loss is one of the early pathologic changes in capillaries of diabetic retinas. Thus, preventing PC loss is beneficial for attenuating vision impairment in patients with DR. Although many studies have revealed the mechanism of PC loss in retinas, little is known about the mechanisms that increase PC survival. We focused on the effect of β-adrenergic receptor agonists (β-agonists) on PC loss in diabetic retinas. In this study, β-agonists increased the cell viability of PCs by increasing PC survival and proliferation. Mechanistically, β-agonist-induced protein kinase B activation in PCs reduced PC apoptosis in response to various stimuli. β2-agonists more potently increased PC survival than β1-agonists. β2-Agonist reduced vascular leakage and PC loss in retinas of mice with streptozotocin-induced diabetes. In cocultures of PCs and ECs, β2-agonists restored the altered permeability and ZO-1 expression in ECs induced by PC loss. We concluded that β-agonists, especially β2-agonists, increase PC survival, thereby preventing diabetes-induced PC loss in retinas. These results provide a potential therapeutic benefit of β-agonists for preventing PC loss in DR.-Yun, J.-H., Jeong, H.-S., Kim, K.-J., Han, M. H., Lee, E. H., Lee, K., Cho, C.-H. β-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation. 10.1096/fj.201700570RR
    MicroRNA-145 Regulates Pathological Retinal Angiogenesis by Suppression of TMOD3. Liu Chi-Hsiu,Wang Zhongxiao,Huang Shuo,Sun Ye,Chen Jing Molecular therapy. Nucleic acids Pathological angiogenesis is a hallmark of various vascular diseases, including vascular eye disorders. Dysregulation of microRNAs (miRNAs), a group of small regulatory RNAs, has been implicated in the regulation of ocular neovascularization. This study investigated the specific role of microRNA-145 (miR-145) in regulating vascular endothelial cell (EC) function and pathological ocular angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Expression of miR-145 was significantly upregulated in OIR mouse retinas compared with room air controls. Treatment with synthetic miR-145 inhibitors drastically decreased levels of pathological neovascularization in OIR, without substantially affecting normal developmental angiogenesis. In cultured human retinal ECs, treatment with miR-145 mimics significantly increased the EC angiogenic function, including proliferation, migration, and tubular formation, whereas miR-145 inhibitors attenuated in vitro angiogenesis. Tropomodulin3 (TMOD3), an actin-capping protein, is a direct miR-145 target and is downregulated in OIR retinas. Treatment with miR-145 mimic led to TMOD3 inhibition, altered actin cytoskeletal architecture, and elongation of ECs. Moreover, inhibition of TMOD3 promoted EC angiogenic function and pathological neovascularization in OIR and abolished the vascular effects of miR-145 inhibitors in vitro and in vivo. Overall, our findings indicate that miR-145 is a novel regulator of TMOD3-dependent cytoskeletal architecture and pathological angiogenesis and a potential target for development of treatments for neovascular eye disorders. 10.1016/j.omtn.2019.03.001
    Morphine promotes neovascularizing retinopathy in sickle transgeneic mice. Gupta Kalpna,Chen Chunsheng,Lutty Gerard A,Hebbel Robert P Blood advances Neovascularizing retinopathy is a significant complication of sickle cell disease (SCD), occurring more frequently in HbSC than HbSS disease. This risk difference is concordant with a divergence of angiogenesis risk, as identified by levels of pro- vs anti-angiogenic factors in the sickle patient's blood. Because our prior studies documented that morphine promotes angiogenesis in both malignancy and wound healing, we tested whether chronic opioid treatment would promote retinopathy in NY1DD sickle transgenic mice. After 10 to 15 months of treatment, sickle mice treated with morphine developed neovascularizing retinopathy to a far greater extent than either of the controls (sickle mice treated with saline and wild-type mice treated identically with morphine). Our dissection of the mechanistic linkage between morphine and retinopathy revealed a complex interplay among morphine engagement with its μ opioid receptor (MOR) on retinal endothelial cells (RECs); morphine-induced production of tumor necrosis factor α and interleukin-6 (IL-6), causing increased expression of both MOR and vascular endothelial growth factor receptor 2 (VEGFR2) on RECs; morphine/MOR engagement transactivating VEGFR2; and convergence of MOR, VEGFR2, and IL-6 activation on JAK/STAT3-dependent REC proliferation and angiogenesis. In the NY1DD mice, the result was increased angiogenesis, seen as neovascularizing retinopathy, similar to the retinal pathology occurring in humans with SCD. Therefore, we conclude that chronic opioid exposure, superimposed on the already angiogenic sickle milieu, might enhance risk for retinopathy. These results provide an additional reason for development and application of opioid alternatives for pain control in SCD. 10.1182/bloodadvances.2018026898
    Nrf2 Activator RS9 Suppresses Pathological Ocular Angiogenesis and Hyperpermeability. Nakamura Shinsuke,Noguchi Tetsuro,Inoue Yuki,Sakurai Shuji,Nishinaka Anri,Hida Yoshifumi,Masuda Tomomi,Nakagami Yasuhiro,Horai Naoto,Tsusaki Hideshi,Hara Hideaki,Shimazawa Masamitsu Investigative ophthalmology & visual science Purpose:Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods:The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results:Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-β, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions:These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability. 10.1167/iovs.18-25745
    RETINAL PIGMENT EPITHELIAL ATROPHY AFTER ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR INJECTIONS FOR RETINAL ANGIOMATOUS PROLIFERATION. Hata Masayuki,Yamashiro Kenji,Oishi Akio,Ooto Sotaro,Tamura Hiroshi,Miyata Manabu,Ueda-Arakawa Naoko,Kuroda Yoshimasa,Takahashi Ayako,Tsujikawa Akitaka,Yoshimura Nagahisa Retina (Philadelphia, Pa.) PURPOSE:To investigate the incidence rate and risk factors for development of retinal pigment epithelial (RPE) atrophy during anti-vascular endothelial growth factor (anti-VEGF) treatment for retinal angiomatous proliferation. METHODS:This study included 46 eyes with treatment-naive retinal angiomatous proliferation. All patients were treated with ranibizumab or aflibercept injections. Color fundus photography, spectral-domain optical coherence tomography, and fundus autofluorescence were evaluated for RPE atrophy diagnosis. Baseline characteristics and gene polymorphisms of ARMS2 A69S, and CFH I62V were analyzed for association with development and progression of RPE atrophy. RESULTS:Among 21 eyes treated with ranibizumab without preexisting RPE atrophy at baseline, 5 eyes (23.8%) developed RPE atrophy at 12 months. Among 20 eyes treated with aflibercept without preexisting RPE atrophy at baseline, 10 eyes (50.0%) developed RPE atrophy at 12 months. Refractile drusen at baseline was associated with RPE atrophy development at 12 months (P = 0.014), and the progression rate of RPE atrophy area was negatively correlated with subfoveal choroidal thickness at baseline (R = -0.595, P = 0.019). Gene polymorphisms were not associated with RPE atrophy. CONCLUSION:Retinal pigment epithelial atrophy developed in 36.6% during 12 months after anti-VEGF treatment for retinal angiomatous proliferation. The presence of refractile drusen at baseline was identified as a novel significant risk factor for RPE atrophy development. 10.1097/IAE.0000000000001457
    Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling. Park-Windhol Cindy,Ng Yin Shan,Yang Jinling,Primo Vincent,Saint-Geniez Magali,D'Amore Patricia A Scientific reports Angiogenesis is central to both normal and pathologic processes. Endothelial cells (ECs) express O-glycoproteins that are believed to play important roles in vascular development and stability. Endomucin-1 (EMCN) is a type I O-glycosylated, sialic-rich glycoprotein, specifically expressed by venous and capillary endothelium. Evidence has pointed to a potential role for EMCN in angiogenesis but it had not been directly investigated. In this study, we examined the role of EMCN in angiogenesis by modulating EMCN levels both in vivo and in vitro. Reduction of EMCN in vivo led to the impairment of angiogenesis during normal retinal development in vivo. To determine the cellular basis of this inhibition, gain- and loss-of-function studies were performed in human retinal EC (HREC) in vitro by EMCN over-expression using adenovirus or EMCN gene knockdown by siRNA. We show that EMCN knockdown reduced migration, inhibited cell growth without compromising cell survival, and suppressed tube morphogenesis of ECs, whereas over-expression of EMCN led to increased migration, proliferation and tube formation. Furthermore, knockdown of EMCN suppressed VEGF-induced signaling as measured by decreased phospho-VEGFR2, phospho-ERK1/2 and phospho-p38-MAPK levels. These results suggest a novel role for EMCN as a potent regulator of angiogenesis and point to its potential as a new therapeutic target for angiogenesis-related diseases. 10.1038/s41598-017-16852-x
    Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis. Boopathy Gandhi T K,Kulkarni Madhura,Ho Sze Yuan,Boey Adrian,Chua Edmond Wei Min,Barathi Veluchamy A,Carney Tom J,Wang Xiaomeng,Hong Wanjin The Journal of biological chemistry Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric-oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity. 10.1074/jbc.M117.794743
    MicroRNA-370 suppresses the retinal capillary endothelial cell growth by targeting KDR gene. Wang X H,Chen L Bratislavske lekarske listy BACKGROUND:Ocular neovascularization (NV) is the one of the major causes of blindness in many ocular diseases. Currently, the administration of anti-VEGF agents has been widely accepted for the clinical management of these devastating diseases. However, the short effective duration and the non-responsive rate due to the protein nature of the anti-VEGF antibodies warrants further investigations to explore alternative angiogenic suppressants. Evidence suggested that microRNA-370 could inhibit the formation of vessels. However, the exact mechanism has not yet been clarified. AIM:To investigate the regulatory role of microRNA-370 in the growth and apoptosis of retinal capillary endothelial cells and association between microRNA-370 and kinase insert domain-containing receptor (KDR) gene. METHODS:The effects of miRNA-370 on cell cycle and apoptosis, as well as the expression of cycle- and apoptosis-related genes, were determined using MTT, TUNEL assay, qRT-PCR, and Western blot. The direct target of miRNA-370 was confirmed using 3' untranslated region (UTR) luciferase reporter assay. RESULTS:The miRNA-370 induced growth inhibition and apoptosis of retinal capillary endothelial cell while the inhibition of miRNA-370 reversed these effects. miRNA-370 upregulated the expression of CyclinD1, p21, p27, FasL, and Bim. Furthermore, miR-370 directly reduced the expression of KDR by targeting its 3'untranslated region. CONCLUSION:MicroRNA-370 inhibits the expression of KDR gene resulting in retinal capillary endothelial cell growth inhibition and apoptosis, which could be of value in the treatment of retinal neovascularization (Tab. 1, Fig. 5, Ref. 27). 10.4149/BLL_2017_040
    Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy. Kobayashi Yoshiyuki,Yoshida Shigeo,Zhou Yedi,Nakama Takahito,Ishikawa Keijiro,Arima Mitsuru,Nakao Shintaro,Sassa Yukio,Takeda Atsunobu,Hisatomi Toshio,Ikeda Yasuhiro,Matsuda Akira,Sonoda Koh-Hei,Ishibashi Tatsuro Molecular vision PURPOSE:We previously demonstrated that tenascin-C was highly expressed in the fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR). However, its role in the pathogenesis of FVMs has not been determined. The purpose of this study was to investigate what role tenascin-C plays in the formation and angiogenesis of FVMs. METHODS:The level of tenascin-C was determined by sandwich enzyme-linked immunosorbent assay in the vitreous samples collected from patients with PDR and with a macular hole as control. The locations of tenascin-C, α- smooth muscle actin (SMA), CD34, glial fibrillary acidic protein (GFAP), and integrin αV in the FVMs from PDR patients were determined by immunohistochemistry. We also measured the in vitro expression of the mRNA and protein of tenascin-C in vascular smooth muscle cells (VSMCs) stimulated by interleukin (IL)-13. The effects of tenascin-C on cell proliferation, migration, and tube formation were determined in human retinal endothelial cells (HRECs) in culture. RESULTS:The mean vitreous levels of tenascin-C were significantly higher in patients with PDR than in patients with a macular hole (p<0.001). Double immunofluorescence analyses of FVMs from PDR patients showed that tenascin-C co-stained FVMs with α-SMA, CD34, and integrin αV but not with GFAP. In addition, IL-13 treatment increased both the expression and secretion of tenascin-C by VSMCs in a dose-dependent manner. Tenascin-C exposure promoted proliferation, migration, and tube formation in HRECs. Tenascin-C neutralizing antibody significantly blocked the tube formation by HRECs exposed to VSMC-IL-13-conditioned medium. CONCLUSIONS:Our findings suggest that tenascin-C is secreted from VSMCs and promotes angiogenesis in the FVMs associated with PDR.
    Vascular Endothelial Growth Factor A and Leptin Expression Associated with Ectopic Proliferation and Retinal Dysplasia in Zebrafish Optic Pathway Tumors. Schultz Laura E,Solin Staci L,Wierson Wesley A,Lovan Janna M,Syrkin-Nikolau Judith,Lincow Deborah E,Severin Andrew J,Sakaguchi Donald S,McGrail Maura Zebrafish In the central nervous system injury induces cellular reprogramming and progenitor proliferation, but the molecular mechanisms that limit regeneration and prevent tumorigenesis are not completely understood. We previously described a zebrafish optic pathway tumor model in which transgenic Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors. Key pathways driving injury-induced glial reprogramming and regeneration contributed to tumor formation. In this study, we examine a time course of proliferation and present new analyses of the Tg(flk1:RFP)is18/+ dysplastic retina and tumor transcriptomes. Retinal dysplasia was first detected in 3-month-old adults, but was not limited to a specific stem cell or progenitor niche. Pathway analyses suggested a decrease in cellular respiration and increased expression of components of Hif1-α, VEGF, mTOR, NFκβ, and multiple interleukin pathways are associated with early retinal dysplasia. Hif-α targets VEGFA (vegfab) and Leptin (lepb) were both highly upregulated in dysplastic retina; however, each showed distinct expression patterns in neurons and glia, respectively. Phospho-S6 immunolabeling indicated that mTOR signaling is activated in multiple cell populations in wild-type retina and in the dysplastic retina and advanced tumor. Our results suggest that multiple pathways may contribute to the continuous proliferation of retinal progenitors and tumor growth in this optic pathway tumor model. Further investigation of these signaling pathways may yield insight into potential mechanisms to control the proliferative response during regeneration in the nervous system. 10.1089/zeb.2016.1366
    BH4-Mediated Enhancement of Endothelial Nitric Oxide Synthase Activity Reduces Hyperoxia-Induced Endothelial Damage and Preserves Vascular Integrity in the Neonate. Edgar Kevin S,Galvin Orla M,Collins Anthony,Katusic Zvonimir S,McDonald Denise M Investigative ophthalmology & visual science Purpose:Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) has important vasoprotective functions that are compromised in the vasodegenerative phase of retinopathy of prematurity, owing to hyperoxia-induced depletion of the essential NOS cofactor BH4. Because modulating eNOS function can be beneficial or detrimental, our aim was to investigate the effect of BH4 supplementation on eNOS function and vascular regression in hyperoxia. Methods:Endothelial-specific eNOS-green fluorescent protein (GFP) overexpressing mice at postnatal day 7 (P7) were exposed to hyperoxia for 48 hours in the presence or absence of supplemental BH4, achieved by administration of sepiapterin, a stable BH4 precursor. Tissue was collected either for retinal flat mounts that were stained with lectin to determine the extent of vessel coverage or for analysis of BH4 by high-performance liquid chromatography, nitrotyrosine (NT) marker by Western blotting, VEGF expression by ELISA, and NOS activity by arginine-to-citrulline conversion. Primary retinal microvascular endothelial cells (RMEC) were similarly treated, and hyperoxia-induced damage was determined. Results:Sepiapterin effectively enhanced BH4 levels in hyperoxia-exposed retinas and brains, elevated NOS activity, and reduced NT-modified protein, leading to reversal of the exacerbated vasoregression observed in the presence of eNOS overexpression. In RMECs, hyperoxia-mediated depletion of BH4 dysregulated the redox balance by reducing nitrite and elevating superoxide and impaired proliferative ability. BH4 supplementation restored normal RMEC proliferation in vitro and also in vivo, providing a mechanistic link with the enhanced vascular coverage in eNOS-GFP retinas. Conclusions:These results demonstrate that BH4 supplementation corrects hyperoxia-induced RMEC dysfunction and preserves vascular integrity by enhancing eNOS function. 10.1167/iovs.16-20523
    LncRNA FENDRR promotes high-glucose-induced proliferation and angiogenesis of human retinal endothelial cells. Shi Yu,Chen Chunsheng,Xu Yidan,Liu Yi,Zhang Hui,Liu Yang Bioscience, biotechnology, and biochemistry The study aimed to investigate the role of lncRNA FENDRR in proliferation and angiogenesis of human retinal endothelial cells (HRECs). HRECs were cultured in high-glucose medium to mimic diabetic retinopathy (DR) model. We overexpressed or knocked down FENDRR in HRECs to evaluate the effect of FENDRR expression on cell proliferation, migration, and capillary morphogenesis of HRECs under either normal glucose or high glucose condition. Results showed that VEGF and FENDRR expression were increased in blood from DR patients compared with the control subjects. Furthermore, high glucose treatment upregulated expression of VEGF and FENDRR secreted from HRECs, in a dose- and time-dependent manner. Importantly, FENDRR overexpression significantly promoted the high-glucose-induced proliferation, migration, capillary morphogenesis, and VEGF expression in HRECs. In contrast, FENDRR knockdown exerted the opposite effects. In conclusion, lncRNA FENDRR promotes the high-glucose-induced proliferation and angiogenesis of HRECs and may serve as a potential target for anti-angiogenic therapy for DR. 10.1080/09168451.2019.1569499
    Sustained and targeted episcleral delivery of celecoxib in a rabbit model of retinal and choroidal neovascularization. Lima Luiz H,Farah Michel E,Gum Glenwood,Ko Pamela,de Carvalho Ricardo A Pontes International journal of retina and vitreous Background:To evaluate the efficacy of selective episcleral delivery of celecoxib formulated in a sustained-release episcleral exoplant on a model of retinal and choroidal neovascularization induced in rabbits by subretinal injection of matrigel combined with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods:Nine New Zealand white rabbits were randomly assigned to three groups (episcleral celecoxib exoplant, intravitreal bevacizumab injection and control group). The bFGF was mixed with matrigel at a concentration of 10 ug/0.1 mL, and VEGF was mixed with matrigel at a concentration of 2 ug/0.1 mL. Animals assigned to celecoxib or intravitreal bevacizumab groups were treated within 03 days from matrigel injection. Fluorescein angiography (FA) and electroretinography (ERG) were performed 5 days, 2, 4 and 8 weeks after matrigel injection. Persistence or regression of three clinical features (subretinal hyperfluorescence, retinal vascular tortuosity and retinal fibrotic spots) was independently evaluated in each study group at all follow-up periods. Statistical analysis using Fisher's exact test was performed to compare the frequency of findings at each time point between treated groups and control. Results:In all study eyes, matrigel induced the appearance of subretinal blebs and the development of retinal and subretinal neovascularization characterized by progressive and late hyperfluorescence on FA. Persistence of subretinal hyperfluorescence was higher in non-treated (control) animals compared to celecoxib ( = 0.0238) treated animals. The mean b-wave amplitude ratios of ERG recordings did not reveal statistically significant differences between the study groups. Control animals retained in average 40% (± 7%) of the pre-treatment recorded b-wave amplitude, compared to 53% (± 29%) after bevacizumab and 53% (± 17%) after celecoxib treatment. Conclusion:In this rabbit model of retinal and subretinal neovascularization, episcleral celecoxib delivery was demonstrated to significantly inhibit neovascularization. It was also noticed, although not statistically significant, an apparent effect of episcleral celecoxib on preventing tractional retinal detachment secondary to epiretinal fibrovascular proliferation. The transscleral delivery of celecoxib combined with sustained-release strategy may have impact in the treatment of retinal and choroidal proliferative diseases. 10.1186/s40942-018-0131-1
    Roles of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase in Angiogenesis: Isoform-Specific Effects. Wang Haibo,Hartnett M Elizabeth Antioxidants (Basel, Switzerland) Angiogenesis is the formation of new blood vessels from preexisting ones and is implicated in physiologic vascular development, pathologic blood vessel growth, and vascular restoration. This is in contrast to vasculogenesis, which is de novo growth of vessels from vascular precursors, or from vascular repair that occurs when circulating endothelial progenitor cells home into an area and develop into blood vessels. The objective of this review is to discuss the isoform-specific role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in physiologic and pathologic angiogenesis and vascular repair, but will not specifically address vasculogenesis. As the major source of reactive oxygen species (ROS) in vascular endothelial cells (ECs), NOX has gained increasing attention in angiogenesis. Activation of NOX leads to events necessary for physiologic and pathologic angiogenesis, including EC migration, proliferation and tube formation. However, activation of different NOX isoforms has different effects in angiogenesis. Activation of NOX2 promotes pathologic angiogenesis and vascular inflammation, but may be beneficial in revascularization in the hindlimb ischemic model. In contrast, activation of NOX4 appears to promote physiologic angiogenesis mainly by protecting the vasculature during ischemia, hypoxia and inflammation and by restoring vascularization, except in models of oxygen-induced retinopathy and diabetes where NOX4 activation leads to pathologic angiogenesis. 10.3390/antiox6020040
    Folic Acid Has a Protective Effect on Retinal Vascular Endothelial Cells against High Glucose. Wang Zhenglin,Xing Wei,Song Yongli,Li Hongli,Liu Yonggang,Wang Yong,Li Chun,Wang Yun,Wu Yan,Han Jing Molecules (Basel, Switzerland) Diabetic retinopathy (DR) is a severe complication of diabetes, which seriously affects the life quality of patients. Because of the damage caused by DR, there is an urgent need to develop effective drugs. Folic acid, a water-soluble vitamin, is one of the vitamin B complexes. Folic acid is widely found in the meat and vegetables. In the clinic, low folic acid levels in the body may have a certain correlation with DR. However, there is no relevant basic research proving a relationship between folic acid levels and DR. The purpose of this study was therefore to investigate whether folic acid has a protective effect on the retinal vascular endothelial cells against high glucose levels. Moreover, the molecular mechanism of action of folic acid was further explored. The results showed that folic acid significantly suppressed the cell viability, tube length, migrated cells and the percentage of BrdU⁺ cells compared with the high glucose group. Moreover, folic acid decreased the mRNA expression of TEAD1 and the protein expression of TEAD1 and YAP1. These findings indicate that folic acid can protect retinal vascular endothelial cells from high glucose-induced injury by regulating the proteins in the Hippo signaling pathway. 10.3390/molecules23092326
    Utilizing network pharmacology to explore the underlying mechanism of in diabetic retinopathy. Piao Chun-Li,Luo Jin-Li,Jin De,Tang Cheng,Wang Li,Lian Feng-Mei,Tong Xiao-Lin Chinese medicine Introduction: (Dan-shen in pinyin), a classic Chinese herb, has been extensively used to treat diabetic retinopathy in clinical practice in China for many years. However, the pharmacological mechanisms of remain vague. The aim of this study was to decrypt the underlying mechanisms of in the treatment of diabetic retinopathy using a systems pharmacology approach. Methods:A network pharmacology-based strategy was proposed to elucidate the underlying multi-component, multi-target, and multi-pathway mode of action of against diabetic retinopathy. First, we collected putative targets of based on the Traditional Chinese Medicine System Pharmacology database and a network of the interactions among the putative targets of and known therapeutic targets of diabetic retinopathy was built. Then, two topological parameters, "degree" and "closeness certainty" were calculated to identify the major targets in the network. Furthermore, the major hubs were imported to the Database for Annotation, Visualization and Integrated Discovery to perform a pathway enrichment analysis. Results:A total of 130 nodes, including 18 putative targets of , were observed to be major hubs in terms of topological importance. The results of pathway enrichment analysis indicated that putative targets of mostly participated in various pathways associated with angiogenesis, protein metabolism, inflammatory response, apoptosis, and cell proliferation. The putative targets of (vascular endothelial growth factor, matrix metalloproteinases, plasminogen, insulin-like growth factor-1, and cyclooxygenase-2) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of on diabetic retinopathy. Conclusions: could alleviate diabetic retinopathy via the molecular mechanisms predicted by network pharmacology. This research demonstrates that the network pharmacology approach can be an effective tool to reveal the mechanisms of traditional Chinese medicine from a holistic perspective. 10.1186/s13020-019-0280-7
    Progress of stem/progenitor cell-based therapy for retinal degeneration. Tang Zhimin,Zhang Yi,Wang Yuyao,Zhang Dandan,Shen Bingqiao,Luo Min,Gu Ping Journal of translational medicine Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future. 10.1186/s12967-017-1183-y
    Compound anisodine affects the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells and brain neural stem cells via the p-ERK1/2/HIF-1α/VEGF pathway. Wang Qun,Gao Shan,Luo Yu,Kang Qian-Yan Experimental and therapeutic medicine As a Traditional Chinese Medicine, compound anisodine (CA) has previously been shown to regulate the vegetative nervous system, improve microcirculation and scavenge reactive oxygen species, and has been commonly utilized as a neuroprotective agent to treat ischemic optic neuropathy and choroidoretinopathy. The present study aimed to investigate the neuroprotective effects of CA on the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells (RPCs) and brain neural stem cells (BNSCs) harvested from neonatal Sprague-Dawley rats. Cells were treated with CA at 0.126, 0.252, 0.505 or 1.010 g/l for four hours prior to or after hypoxia (<1% oxygen) for four h, followed by re-oxygenation for four hours; a normal control group and a CA-untreated hypoxia model group were also included. An MTT assay demonstrated that the cell viability was markedly improved following treatment with 0.126-1.010 g/l CA, compared with that in the hypoxia model group (P<0.05). Bromodeoxyuridine (BrdU) immunocytochemical staining and flow cytometry indicated that after culture in hypoxia for 4 h, the number of BrdU RPCs and BNSCs was significant decreased, as well as the cell population in S+G2 phase of the cell cycle, which was significantly attenuated by treatment with 1.010 g/l CA for 4 h prior to hypoxia (P<0.05). Furthermore, laser scanning confocal microscopy showed that the intracellular calcium concentration in hypoxia-cultured RPCs and BNSCs was markedly increased, which was attenuated by 0.126-1.010 g/l CA in a concentration-dependent manner (P<0.05). Furthermore, western blot analysis demonstrated that after hypoxia, the protein levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were upregulated in RPCs and BNSCs, whereas phosphorylated extracellular signal-regulated kinase (phospho-ERK 1/2) and Cyclin D1 were downregulated; of note, treatment with 1.010 g/l CA significantly attenuated these changes (P<0.05). The results of the present study suggested that CA may improve the proliferation and inhibit calcium overload in hypoxia-induced RPCs and BNSCs by altering the protein levels of Cyclin D1 as well as signaling through the p-ERK1/2/HIF-1α/VEGF pathway. 10.3892/etm.2017.4528
    AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro. Wu Wenyi,Duan Yajian,Ma Gaoen,Zhou Guohong,Park-Windhol Cindy,D'Amore Patricia A,Lei Hetian Investigative ophthalmology & visual science Purpose:Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. Methods:The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. Results:AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. Conclusions:AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis. 10.1167/iovs.17-21902
    Fzd7 (Frizzled-7) Expressed by Endothelial Cells Controls Blood Vessel Formation Through Wnt/β-Catenin Canonical Signaling. Peghaire Claire,Bats Marie Lise,Sewduth Raj,Jeanningros Sylvie,Jaspard Beatrice,Couffinhal Thierry,Duplàa Cécile,Dufourcq Pascale Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Vessel formation requires precise orchestration of a series of morphometric and molecular events controlled by a multitude of angiogenic factors and morphogens. Wnt/frizzled signaling is required for proper vascular formation. In this study, we investigated the role of the Fzd7 (frizzled-7) receptor in retinal vascular development and its relationship with the Wnt/β-catenin canonical pathway and Notch signaling. APPROACH AND RESULTS:Using transgenic mice, we demonstrated that Fzd7 is required for postnatal vascular formation. Endothelial cell (EC) deletion of fzd7 (fzd7) delayed retinal plexus formation because of an impairment in tip cell phenotype and a decrease in stalk cell proliferation. Dvl (dishevelled) proteins are a main component of Wnt signaling and play a functionally redundant role. We found that Dvl3 depletion in dvl1 mice mimicked the fzd7 vascular phenotype and demonstrated that Fzd7 acted via β-catenin activation by showing that LiCl treatment rescued impairment in tip and stalk cell phenotypes induced in fzd7 mutants. Deletion of fzd7 or Dvl1/3 induced a strong decrease in Wnt canonical genes and Notch partners' expression. Genetic and pharmacological rescue strategies demonstrated that Fzd7 acted via β-catenin activation, upstream of Notch signaling to control Dll4 and Jagged1 EC expression. CONCLUSIONS:Fzd7 expressed by EC drives postnatal angiogenesis via activation of Dvl/β-catenin signaling and can control the integrative interaction of Wnt and Notch signaling during postnatal angiogenesis. 10.1161/ATVBAHA.116.307926
    [Diabetic retinopathy: pathogenesis and therapeutic implications]. Pelikánová Terezie Vnitrni lekarstvi UNLABELLED:Diabetic retinopathy (DR) develops in patients with both type 1 and type 2 diabetes and is the major cause of vision loss and blindness in the working population. The main risk factor of DR is hyperglycemia accompanied by enhanced mitochondrial production of reactive oxygen species and oxidative stress, formation of advanced glycation end products (AGE) and hexosamines, increase in polyol metabolism of glucose. The severity of vascular injury depends on the individual genetic background and is modified by other epigenetic, metabolic and haemodynamic factors, including hypertension, dyslipidemia and oxidative stress. In diabetes, damage to the retina occurs in the vasculature (endothelial cells and pericytes), neurons and glia, pigment epithelial cells and infiltrating immunocompetent cells: monocytes, granulocytes, lymfocytes. These activated cells change the production pattern of a number of mediators such as growth factors, proinflammatory cytokines, vasoactive molecules, coagulation factors and adhesion molecules resulting in increased blood flow, increased capillary permeability, proliferation of extracellular matrix and thickening of basal membranes, altered cell turnover (apoptosis, proliferation, hypertrophy), procoagulant and proaggregant pattern, and finally in angiogenesis and tissue remodelling. Brain, liver, adipose tissue, GUT, skeletal muscle and other tissues could be another source of mediators. Therapeutic approaches used for patients with or at risk for diabetic retinopathy include drug therapy to reduce modifiable risk factors, laser photocoagulation, intravitreous administration of anti-VEGF agents/steroids and intraocular surgery. Screening plays an important role in early detection and intervention to prevent the progression of diabetic retinopathy. Described insights into pathophysiological mechanisms responsible for DR, could help in the development of more targeted approach for prevention and treatment of diabetic retinopathy. KEY WORDS:anti-VEGF - diabetic dyslipidemia - growth factors - hyperglycemia - hypertension - microRNA - oxidative stress - retinopathy.
    Therapies targeting Frizzled-7/β-catenin pathway prevent the development of pathological angiogenesis in an ischemic retinopathy model. Bats Marie-Lise,Bougaran Pauline,Peghaire Claire,Gueniot Florian,Abelanet Alice,Chan Hélène,Séguy Camille,Jeanningros Sylvie,Jaspard-Vinassa Béatrice,Couffinhal Thierry,Duplàa Cécile,Dufourcq Pascale FASEB journal : official publication of the Federation of American Societies for Experimental Biology Retinopathies remain major causes of visual impairment in diabetic patients and premature infants. Introduction of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) has transformed therapy for these proliferative retinopathies. However, limitations associated with anti-VEGF medications require to unravel new pathways of vessel growth to identify potential drug targets. Here, we investigated the role of Wnt/Frizzled-7 (Fzd7) pathway in a mouse model of oxygen-induced retinopathy (OIR). Using transgenic mice, which enabled endothelium-specific and time-specific Fzd7 deletion, we demonstrated that Fzd7 controls both vaso-obliteration and neovascular phases (NV). Deletion of Fzd7 at P12, after the ischemic phase of OIR, prevented formation of aberrant neovessels into the vitreous by suppressing proliferation of endothelial cells (EC) in tufts. Next we validated in vitro two Frd7 blocking strategies: a monoclonal antibody (mAbFzd7) against Fzd7 and a soluble Fzd7 receptor (CRD). In vivo a single intravitreal microinjection of mAbFzd7 or CRD significantly attenuated retinal neovascularization (NV) in mice with OIR. Molecular analysis revealed that Fzd7 may act through the activation of Wnt/β-catenin and Jagged1 expression to control EC proliferation in extra-retinal neovessels. We identified Fzd7/β-catenin signaling as new regulator of pathological retinal NV. Fzd7 appears to be a potent pharmacological target to prevent or treat aberrant angiogenesis of ischemic retinopathies. 10.1096/fj.201901886R
    Asymmetric dimethylarginine contributes to retinal neovascularization of diabetic retinopathy through EphrinB2 pathway. Du Mei-Rong,Yan Li,Li Nian-Sheng,Wang Yu-Jie,Zhou Ting,Jiang Jun-Lin Vascular pharmacology Diabetic retinopathy (DR) is a leading cause of vision loss with retinal neovascularization. This study aims to investigate whether Asymmetric dimethylarginine (ADMA) impacts the pathogenesis of DR via focusing on promoting retinal neovascularization and its underlying molecular mechanisms. Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ) for 20 weeks. ADMA levels in aqueous and the influence of hypoxia on ADMA and angiogenesis in RF/6A cells were examined. The effects and underlying molecular mechanisms of ADMA on neovascularization of RF/6A cells were further evaluated by administration of ADMA, DDAH siRNA or ephrinB2 siRNA. Results showed that ADMA levels were elevated in both aqueous from diabetic rats and culture medium in RF/6A cells pretreated with hypoxia. Administration of ADMA directly promoted proliferation, migration, adhesion and tube formation of RF/6A cells, which was further confirmed by DDAH1 siRNA or DDAH2 siRNA. In addition, ephrinB2 expression was increased under diabetic conditions, and the angiogenic effects of ADMA were blocked by ephrinB2 siRNA. In conclusion, ADMA contributes to the neovascularization of retina in diabetic mellitus, which is regulated by ephrinB2. 10.1016/j.vph.2018.05.004
    Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway. Ha Jung Min,Jin Seo Yeon,Lee Hye Sun,Shin Hwa Kyoung,Lee Dong Hyung,Song Sang Heon,Kim Chi Dae,Bae Sun Sik The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis. 10.4196/kjpp.2016.20.5.533
    Antiangiogenic effect of betaine on pathologic retinal neovascularization via suppression of reactive oxygen species mediated vascular endothelial growth factor signaling. Park Sung Wook,Jun Hyoung Oh,Kwon Euna,Yun Jun-Won,Kim Jin Hyoung,Park Young-Jun,Kang Byeong-Cheol,Kim Jeong Hun Vascular pharmacology Reactive oxygen species (ROS) as well as vascular endothelial growth factor (VEGF) play important roles in pathologic retinal neovascularization. We investigated whether betaine inhibits pathologic retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR). Betaine was intravitreally injected in OIR mice at postnatal day (P) 14. At P17, the neovascular tufts area in OIR retina was analyzed. Intravitreal injection of betaine (200μM) effectively reduced the neovascular tufts area in OIR retina (68.0±6.7% of the control eyes, P<0.05). Even in a high concentration (2mM), betaine never induced any retinal toxicity or cytotoxicity. Betaine significantly inhibited VEGF-induced proliferation, migration, and tube formation in human retinal microvascular endothelial cells (HRMECs). Betaine suppressed VEGF-induced VEGFR-2, Akt and ERK phosphorylation in HRMECs. In human brain astrocytes, betaine reduced tBH-induced ROS production, and subsequently attenuated tBH-induced VEGFA mRNA transcription via suppression of ROS. Our data suggest that betaine has an anti-angiogenic effect on pathologic retinal neovascularization via suppression of ROS mediated VEGF signaling. Betaine could be a potent anti-angiogenic agent to treat pathologic retinal neovascularization. 10.1016/j.vph.2016.07.007
    MiR-7 regulates the PI3K/AKT/VEGF pathway of retinal capillary endothelial cell and retinal pericytes in diabetic rat model through IRS-1 and inhibits cell proliferation. Cao Y-L,Liu D-J,Zhang H-G European review for medical and pharmacological sciences OBJECTIVE:To investigate the role of miR-7 in diabetic retinopathy and the underlying mechanism. MATERIALS AND METHODS:The rat model of diabetic retinopathy (DR) was established. After that, the endothelial cell (EC) and retinal pericyte (RP) were isolated. QRT-PCR was used to detect the expression of miR-7 and insulin receptor substrate-1 (IRS-1) in ECs and RPs cells while the protein level of IRS1 was detected by Western blot. miR-7 mimic and miR-7 inhibitor were transfected to achieve miR-7 overexpression or knockdown. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay after miR-7 overexpression or knockdown. Besides, the expression levels of PI3K, AKT, and VEGF were detected by Western Blot. The luciferase reporter assay was performed to investigate whether miR-7 could be combined with IRS-1. Conversely, whether miR-7 could affect IRS-1 was also verified. RESULTS:miR-7 expression was significantly decreased in ECs and RPs of the experimental group compared with the control group, while the mRNA and protein levels of IRS-1 were increased. The CCK-8 assay showed that overexpression of miR-7 decreased the cell activity in ECs and RPs. In contrast, knock-down of miR-7 could increase the cell viability. Besides, Western blot showed that after overexpression of miR-7, the expressions of PI3K, AKT, and VEGF in ECs and RPs cells were down-regulated. Meanwhile, miR-7 knockdown upregulated the protein levels of PI3K, AKT, and VEGF. The luciferase reporter assay suggested that the 3'UTR region of IRS-1 could be combined with miR-7, which may be the downstream target gene for miR-7. Moreover, knockdown of IRS-1 could reverse the effect of the miR-7 inhibitor on cell proliferation in the diabetic model. CONCLUSIONS:MiR-7 was lowly expressed in ECs and RPs cells. Overexpression of miR-7 can down-regulate the expression levels of PI3K, AKT, and VEGF by down-regulating its downstream target gene IRS-1, and ultimately inhibit the proliferation of retinal cells. 10.26355/eurrev_201807_15493
    ALK1 Loss Results in Vascular Hyperplasia in Mice and Humans Through PI3K Activation. Alsina-Sanchís Elisenda,García-Ibáñez Yaiza,Figueiredo Ana M,Riera-Domingo Carla,Figueras Agnès,Matias-Guiu Xavier,Casanovas Oriol,Botella Luisa M,Pujana Miquel A,Riera-Mestre Antoni,Graupera Mariona,Viñals Francesc Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-β (transforming growth factor-β) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia-a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia. APPROACH AND RESULTS:In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1 mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1 retinas and in turn normalize the vasculature. CONCLUSIONS:Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia. 10.1161/ATVBAHA.118.310760
    Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin. Qiu Feng,Tong Huijuan,Wang Yawen,Tao Jun,Wang Hailin,Chen Lei Bioscience, biotechnology, and biochemistry The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin. 10.1080/09168451.2018.1459179
    NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy. Dubrac Alexandre,Künzel Steffen E,Künzel Sandrine H,Li Jinyu,Chandran Rachana Radhamani,Martin Kathleen,Greif Daniel M,Adams Ralf H,Eichmann Anne Nature communications Pericytes are mural cells that surround capillaries and control angiogenesis and capillary barrier function. During sprouting angiogenesis, endothelial cell-derived platelet-derived growth factor-B (PDGF-B) regulates pericyte proliferation and migration via the platelet-derived growth factor receptor-β (PDGFRβ). PDGF-B overexpression has been associated with proliferative retinopathy, but the underlying mechanisms remain poorly understood. Here we show that abnormal, α-SMA-expressing pericytes cover angiogenic sprouts and pathological neovascular tufts (NVTs) in a mouse model of oxygen-induced retinopathy. Genetic lineage tracing demonstrates that pericytes acquire α-SMA expression during NVT formation. Pericyte depletion through inducible endothelial-specific knockout of Pdgf-b decreases NVT formation and impairs revascularization. Inactivation of the NCK1 and NCK2 adaptor proteins inhibits pericyte migration by preventing PDGF-B-induced phosphorylation of PDGFRβ at Y1009 and PAK activation. Loss of Nck1 and Nck2 in mural cells prevents NVT formation and vascular leakage and promotes revascularization, suggesting PDGFRβ-Y1009/NCK signaling as a potential target for the treatment of retinopathies. 10.1038/s41467-018-05926-7
    6'-Sialylgalactose inhibits vascular endothelial growth factor receptor 2-mediated angiogenesis. Chung Tae-Wook,Kim Eun-Yeong,Choi Hee-Jung,Han Chang Woo,Jang Se Bok,Kim Keuk-Jun,Jin Ling,Koh Young Jun,Ha Ki-Tae Experimental & molecular medicine Angiogenesis should be precisely regulated because disordered neovascularization is involved in the aggravation of multiple diseases. The vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2) axis is crucial for controlling angiogenic responses in vascular endothelial cells (ECs). Therefore, inactivating VEGFR-2 signaling may effectively suppress aberrant angiogenesis and alleviate related symptoms. In this study, we performed virtual screening, identified the synthetic disaccharide 6'-sialylgalactose (6SG) as a potent VEGFR-2-binding compound and verified its high binding affinity by Biacore assay. 6SG effectively suppressed VEGF-A-induced VEGFR-2 phosphorylation and subsequent in vitro angiogenesis in HUVECs without inducing cytotoxicity. 6SG also inhibited VEGF-A-induced extracellular-regulated kinase (ERK)/Akt activation and actin stress fiber formation in HUVECs. We demonstrated that 6SG inhibited retinal angiogenesis in a mouse model of retinopathy of prematurity and tumor angiogenesis in a xenograft mouse model. Our results suggest a potential therapeutic benefit of 6SG in inhibiting angiogenesis in proangiogenic diseases, such as retinopathy and cancer. 10.1038/s12276-019-0311-6
    Somatolactogens and diabetic retinopathy. Bermea Kevin Christian,Rodríguez-García Alejandro,Tsin Andrew,Barrera-Saldaña Hugo Alberto Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society IMPORTANCE:Diabetic retinopathy (DR) is one of the most common of all diabetic complications. The number of people with DR in the United States is expected to increase to 16 million by 2050. DR is the leading cause of blindness among working-age adults in many different countries, including the United States. In later DR stages, neovascularization is associated with extensive retinal capillary non-perfusion and vitreo-proliferation leading to retinal detachment. This neovascularization is orchestrated by an imbalance of growth factors in the retina from which somatolactogens (pituitary growth hormone, GH-N; placental growth hormone, GH-V; prolactin, PRL; and placental lactogen, PL, also referred as chorionic somatomammotropin, CSH), may play an important role. OBSERVATIONS:Somatolactogens are a group of hormones that share many structural and functional features. They are important for physiological changes in pregnancy, for adequate development of the fetus, and in the case of GH-N, for promoting growth after birth. GH-N is synthesized by the anterior pituitary, GH-V and PL are secreted by the placenta, whereas, PRL is synthesized by the anterior pituitary and uterine decidua. However, in recent years the expression of GH-N and PRL and their receptors have been detected in other tissues including the retina, acting as neuroprotective and pro-angiogenic agents. The relationship of GH-N and diabetic retinopathy (DR) was established many years ago when it was observed that its deficiency was related to regression of DR while an increase in serum levels of GH-N, GH-V, and PL promoted DR. While more studies are needed to define the potential implications of GH-V and PL in DR pathogenesis, it has been demonstrated that GH-N and PRL participate in DR by enhancing neovascularization. Some PRL isoforms, however, have shown an anti-angiogenic activity rather than pro-angiogenesis and appears to be PRL's main role in the regulation of retinal vasculature. CONCLUSIONS:Somatolactogens are a group of hormones with a significant role in neuroprotection and angiogenesis regulation in the eye. Understanding the mechanisms of angiogenesis regulation by somatolactogens will potentially lead to the development of new drugs for DR. 10.1016/j.ghir.2018.02.002
    Transthyretin Upregulates Long Non-Coding RNA MEG3 by Affecting PABPC1 in Diabetic Retinopathy. Fan Guangming,Gu Yu,Zhang Jiaojiao,Xin Yu,Shao Jun,Giampieri Francesca,Battino Maurizio International journal of molecular sciences The aim of the study was to demonstrate how transthyretin (TTR) could affect long non-coding RNA (lncRNA) of maternally expressed gene 3 (MEG3) and play important roles in diabetic retinopathy (DR). A DR model in C57BL/6 mice was established after intraperitoneal injection of streptozotocin (STZ). After intravitreal injection with TTR pAAV vector, MEG3 short hairpin RNA (shRNA), scrambled shRNA, or MEG3, retinal imaging, retinal trypsin digestion, and fundus vascular permeability tests were performed. Cell counting kit-8 (CCK8), transwell, and Matrigel assays were employed to detect the proliferation and migration of human retinal microvascular endothelial cells (hRECs). The binding between long non-coding RNA of maternally expressed gene 3 (lncRNA-MEG3) and microRNA-223-3p (miR-223-3p) was observed by using luciferase reporter assays, while co-immunoprecipitation (co-IP) was employed to confirm the interaction between TTR and the target. In the DR mice model, retinal vascular leakage and angiogenesis were repressed by overexpressing TTR. In vitro the added TTR promoted the level of lncRNA-MEG3 by interacting with poly (A) binding protein cytoplasmic 1 (PABPC1), and then repressed proliferation and angiogenesis of hRECs. In vivo, silencing or overexpressing lncRNA-MEG3 significantly affected retinal vascular phenotypes. Additionally, the interaction between lncRNA-MEG3 and miR-223-3p was confirmed, and silencing of miR-223-3p revealed similar effects on hRECs as overexpression of lncRNA-MEG3. In summary, in the DR environment, TTR might affect the lncRNA MEG3/miR-223-3p axis by the direct binding with PABPC1, and finally repress retinal vessel proliferation. 10.3390/ijms20246313
    Abscisic acid - an anti-angiogenic phytohormone that modulates the phenotypical plasticity of endothelial cells and macrophages. Chaqour Julienne,Lee Sangmi,Ravichandra Aashreya,Chaqour Brahim Journal of cell science Abscisic acid (ABA) has shown anti-inflammatory and immunoregulatory properties in preclinical models of diabetes and inflammation. Herein, we studied the effects of ABA on angiogenesis, a strictly controlled process that, when dysregulated, leads to severe angiogenic disorders including vascular overgrowth, exudation, cellular inflammation and organ dysfunction. By using a 3D sprouting assay, we show that ABA effectively inhibits migration, growth and expansion of endothelial tubes without affecting cell viability. Analyses of the retinal vasculature in developing normoxic and hyperoxic mice challenged by oxygen toxicity reveal that exogenously administered ABA stunts the development and regeneration of blood vessels. In these models, ABA downregulates endothelial cell (EC)-specific growth and migratory genes, interferes with tip and stalk cell specification, and hinders the function of filopodial protrusions required for precise guidance of vascular sprouts. In addition, ABA skews macrophage polarization towards the M1 phenotype characterized by anti-angiogenic marker expression. In accordance with this, ABA treatment accelerates macrophage-induced programmed regression of fetal blood vessels. These findings reveal protective functions of ABA against neovascular growth through modulation of EC and macrophage plasticity, suggesting the potential utility of ABA as a treatment in vasoproliferative diseases. 10.1242/jcs.210492
    Neurofibromin Deficiency Induces Endothelial Cell Proliferation and Retinal Neovascularization. Zhang Hanfang,Hudson Farlyn Z,Xu Zhimin,Tritz Rebekah,Rojas Modesto,Patel Chintan,Haigh Stephen B,Bordán Zsuzsanna,Ingram David A,Fulton David J,Weintraub Neal L,Caldwell Ruth B,Stansfield Brian K Investigative ophthalmology & visual science Purpose:Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization. Methods:Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching. Results:Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas. Conclusions:These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial. 10.1167/iovs.17-22588
    Transthyretin represses neovascularization in diabetic retinopathy. Shao Jun,Yao Yong Molecular vision PURPOSE:The apoptosis of human umbilical vein endothelial cells has been reportedly induced by the protein transthyretin (TTR). In human ocular tissue, TTR is generally considered to be secreted mainly by retinal pigment epithelial cells (hRPECs); however, whether TTR affects the development of neovascularization in diabetic retinopathy (DR) remains unclear. METHODS:Natural and simulated DR media were used to culture human retinal microvascular endothelial cells (hRECs). Hyperglycemia was simulated by increasing the glucose concentration from 5.5 mM up to 25 mM, while hypoxia was induced with 200 µM CoCl. To understand the effects of TTR on hRECs, cell proliferation was investigated under natural and DR conditions. Overexpression of TTR, an in vitro wound-healing assay, and a tube formation assay were employed to study the repression of TTR on hRECs. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study the mRNA levels of DR-related genes, such as , , , and . RESULTS:The proliferation of hRECs was significantly decreased in the simulated hyperglycemic and hypoxic DR environments. The cells were further repressed by added exogenous or endogenous TTR only under hyperglycemic conditions. The in vitro migration and tube formation processes of the hRECs were inhibited with TTR; furthermore, in the hyperglycemia and hyperglycemia/hypoxia environments, the levels of and mRNA were enhanced with exogenous TTR, while those of , and were repressed. CONCLUSIONS:In hyperglycemia, the proliferation, migration, and neovascularization of hRECs were significantly inhibited by TTR. The key genes for DR neovascularization, including , , , and , were regulated by TTR. Under DR conditions, TTR significantly represses neovascularization by inhibiting the proliferation, migration and tube formation of hRECs.
    Serum miR-122 levels correlate with diabetic retinopathy. Pastukh Nina,Meerson Ari,Kalish Dorina,Jabaly Hanin,Blum Arnon Clinical and experimental medicine Diabetic retinopathy is the most severe ocular complication of diabetes and may lead to visual disability and blindness. Proliferative diabetic retinopathy (PDR) is characterized by ischemia-induced neovascularization with associated complications. An association was established between the presence of PDR, cardiovascular disease, and mortality among patients with type 1 diabetes mellitus and type 2 diabetes mellitus in epidemiological studies. However, the mechanism underlying increased cardiovascular risk in patients with PDR is still unknown. In recent years, a group of miRNAs has been linked to the pathology of diabetes mellitus. Besides, miRNAs in biofluids such as serum have been suggested as potential minimally invasive biomarkers of diabetes and vascular complications. This was a prospective study that recruited 40 human subjects: 10 healthy subjects, 10 with diabetes but without retinopathy (NDR), 10 with diabetic non-proliferative retinopathy (NPDR), and 10 with proliferative diabetic retinopathy (PDR). To examine whether serum miRNAs show altered levels at different stages of diabetic retinopathy, seven specific miRNA candidates (miR-126-3p, miR-130a-3p, miR-21-1, let-7f-5p, miR-122, miR-30c and miR-451a) were measured by qRT-PCR in RNA isolated from sera of all subjects. miR-122 levels increased in parallel with retinopathy severity: from healthy controls to NDR and from NDR to NPDR. However, when the disease progressed to PDR a marked decrease in miR-122 level was noted. This decrease was significant both compared to NPDR samples (p = 0.016) and to all non-PDR samples (p = 0.0002). Additionally, a positive trend was observed comparing miR-122 levels and the number of endothelial progenitor cells in the sera of all subjects. A significant increase in miR-122 was found in patients with diabetic retinopathy that may be related to its role in preventing angiogenesis and proliferation. The dramatic decline in patients with PDR may represent an inhibition or exhaustion of the anti-angiogenic anti-proliferative defense system. Further studies are needed to understand whether miRNA-122 has a role in the pathogenesis of diabetic retinopathy. 10.1007/s10238-019-00546-x
    Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus. Shan Kun,Liu Chang,Liu Bai-Hui,Chen Xue,Dong Rui,Liu Xin,Zhang Yang-Yang,Liu Ban,Zhang Shu-Jie,Wang Jia-Jian,Zhang Sheng-Hai,Wu Ji-Hong,Zhao Chen,Yan Biao Circulation BACKGROUND:The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS:Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS:circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS:The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy. 10.1161/CIRCULATIONAHA.117.029004
    Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization. McAnally Danielle,Siddiquee Khandaker,Gomaa Ahmed,Szabo Andras,Vasile Stefan,Maloney Patrick R,Divlianska Daniela B,Peddibhotla Satyamaheshwar,Morfa Camilo J,Hershberger Paul,Falter Rebecca,Williamson Robert,Terry David B,Farjo Rafal,Pinkerton Anthony B,Qi Xiaping,Quigley Judith,Boulton Michael E,Grant Maria B,Smith Layton H PloS one Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization. While anti-VGEF therapies have been successful, up to 30% of patients are non-responsive. Therefore, there is a need for new therapeutic targets, and small molecule inhibitors of angiogenesis to complement existing treatments. Apelin and its receptor have recently been shown to play a key role in both developmental and pathological angiogenesis in the eye. Through a cell-based high-throughput screen, we identified 4-aminoquinoline antimalarial drugs as potent selective antagonists of APJ. The prototypical 4-aminoquinoline, amodiaquine was found to be a selective, non-competitive APJ antagonist that inhibited apelin signaling in a concentration-dependent manner. Additionally, amodiaquine suppressed both apelin-and VGEF-induced endothelial tube formation. Intravitreal amodaiquine significantly reduced choroidal neovascularization (CNV) lesion volume in the laser-induced CNV mouse model, and showed no signs of ocular toxicity at the highest doses tested. This work firmly establishes APJ as a novel, chemically tractable therapeutic target for the treatment of ocular neovascularization, and that amodiaquine is a potential candidate for repurposing and further toxicological, and pharmacokinetic evaluation in the clinic. 10.1371/journal.pone.0202436
    Proteomic Biomarkers of Retinal Inflammation in Diabetic Retinopathy. Youngblood Hannah,Robinson Rebekah,Sharma Ashok,Sharma Shruti International journal of molecular sciences Diabetic retinopathy (DR), a sight-threatening neurovasculopathy, is the leading cause of irreversible blindness in the developed world. DR arises as the result of prolonged hyperglycemia and is characterized by leaky retinal vasculature, retinal ischemia, retinal inflammation, angiogenesis, and neovascularization. The number of DR patients is growing with an increase in the elderly population, and therapeutic approaches are limited, therefore, new therapies to prevent retinal injury and enhance repair are a critical unmet need. Besides vascular endothelial growth factor (VEGF)-induced vascular proliferation, several other mechanisms are important in the pathogenesis of diabetic retinopathy, including vascular inflammation. Thus, combining anti-VEGF therapy with other new therapies targeting these pathophysiological pathways of DR may further optimize treatment outcomes. Technological advancements have allowed for high-throughput proteomic studies examining biofluids such as aqueous humor, vitreous humor, tear, and serum. Many DR biomarkers have been identified, especially proteins involved in retinal inflammatory processes. This review attempts to summarize the proteomic biomarkers of DR-associated retinal inflammation identified over the last several years. 10.3390/ijms20194755
    53BP1 Deficiency Promotes Pathological Neovascularization in Proliferative Retinopathy. Troullinaki Maria,Garcia-Martin Ruben,Sprott David,Klotzsche-von Ameln Anne,Grossklaus Sylvia,Mitroulis Ioannis,Chavakis Triantafyllos,Economopoulou Matina Thrombosis and haemostasis The replication stress inflicted on retinal endothelial cells (ECs) in the context of hypoxia-induced pathological neovascularization during proliferative retinopathy is linked with activation of the deoxyribonucleic acid (DNA) repair response. Here, we studied the effect of deficiency of the DNA damage response adaptor 53BP1, which is an antagonist of homologous recombination (HR), in the context of proliferative retinopathy. In the model of retinopathy of prematurity (ROP), 53BP1-deficient mice displayed increased hypoxia-driven pathological neovascularization and tuft formation, accompanied by increased EC proliferation and reduced EC apoptosis, as compared with 53BP1-sufficient mice. In contrast, physiological retina angiogenesis was not affected by 53BP1 deficiency. Knockdown of 53BP1 in ECs in vitro also resulted in enhanced proliferation and reduced apoptosis of the cells under hypoxic conditions. Additionally, upon 53BP1 knockdown, ECs displayed increased HR rate in hypoxia. Consistently, treatment with an HR inhibitor reversed the hyper-proliferative angiogenic phenotype associated with 53BP1 deficiency in ROP. Thus, by unleashing HR, 53BP1 deletion increases pathological EC proliferation and neovascularization in the context of ROP. Our data shed light to a previously unknown interaction between the DNA repair response and pathological neovascularization in the retina. 10.1055/s-0038-1676966
    The Effects of the CXCR4 Antagonist, AMD3465, on Human Retinal Vascular Endothelial Cells (hRVECs) in a High Glucose Model of Diabetic Retinopathy. Wu Di,Jin Li,Xu Hongshuang Medical science monitor : international medical journal of experimental and clinical research BACKGROUND High blood glucose levels in diabetes result in retinal angiogenesis, which is the key feature of diabetic retinopathy. This study aimed to investigate the effects of the CXCR4 antagonist, AMD3465, on human retinal vascular endothelial cells (hRVECs) [i]in vitro[/i]. MATERIAL AND METHODS Cell viability and the protein expression levels of CXCR4 and stromal cell-derived factor 1 (SDF-1) were evaluated in high glucose (HG)-treated human retinal vascular endothelial cells (hRVECs). The cell counting kit 8 (CCK-8) assay, the colony formation assay, immunofluorescence, and Western blot were used to investigate the effects of AMD3465 on hRVEC cell viability, colony formation, cell proliferation, and expression of CXCR4 and SDF-1. Cell apoptosis and angiogenesis were assessed by flow cytometry and Western blot. RESULTS Treatment with high glucose reduced the viability of hRVECs and increased the protein expression levels of CXCR4 and SDF-1. Following treatment with AMD3465, the colony formation capacity and cell proliferation in hRVECs increased, and there was a significant reduction in apoptosis rate compared with the untreated cells. AMD3465 significantly reduced the expression of angiogenesis-associated proteins, including ICAM1, VCAM1, VEGF, and AngII. AMD3465 significantly reduced the protein expression levels of TNF-α, IL-1β, NF-κB, and p-p65. CONCLUSIONS The CXCR4 antagonist, AMD3465, reduced apoptosis of HG-treated hRVECs in an [i]in vitro[/i] model of diabetic retinopathy. 10.12659/MSM.917186
    The Role of Hypoxia, Hypoxia-Inducible Factor (HIF), and VEGF in Retinal Angiomatous Proliferation. Barben Maya,Samardzija Marijana,Grimm Christian Advances in experimental medicine and biology In industrialized countries, age-related macular degeneration (AMD) is the leading cause of blindness in elderly people. Hallmarks of the non-neovascular (dry) form of AMD are the formation of drusen and geographic atrophy, whereas the exudative (wet) form of the disease is characterized by invading blood vessels. In retinal angiomatous proliferation (RAP), a special form of wet AMD, intraretinal vessels grow from the deep plexus into the subretinal space. Little is known about the mechanisms leading to intraretinal neovascularization, but age-related changes such as reduction of choroidal blood flow, accumulation of drusen, and thickening of the Bruch's membrane may lead to reduced oxygen availability in photoreceptors. Such a chronic hypoxic situation may induce several cellular response pathways including the stabilization of hypoxia-inducible factors (HIFs) and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Here, we discuss the potential contribution of hypoxia and HIFs in RAP disease pathology and in some mouse models for subretinal neovascularization. 10.1007/978-3-319-75402-4_22
    The Proinflammatory and Proangiogenic Macrophage Migration Inhibitory Factor Is a Potential Regulator in Proliferative Diabetic Retinopathy. Abu El-Asrar Ahmed M,Ahmad Ajmal,Siddiquei Mohammad Mairaj,De Zutter Alexandra,Allegaert Eef,Gikandi Priscilla W,De Hertogh Gert,Van Damme Jo,Opdenakker Ghislain,Struyf Sofie Frontiers in immunology The macrophage migration inhibitory factor (MIF)/CD74 signaling pathway is strongly implicated in inflammation and angiogenesis. We investigated the expression of MIF and its receptor CD74 in proliferative diabetic retinopathy (PDR) to reveal a possible role of this pathway in the pathogenesis of PDR. Levels of MIF, soluble (s)CD74, soluble intercellular adhesion molecule-1 (sICAM-1) and vascular endothelial growth factor (VEGF) were significantly increased in the vitreous from patients with PDR compared to nondiabetic control samples. We detected significant positive correlations between the levels of MIF and the levels of sICAM-1 ( = 0.43; = 0.001) and VEGF ( = 0.7; < 0.001). Through immunohistochemical analysis of PDR epiretinal membranes, significant positive correlations were also found between microvessel density (CD31 expression) and the numbers of blood vessels expressing MIF ( = 0.56; = 0.045) and stromal cells expressing MIF ( = 0.79; = 0.001) and CD74 ( = 0.59; = 0.045). Similar to VEGF, MIF was induced in Müller cells cultured under hypoxic conditions and MIF induced phosphorylation of ERK1/2 and VEGF production in Müller cells. Intravitreal administration of MIF in normal rats induced increased retinal vascular permeability and significant upregulation of phospho-ERK1/2, NF-κB, ICAM-1 and vascular cell adhesion molecule-1 expression in the retina. MIF induced migration and proliferation of human retinal microvascular endothelial cells. These results suggest that MIF/CD74 signaling is involved in PDR angiogenesis. 10.3389/fimmu.2019.02752
    A proposal for early and personalized treatment of diabetic retinopathy based on clinical pathophysiology and molecular phenotyping. Gardner Thomas W,Sundstrom Jeffrey M Vision research This paper presents a new approach to the prevention and treatment of early stage diabetic retinopathy before vision is severely impaired. This approach includes two major steps. The first step is to understand the mechanisms of vision impairment and classify diabetic retinopathy on the basis of pathophysiologic adaptations, rather than on the presence of advanced pathologic lesions, as defined by current clinical practice conventions. The second step is to develop patient-specific molecular diagnoses of diabetic retinopathy so that patients can be treated based on their individual characteristics, a process analogous to the individualized diagnosis and treatment of cancer patients. This step is illustrated by proteomic analysis of vitreous fluid that reveals evidence of neuroretinal degeneration and inflammation, as well as vascular proliferation. Together, these steps may lead to improved means to preserve vision in the ever-increasing number of patients with diabetes worldwide. 10.1016/j.visres.2017.03.006
    Cyp1b1-deficient retinal astrocytes are more proliferative and migratory and are protected from oxidative stress and inflammation. Falero-Perez Juliana,Sorenson Christine M,Sheibani Nader American journal of physiology. Cell physiology Astrocytes (ACs) are the most abundant cells in the central nervous system. Retinal ACs play an important role in maintaining the integrity of retinal neurovascular function, and their dysfunction contributes to the pathogenesis of various eye diseases including diabetic retinopathy. Cytochrome P450 1B1 (CYP1B1) expression in the neurovascular structures of the central nervous system including ACs has been reported. We previously showed that CYP1B1 expression is a key regulator of redox homeostasis in retinal vascular cells. Its deficiency in mice resulted in increased oxidative stress and attenuation of angiogenesis in vivo and proangiogenic activity of retinal vascular cells in vitro. Here, using retinal ACs prepared from wild-type () and -deficient () mice, we determined the impact of Cyp1b1 expression on retinal AC function. We showed that retinal ACs were more proliferative and migratory. These cells also produced increased amounts of fibronectin and its receptors, αβ- and αβ-integrin. These results were consistent with the increased adhesive properties of ACs and their lack of ability to form a network in Matrigel. This was reversed by reexpression of Cyp1b1 in ACs. Although no significant changes were observed in Akt/SRC/MAPK signaling pathways, production of inflammatory mediators bone morphogenetic protein-7 (BMP-7) and monocyte chemoattractant protein-1 (MCP-1) was decreased in ACs. ACs also showed increased levels of connexin 43 phosphorylation and cluster of differentiation 38 expression when challenged with HO. These results are consistent with increased proliferation and diminished oxidative stress in cells. Thus, Cyp1b1 expression in ACs plays an important role in retinal neurovascular homeostasis. 10.1152/ajpcell.00021.2019
    Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy. Dong Yoko,Noda Kousuke,Murata Miyuki,Yoshida Shiho,Saito Wataru,Kanda Atsuhiro,Ishida Susumu Current eye research PURPOSE:To determine the presence of N-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR). METHODS:Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells. RESULTS:In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (α-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells. CONCLUSIONS:The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR. 10.3109/02713683.2016.1150491
    Preclinical Evaluation and Optimization of a Cell Therapy Using Human Cord Blood-Derived Endothelial Colony-Forming Cells for Ischemic Retinopathies. Reid Emma,Guduric-Fuchs Jasenka,O'Neill Christina L,Allen Lynsey-Dawn,Chambers Sarah E J,Stitt Alan W,Medina Reinhold J Stem cells translational medicine Cell therapy using endothelial progenitors holds promise for vascular repair in ischemic retinopathies. Using a well-defined subpopulation of human cord blood-derived endothelial progenitors known as endothelial colony-forming cells (ECFCs), we have evaluated essential requirements for further development of this cell therapy targeting the ischemic retina, including dose response, delivery route, and toxicity. First, to evaluate therapeutic efficacy relating to cell dose, ECFCs were injected into the vitreous of mice with oxygen-induced retinopathy. Using angiography and histology, we found that intravitreal delivery of low dose (1 × 10 ) ECFCs was as effective as higher cell doses (1 × 10 , 1 × 10 ) in promoting vascular repair. Second, injection into the common carotid artery was tested as an alternative, systemic delivery route. Intracarotid ECFC delivery conferred therapeutic benefit which was comparable to intravitreal delivery using the same ECFC dose (1 × 10 ), although there were fewer human cells observed in the retinal vasculature following systemic delivery. Third, cell immunogenicity was evaluated by injecting ECFCs into the vitreous of healthy adult mice. Assessment of murine ocular tissues identified injected cells in the vitreous, while demonstrating integrity of the host retina. In addition, ECFCs did not invade into the retina, but remained in the vitreous, where they eventually underwent cell death within 3 days of delivery without evoking an inflammatory response. Human specific Alu sequences were not found in healthy mouse retinas after 3 days of ECFC delivery. These findings provide supportive preclinical evidence for the development of ECFCs as an efficacious cell product for ischemic retinopathies. Stem Cells Translational Medicine 2018;7:59-67. 10.1002/sctm.17-0187
    Laminin-Dependent Interaction between Astrocytes and Microglia: A Role in Retinal Angiogenesis. Biswas Saptarshi,Bachay Galina,Chu Julianne,Hunter Dale D,Brunken William J The American journal of pathology Retinal vascular diseases are among the leading causes of acquired blindness. In recent years, retinal microglia have been shown to influence vascular branching density and endothelial cell proliferation. However, how microglial recruitment and activation are regulated during development remains unclear. We hypothesized that microglial recruitment, activation, and down-stream signaling are modulated by components of the mural basement membrane. We used a reverse genetic approach to disrupt laminin expression in the vascular basement membrane and demonstrate that microglia respond to the mural basement membrane in an isoform-specific manner. Microglial density is significantly increased in the laminin γ3-null (Lamc3) retinal superficial vascular plexus and consequently the vascular branching density is increased. Microglia also respond to astrocyte-derived matrices and become hyperactivated in the Lamc3 retina or when tested in vitro with cell-derived matrix. Pharmacological activation of microglia in the wild-type retina produced an Lamc3-like vascular phenotype, whereas pharmacological blocking of microglial activation in the Lamc3 retina rescued the wild-type vascular phenotype. On the molecular level, microglial transforming growth factor-β1 expression is down-regulated in the Lamc3 retina, and SMAD signaling decreased in endothelial cells with a consequent increase in endothelial proliferation. The reverse effects were seen in the Lamb2 retina. Together, our results demonstrate a novel mechanism by which laminins modulate vascular branching and endothelial cell proliferation during retinal angiogenesis. 10.1016/j.ajpath.2017.05.016
    MicroRNA-9 inhibits retinal neovascularization in rats with diabetic retinopathy by targeting vascular endothelial growth factor A. Liu Wen-Lan Journal of cellular biochemistry Diabetic retinopathy (DR) is a leading cause of adult visual impairment and loss. This study aims to explore the effects of microRNA-9 (miR-9) on retinal neovascularization during DR by targeting the vascular endothelial growth factor A (VEGFA). DR rat models were successfully established. Retinal microvascular endothelial cells (RMECs) of DR rats were isolated and treated with miR-9 mimic, miR-9 inhibitor or small interfering RNA (siRNA)-VEGFA. The expressions of miR-9, VEGFA, and cluster of differentiation 31 (CD31) of the rats' tissues and cells were examined. The targeting relationship between miR-9 and VEGFA was testified. The tubule formation, the cell proliferation and the periodic distribution and apoptosis were evaluated after transfection. In the retinal tissues of DR rats, miR-9 expression decreased while the expression of VEGFA and CD31 increased. Notably, miR-9 targeted and inhibited VEGFA expression. In response to the treatment of miR-9 mimic and siRNA-VEGFA, a reduction was identified in CD31 expression, tubule formation, and proliferation of RMECs and cell ratio in the S phase, but an increase was observed in apoptosis rate of RMECs. The treatment of miR-9 inhibitor reversed the manifestations. Our study demonstrated that miR-9 could inhibit retinal neovascularization of DR and tubule formation, and promote apoptosis in RMECs by targeting VEGFA. 10.1002/jcb.28081
    microRNA-181a inhibits ocular neovascularization by interfering with vascular endothelial growth factor expression. Yang Chun,Tahiri Houda,Cai Chenrongrong,Gu Muqing,Gagnon Carmen,Hardy Pierre Cardiovascular therapeutics AIM:Excess angiogenesis or neovascularization plays a key role in the pathophysiology of several ocular diseases such as retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration. microRNA-181a (miR-181a) was found highly expressed in retina and choroidal tissues. This study intends to investigate the role of miR-181a in the regulation of ocular neovascularization in different pathophysiological conditions. METHOD:We performed the RNA sequence to identify the microRNAs components of anti-angiogenic lymphocyte-derived microparticles (LMPs). The effect of miR-181a on human retinal endothelial cells proliferation was assessed in vitro. The impact of miR-181a on angiogenesis was confirmed using in vitro angiogenesis assay, ex vivo choroidal explant, and in vivo retinal neovascularization. The expression of major angiogenic factors was assessed by real-time qPCR. RESULTS:RNA sequence revealed that miR-181a is selectively enriched in LMPs. Importantly, the inhibition of miR-181a significantly abrogated the effect of LMPs on endothelial viability, but overexpression of miR-181a reduced endothelial cell viability in a dose-dependent manner. miR-181a strongly inhibited in vitro angiogenesis and ex vivo choroidal neovascularization. The strong anti-angiogenic effect of miR-181a was also displayed on the retinal neovascularization of the in vivo mouse model of oxygen-induced retinopathy. In keeping with its effect, several angiogenesis-related genes were dysregulated in the miR-181a overexpressed endothelial cells. CONCLUSION:These data may open unexpected avenues for the development of miR-181a as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of neovascular diseases. 10.1111/1755-5922.12329
    RNA sequencing identified specific circulating miRNA biomarkers for early detection of diabetes retinopathy. Liang Zhen,Gao Kai P,Wang Yi X,Liu Zi C,Tian Li,Yang Xin Z,Ding Jing Y,Wu Wei T,Yang Wen H,Li Yi L,Zhang Ze B,Zhai Ri H American journal of physiology. Endocrinology and metabolism Diabetic retinopathy (DR) is the leading cause of blindness in patients with diabetes. However, biomarkers for early detection of DR are still lacking. MicroRNAs (miRNAs) regulate multiple biological functions and are often deregulated in DR. We aimed to investigate whether circulating miRNAs can be used as biomarkers of early-stage DR. We used RNA-seq and qRT-PCR to identify differential serum miRNAs in patients with type 2 diabetes mellitus with DR (T2DM-DR), T2DM without DR (T2DM-no-DR), and healthy controls. We validated differential circulating miRNAs in two phases using qRT-PCR assays. RNA-seq analysis identified 7 differential circulating miRNAs between T2DM-DR and T2DM-no-DR and 47 differential miRNAs between T2DM-DR and healthy subjects. Two-stage analysis verified that a profile of five serum miRNAs (hsa-let-7a-5p, hsa-miR-novel-chr5_15976, hsa-miR-28-3p, has-miR-151a-5p, has-miR-148a-3p) was significantly associated with T2DM-DR. Receiver-operator-characteristic analyses showed that a panel of three miRNAs (hsa-let-7a-5p, hsa-miR-novel-chr5_15976, and hsa-miR-28-3p) presented 0.92 sensitivity and 0.94 specificity for distinguishing T2DM-DR from T2DM-no-DR, and 0.93 sensitivity and 0.86 specificity for differentiating early-stage T2DM-DR (NPDR) from late-stage DR (PDR). Lentivirus-mediated overexpression of hsa-let-7a-5p in human retinal microvascular endothelial cells (HRMECs) significantly promoted proliferation rates of HRMECs. In conclusion, the three-miRNA signature from serum may serve as a noninvasive diagnostic biomarker for DR. Furthermore, we showed that DR-associated miRNAs may be involved in the pathogenesis of DR, at least in part, through modifying proliferation of HRMECs. 10.1152/ajpendo.00021.2018
    Hyperglycemia Induces Neutrophil Extracellular Traps Formation Through an NADPH Oxidase-Dependent Pathway in Diabetic Retinopathy. Wang Luoziyi,Zhou Xin,Yin Yizhou,Mai Yuxin,Wang Desai,Zhang Xuedong Frontiers in immunology Neutrophil extracellular traps (NETs), the product of NETosis, is found to localize pathogens and crystals in immune response. Recent studies have found that excessive NETs lead to disease conditions such as diabetes and its complications like diabetic retinopathy (DR). However, the correlation between NETs and high glucose or DR remains unclear. Here, we found NETs level was significantly increased in the serum of diabetic patients, especially in proliferation diabetic retinopathy (PDR) patients. High glucose dramatically increased NETs production in diabetic individuals with time prolonging. The activation of NADPH oxidase was involved in the NETs process which is triggered by high glucose. Moreover, we verified the infiltration of neutrophils in the eyes and adhesion to vascular endothelial cells in diabetic rat models. NETs formation was observed in the vitreous bodies and retinas of diabetic individuals, which indicates NETs may play a role in the pathogenesis of diabetic retinopathy. Furthermore, anti-VEGF therapy downregulates NETs production indicating that NADPH oxidase-derived ROS may be another signaling pathway involved in anti-VEGF therapy. 10.3389/fimmu.2018.03076
    Gambogic acid ameliorates diabetes-induced proliferative retinopathy through inhibition of the HIF-1α/VEGF expression via targeting PI3K/AKT pathway. Cui Jianyi,Gong Rui,Hu Shuiqing,Cai Ling,Chen Lei Life sciences AIMS:Gambogic acid (GA) is one of active components of Chinese medicine gamboges resin. Diabetic retinopathy (DR) is a most serious microvascular complication of diabetes and also the leading cause of blindness. The aim of this study is to evaluate the beneficial effect of GA on diabetes-induced retinal angiogenesis and further explore the potential mechanisms. MATERIAL AND METHODS:High glucose (HG)-treated RF/6A cells and STZ-induced diabetic mice were used as in vitro and in vivo models. Then the effects of GA on proliferation, migration and tube formation in RF/6A cells and pathomorphological changes in STZ-induced diabetic mice were determined. The activation of HIF-1α/VEGF and PI3K/AKT signaling pathways was assessed by various molecular biological experiments. KEY FINDINGS:According to our results, GA inhibited HG-induced proliferation, migration and tube formation in choroid-retinal endothelial RF/6A cells. The upregulation of HIF-1α and VEGF induced by HG in RF/6A cells was restrained by GA treatment significantly. Moreover, GA suppressed retinal pathomorphological changes and angiogenesis in STZ-induced diabetic mice in vivo, and also inhibited the activation of HIF-1α/VEGF pathway induced by diabetics. Finally, GA suppressed the activation of PI3K/AKT signaling pathway in STZ-induced diabetic mice in vivo and in HG-induced RF/6A cells in vitro. Further activation of PI3K/AKT pathway by IGF-1 restrained the beneficial effect of GA in RF/6A cells. SIGNIFICANCE:Our results provide evidence that GA may ameliorate diabetes-induced retinal angiogenesis, which are proofs that GA may be developed as a potential drug for treating DR. 10.1016/j.lfs.2017.11.007
    Preoperative Timing of Intravitreal Bevacizumab Injection for Proliferative Diabetic Retinopathy Patients. Feng Jing,Li Bing,Wen Jing,Jiang Yanrong Ophthalmic research OBJECTIVE:To evaluate the changes in aqueous concentrations of inflammatory cytokines and fibrosis-related factors, and to detect the expression of vascular endothelial growth factor (VEGF) and proliferating cells in fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR) after injection of intravitreal bevacizumab (IVB). METHODS:Forty-two eyes of 42 patients with PDR, including 28 eyes that received IVB (1.25 mg) 2, 5, and 14 days before pars plana vitrectomy (PPV), and 14 eyes without IVB, were enrolled, in addition to 10 eyes of 10 patients with nondiabetic ocular diseases. Aqueous concentrations of inflammatory cytokines and fibrosis-related factors were analyzed by a multiplex bead assay. Fluorescence immunostaining was performed to examine the expression of VEGF and proliferating cells in the excised epiretinal membranes. RESULTS:PDR eyes without IVB had the highest vitreous VEGF levels, and the level was statistically significant compared with that of PDR eyes that received IVB 2 days before surgery, PDR eyes that received IVB 5 days before surgery, and nondiabetic eyes (p = 0.011, p = 0.012, and p < 0.001, respectively). The expression of fibroblastic cells and connective tissue growth factor increased in epiretinal FVMs of the IVB group 21 days after treatment. CONCLUSIONS:IVB injection may lead to a decrease in the intraocular concentrations of VEGF after 2-5 days and induce the formation of proliferation after 21 days, which suggests that PPV in PDR patients should take place within 1 week of the administration of preoperative IVB. 10.1159/000493640
    MicroRNA-34a attenuates VEGF-mediated retinal angiogenesis via targeting Notch1. Shi Shaoyang,Jin Yong,Song Haishan,Chen Xiaolong Biochemistry and cell biology = Biochimie et biologie cellulaire Pathological angiogenesis in the retina is one of the main ocular diseases closely associated with vision loss. This work investigated the roles of microRNA-34a (miR-34a) and its potential target Notch1, in retinal angiogenesis. For this we used oxygen-induced retinopathy (OIR) rats and human retinal microvascular endothelial cells (HRMECs) stimulated with vascular endothelial growth factor (VEGF). We performed hematoxylin-eosin staining, Western blot for VEGF, and immunofluorescence staining for CD31 to verify the establishment of our OIR model. We observed down-regulation of miR-34a, and up-regulation of Notch1 and Hey1 in retinas from OIR rats. We found similar results with the VEGF-stimulated HRMECs. By performing MTT assay, cell scratch assay, tube formation assay, and by detecting the expression of matrix-metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitors of metalloproteinases-1 (TIMP-1), and TIMP-2, we found that transfection of miR-34a ameliorated VEGF-mediated angiogenesis of HRMECs. We further observed that siRNA-induced gene silencing of Notch1 prevented VEGF-induced angiogenesis via regulating cell proliferation, cell migration, and tube formation of HRMECs. Additionally, activation of Notch1 by transfection of Notch1 plasmid attenuated the inhibitory effects of miR-34a on tube formation, in the present of VEGF. Results from our dual-luciferase reporter gene assay suggested that miR-34a targets Notch1. In summary, our data demonstrate that miR-34a attenuates retinal angiogenesis via targeting Notch1. 10.1139/bcb-2018-0304
    Tissue regeneration using endothelial colony-forming cells: promising cells for vascular repair. Banno Kimihiko,Yoder Mervin C Pediatric research Repairing and rebuilding damaged tissue in diseased human subjects remains a daunting challenge for clinical medicine. Proper vascular formation that serves to deliver blood-borne nutrients and adequate levels of oxygen and to remove wastes is critical for successful tissue regeneration. Endothelial colony-forming cells (ECFC) represent a promising cell source for revascularization of damaged tissue. ECFCs are identified by displaying a hierarchy of clonal proliferative potential and by pronounced postnatal vascularization ability in vivo. In this review, we provide a brief overview of human ECFC isolation and characterization, a survey of a number of animal models of human disease in which ECFCs have been shown to have prominent roles in tissue repair, and a summary of current challenges that must be overcome before moving ECFC into human subjects as a cell therapy. 10.1038/pr.2017.231
    Prion protein is essential for diabetic retinopathy-associated neovascularization. Zhu Lingyan,Xu Jixiong,Liu Ying,Gong Tian,Liu Jianying,Huang Qiong,Fischbach Shane,Zou Wenquan,Xiao Xiangwei Angiogenesis Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and pathological proliferation of retinal vessels, often progresses to vision loss. Vascular endothelial growth factor (VEGF) signaling plays a pivotal role in the development of DR, but the exact underlying molecular mechanisms remain ill-defined. Cellular prion protein (PrP) is a surface protein expressed by vascular endothelial cells, and the increased expression of PrP is associated with physiological and pathological vascularization. Nevertheless, a role for PrP in the development of DR has not been appreciated. Here, we addressed this question. We found that the development of streptozocin (STZ)-induced DR, but not the STZ-induced hyperglycemia/diabetes itself, was significantly attenuated in PrP-KO mice, compared to control wildtype (WT) mice, evident by measurement of retinal vascular leakage, retinal neovascularization, a retinopathy score and visual acuity assessment. Moreover, the attenuation of DR severity seemingly resulted from attenuation of retinal neovascularization via VEGF/ras/rac signaling. Together, our study suggests a previously unappreciated role for PrP in the development of DR. 10.1007/s10456-018-9619-4
    Interplay between CCN1 and Wnt5a in endothelial cells and pericytes determines the angiogenic outcome in a model of ischemic retinopathy. Lee Sangmi,Elaskandrany Menna,Lau Lester F,Lazzaro Douglas,Grant Maria B,Chaqour Brahim Scientific reports CYR61-CTGF-NOV (CCN)1 is a dynamically expressed extracellular matrix (ECM) protein with critical functions in cardiovascular development and tissue repair. Angiogenic endothelial cells (ECs) are a major cellular source of CCN1 which, once secreted, associates with the ECM and the cell surface and tightly controls the bidirectional flow of information between cells and the surrounding matrix. Endothelium-specific CCN1 deletion in mice using a cre/lox strategy induces EC hyperplasia and causes blood vessels to coalesce into large flat hyperplastic sinuses with no distinctive hierarchical organization. This is consistent with the role of CCN1 as a negative feedback regulator of vascular endothelial growth factor (VEGF) receptor activation. In the mouse model of oxygen-induced retinopathy (OIR), pericytes become the predominant CCN1 producing cells. Pericyte-specific deletion of CCN1 significantly decreases pathological retinal neovascularization following OIR. CCN1 induces the expression of the non-canonical Wnt5a in pericyte but not in EC cultures. In turn, exogenous Wnt5a inhibits CCN1 gene expression, induces EC proliferation and increases hypersprouting. Concordantly, treatment of mice with TNP470, a non-canonical Wnt5a inhibitor, reestablishes endothelial expression of CCN1 and significantly decreases pathological neovascular growth in OIR. Our data highlight the significance of CCN1-EC and CCN1-pericyte communication signals in driving physiological and pathological angiogenesis. 10.1038/s41598-017-01585-8
    Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis. O'Leary Caitriona,McGahon Mary K,Ashraf Sadaf,McNaughten Jennifer,Friedel Thomas,Cincolà Patrizia,Barabas Peter,Fernandez Jose A,Stitt Alan W,McGeown J Graham,Curtis Tim M Investigative ophthalmology & visual science Purpose:We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis. Methods:Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording. Results:TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model. Conclusions:TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina. 10.1167/iovs.18-26344
    The microenvironment of proliferative diabetic retinopathy supports lymphatic neovascularization. Gucciardo Erika,Loukovaara Sirpa,Korhonen Ani,Repo Pauliina,Martins Beatriz,Vihinen Helena,Jokitalo Eija,Lehti Kaisa The Journal of pathology Proliferative diabetic retinopathy (PDR) is a major diabetic microvascular complication characterized by pathological angiogenesis. Several retinopathy animal models have been developed to study the disease mechanisms and putative targets. However, knowledge on the human proliferative disease remains incomplete, relying on steady-state results from thin histological neovascular tissue sections and vitreous samples. New translational models are thus required to comprehensively understand the disease pathophysiology and develop improved therapeutic interventions. We describe here a clinically relevant model, whereby the native multicellular PDR landscape and neo(fibro)vascular processes can be analysed ex vivo and related to clinical data. As characterized by three-dimensional whole-mount immunofluorescence and electron microscopy, heterogeneity in patient-derived PDR neovascular tissues included discontinuous capillaries coupled with aberrantly differentiated, lymphatic-like and tortuous endothelia. Spatially confined apoptosis and proliferation coexisted with inflammatory cell infiltration and unique vascular islet formation. Ex vivo-cultured explants retained multicellularity, islet patterning and capillary or fibrotic outgrowth in response to vitreoretinal factors. Strikingly, PDR neovascular tissues, whose matched vitreous samples enhanced lymphatic endothelial cell sprouting, contained lymphatic-like capillaries in vivo and developed Prox1 capillaries and sprouts with lymphatic endothelial ultrastructures ex vivo. Among multiple vitreal components, vascular endothelial growth factor C was one factor found at lymphatic endothelium-activating concentrations. These results indicate that the ischaemia-induced and inflammation-induced human PDR microenvironment supports pathological neolymphovascularization, providing a new concept regarding PDR mechanisms and targeting options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. 10.1002/path.5070
    Inhibition of Pathological Retinal Neovascularization by a Small Peptide Derived from Human Tissue-Type Plasminogen Kringle 2. Sun Qian,Shen Yinchen,Su Li,Xu Xun Frontiers in pharmacology Retinal neovascularization is a hallmark pathological process of numerous ocular diseases which comprise the most common causes of blindness and affect millions of people from infants to the elderly. Compared to large proteins, small peptides have advantages for therapeutic application in ocular diseases, especially for retinal diseases. In this study, we investigated a small peptide derived from human tissue-type plasminogen kringle 2 (t-PA kringle 2), named TKII-12, and investigated the effect of TKII-12 on various aspects of vascular endothelial growth factor (VEGF)-induced angiogenesis and . Our results showed that TKII-12 effectively inhibited VEGF-induced human retinal microvascular endothelial cell proliferation, migration and tube formation on Matrigel dose-dependently as well as sequence-dependently. TKII-12 inhibited VEGF-induced formation of actin stress fibers and focal adhesions in vascular endothelial cells. Moreover, TKII-12 effectively inhibited retinal neovascularization in a mouse oxygen-induced retinopathy (OIR) model. Our study demonstrated that TKII-12 could effectively inhibit retinal angiogenesis and by eliminating the formation of focal adhesion complexes and the organization of actin stress fibers. TKII-12 can serve as a prototype for retinal angiogenesis inhibitory drug development. 10.3389/fphar.2019.01639
    Retinal expression of small non-coding RNAs in a murine model of proliferative retinopathy. Liu Chi-Hsiu,Wang Zhongxiao,Sun Ye,SanGiovanni John Paul,Chen Jing Scientific reports Ocular neovascularization is a leading cause of blindness in proliferative retinopathy. Small non-coding RNAs (sncRNAs) play critical roles in both vascular and neuronal development of the retina through post-transcriptional regulation of target gene expression. To identify the function and therapeutic potential of sncRNAs in retinopathy, we assessed the expression profile of retinal sncRNAs in a mouse model of oxygen-induced retinopathy (OIR) with pathologic proliferation of neovessels. Approximately 2% of all analyzed sncRNAs were significantly altered in OIR retinas compared with normoxic controls. Twenty three microRNAs with substantial up- or down-regulation were identified, including miR-351, -762, -210, 145, -155, -129-5p, -150, -203, and -375, which were further analyzed for their potential target genes in angiogenic, hypoxic, and immune response-related pathways. In addition, nineteen small nucleolar RNAs also revealed differential expression in OIR retinas compared with control retinas. A decrease of overall microRNA expression in OIR retinas was consistent with reduced microRNA processing enzyme Dicer, and increased expression of Alu element in OIR. Together, our findings elucidated a group of differentially expressed sncRNAs in a murine model of proliferative retinopathy. These sncRNAs may exert critical post-transcriptional regulatory roles in regulating pathological neovascularization in eye diseases. 10.1038/srep33947
    The role of anti-vascular endothelial growth factor (anti-VEGF) in the management of proliferative diabetic retinopathy. Zhao Yue,Singh Rishi P Drugs in context Diabetes is a major cause of visual impairment among working-age adults in the United States. The proliferative form of diabetic retinopathy is associated with severe vision loss (acuity <5/200). The standard treatment in proliferative diabetic retinopathy (PDR) is panretinal photocoagulation (PRP), which is effective but has established side effects such as peripheral visual-field constraints. Vascular endothelial growth factor (VEGF) is thought to drive the process of vascular proliferation. Drugs targeting VEGF (anti-VEGF) have been studied extensively in diabetic macular edema (DME), and results have shown that diabetic retinopathy regresses with anti-VEGF treatment. Recent studies show that anti-VEGF is not inferior to PRP for PDR while treatment is maintained, though recurrence rate when anti-VEGF treatment is stopped is unclear. In vitreous hemorrhage where PRP cannot be performed, use of anti-VEGF medications can treat underlying PDR and delay or reduce need for vitrectomy. Limitations of anti-VEGF treatment, however, require careful patient selection and monitoring. This review discusses recent clinical trials and guidelines for anti-VEGF use in PDR. 10.7573/dic.212532
    Astragalus root extract inhibits retinal cell apoptosis and repairs damaged retinal neovascularization in retinopathy of prematurity. Liu Xiaojun,Wang Bin,Sun Yongfa,Jia Yutao,Xu Zhaoying Cell cycle (Georgetown, Tex.) Since the functions of Astragalus root extract in retinopathy remain to be unraveled, this study is performed to elucidate whether Astragalus root extract functions in retinal cell apoptosis and angiogenesis in retinopathy of prematurity (ROP). Newborn mice were selected for establishing mice models of oxygen-induced retinopathy (OIR), which were treated with high-, medium- or low-Astragalus root extract. Evans Blue (EB) was perfused to detect the blood retinal barrier. Additionally, the vascular morphology, number of endothelial cell nuclei of neovascularization, proliferation of blood vessels, ultrastructural changes were determined via a series of assays. Moreover, levels of reactive oxygen species (ROS), expression of other factors such as VEGF, PEDF, IGF-1, HIF-1α, Bax, Bcl-2, eNOS, nNOS, and iNOS were detected. Astragalus root extract was found to protect blood-retinal barrier in the OIR model mice through repairing the structure and morphology of retina, inhibiting ROS production, retinal cell apoptosis, as well as improving retinal vascular angiogenesis. Astragalus root extract was also found to decrease VEGF and HIF-1α expression, but enhance PEDF and IGF-1 expression in the OIR model mice, thereby protecting retinas in ROP. This study highlights that Astragalus root extract is able to suppress retinal cell apoptosis and repair damaged retinal neovascularization in ROP, which provides basis for ROP therapy. 10.1080/15384101.2019.1669998
    Dual blockade of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) exhibits potent anti-angiogenic effects. Li Dong,Xie Kun,Zhang Longzhen,Yao Xuejing,Li Hongwen,Xu Qiaoyu,Wang Xin,Jiang Jing,Fang Jianmin Cancer letters Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF or FGF-2) are potent pro-angiogenic factors and play a critical role in cancer development and progression. Clinical anti-VEGF therapy trials had a major challenge due to upregulated expression of other pro-angiogenic factor, like FGF-2. This study developed a novel chimeric decoy receptor VF-Trap fusion protein to simultaneously block activity of both VEGF and FGF pathways in order to achieve an additive or synergistic anti-tumor effect. Our in vitro data showed that VF-Trap potently blocked proliferation and migration of both VEGF- and FGF-2-induced vascular endothelial cells. In animal models, treatment of xenograft tumors with VF-Trap resulted in significant inhibition of tumor growth compared to blockage of the single molecule, like VEGF or FGF blocker. In addition, VF-Trap was also more potent in inhibition of ocular angiogenesis in a mouse oxygen-induced retinopathy (OIR) model. These data demonstrated the potent anti-angiogenic effects of this novel VF-Trap fusion protein on blockage of VEGF and FGF-2 activity in vitro and in animal models. Further study will assess its effects in clinic as a therapeutic agent for angiogenesis-related disorders, such as cancer and ocular vascular diseases. 10.1016/j.canlet.2016.04.036
    Enhancing Retinal Endothelial Glycolysis by Inhibiting UCP2 Promotes Physiologic Retinal Vascular Development in a Model of Retinopathy of Prematurity. Han Xiaokun,Kong Jun,Hartnett M Elizabeth,Wang Haibo Investigative ophthalmology & visual science Purpose:We address the hypothesis that uncoupling protein 2 (UCP2), a cellular glucose regulator, delays physiologic retinal vascular development (PRVD) by interfering with glucose uptake through glucose transporter 1 (Glut1). Methods:In the rat 50/10 oxygen-induced retinopathy (OIR) model, retinal Glut1 and UCP2 were measured and compared to room air (RA)-raised pups at postnatal day 14 (p14). Pups in OIR and RA received intraperitoneal genipin, an UCP2 inhibitor, or control every other day from p3 until p13. Analyses at p14 included avascular/total retinal area (AVA), Western blots of retinal UCP2 and Glut1, and immunostaining of Glut1 in retinal cryosections. Intravitreal neovascular/total retinal area (IVNV) was analyzed at p18, and electroretinograms were performed at p26. Glut1 and phosphorylated VEGFR2 (p-VEGFR2), glucose uptake, adenosine triphosphate (ATP) production, and cell proliferation were measured in human retinal microvascular endothelial cells (hRMVECs) pretreated with genipin or transfected with UCP2siRNA, Glut1siRNA, or control siRNA when incubated with VEGF or PBS. Results:At p14, OIR pups had increased AVA with decreased Glut1 and increased UCP2 in the retina compared to RA retinas. Intraperitoneal genipin increased retinal Glut1 and reduced AVA. Compared to control, treatment with genipin or knockdown of UCP2 significantly increased Glut1, glucose uptake, ATP production, VEGF-induced p-VEGFR2 and cell proliferation in hRMVECs. Knockdown of Glut1 inhibited VEGF-induced p-VEGFR2. Genipin-treated OIR pups with decreased AVA at p14 had reduced IVNV at p18 and increased amplitudes in a- and b-waves at p26. Conclusions:Extending PRVD by increasing retinal endothelial glucose uptake may represent a strategy to prevent severe retinopathy of prematurity and vision loss. 10.1167/iovs.19-26553
    ANGPTL-4 induces diabetic retinal inflammation by activating Profilin-1. Lu Qianyi,Lu Peirong,Chen Wei,Lu Li,Zheng Zhi Experimental eye research Diabetic retinopathy (DR), the most common cause of irreversible blindness in working-age adults, results in central vision loss that is caused by microvascular damage to the inner lining of the back of the eye, the retina. The aim of this work was to assess the temporal relationships between angiopoietin-like protein-4 (ANGPTL-4), a novel adipocytokine factor, and diabetic retinal inflammation and microvascular dysfunction. The downstream pathway(s) and upstream mediator(s) of ANGPTL-4 were then determined under high glucose (HG) conditions. Diabetic rats and control animals were randomly assigned to receive hypoxia inducible factor-1 alpha (HIF-1α) blockade (doxorubicin or shRNA) or vehicle for 8 weeks. Human retinal microvascular endothelial cells (HRMECs) were incubated with normal or high glucose, with or without blockade or recombinant proteins, for ANGPTL-4, HIF-1α, and vascular endothelial growth factor (VEGF). The levels of ANGPTL-4, profilin-1, HIF-1α, VEGF, interleukin 1 beta (IL-1β), IL-6, and intercellular adherent molecule 1 (ICAM-1) in the rat retinas and HRMEC extracts were examined by Western blotting and real-time RT-PCR. The levels of ANGPTL-4, profilin-1, HIF-1α, and VEGF protein and mRNA were significantly higher in the diabetic rats and HG-exposed HRMECs. ANGPTL-4 was a potent modulator of increased inflammation, permeability, and angiogenesis via activation of the profilin-1 signaling pathway. Our results showed that ANGPTL-4 upregulation was induced by HG, which was dependent on HIF-1α activation that was also triggered by HG, both in vivo and in vitro. Our results suggest that targeting ANGPTL-4, alone or in combination with profilin-1, may be an effective therapeutic strategy and diagnostic screening biomarker for proliferative diabetic retinopathy and other vitreous-retinal inflammatory diseases. 10.1016/j.exer.2017.10.009
    Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization. Inoue Yuki,Shimazawa Masamitsu,Nakamura Shinsuke,Takata Shinsuke,Hashimoto Yuhei,Izawa Hiroshi,Masuda Tomomi,Tsuruma Kazuhiro,Sakaue Tomohisa,Nakayama Hironao,Higashiyama Shigeki,Hara Hideaki Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis. APPROACH AND RESULTS:We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. CONCLUSIONS:These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration. 10.1161/ATVBAHA.117.310337
    The Role of Microglia in Diabetic Retinopathy: Inflammation, Microvasculature Defects and Neurodegeneration. Altmann Christine,Schmidt Mirko H H International journal of molecular sciences Diabetic retinopathy is a common complication of diabetes mellitus, which appears in one third of all diabetic patients and is a prominent cause of vision loss. First discovered as a microvascular disease, intensive research in the field identified inflammation and neurodegeneration to be part of diabetic retinopathy. Microglia, the resident monocytes of the retina, are activated due to a complex interplay between the different cell types of the retina and diverse pathological pathways. The trigger for developing diabetic retinopathy is diabetes-induced hyperglycemia, accompanied by leukostasis and vascular leakages. Transcriptional changes in activated microglia, mediated via the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and extracellular signal-regulated kinase (ERK) signaling pathways, results in release of various pro-inflammatory mediators, including cytokines, chemokines, caspases and glutamate. Activated microglia additionally increased proliferation and migration. Among other consequences, these changes in microglia severely affected retinal neurons, causing increased apoptosis and subsequent thinning of the nerve fiber layer, resulting in visual loss. New potential therapeutics need to interfere with these diabetic complications even before changes in the retina are diagnosed, to prevent neuronal apoptosis and blindness in patients. 10.3390/ijms19010110
    The Suppression of Kallistatin on High-Glucose-Induced Proliferation of Retinal Endothelial Cells in Diabetic Retinopathy. Xing Qian,Zhang Guowei,Kang Lihua,Wu Jian,Chen Hui,Liu Gaoqin,Zhu Rongrong,Guan Huaijin,Lu Peirong Ophthalmic research BACKGROUND:Diabetic retinopathy (DR) is a severe ocular complication of diabetes. Kallistatin has multiple biological functions including anti-inflammation and antiangiogenesis. Our aim was to detect the level of kallistatin in the vitreous of proliferative DR (PDR) and its effect on proliferation, migration, and tube formation of human retinal endothelial cells (HRECs) under high glucose in an in vitro model. METHODS:Vitreous humor samples were obtained through pars plana vitrectomy from 7 nondiabetic patients with idiopathic macular holes or idiopathic preretinal membranes and 10 PDR patients. The vitreous levels of kallistatin were measured by ELISA. HRECs were cultured with different concentrations of glucose and 1,000 nM kallistatin. The proliferation of HRECs was evaluated by a Cell Counting Kit-8 assay. Cell migration was assessed by using Transwell chambers. Cell sprouting was detected by tube formation assay. The RNA interference technique was used to create the knockdown of the kallistatin gene in HRECs for evaluating its effect on the proliferation, migration, and tube formation of HRECs. RESULTS:The vitreous levels of kallistatin were significantly lower in PDR patients in comparison with nondiabetic control patients (p < 0.05). Compared with 5 mM of normal glucose treatment, high glucose (30 mM) in culture significantly increased the proliferation and migration of HRECs, which was attenuated by 1,000 nM kallistatin. In addition, 1,000 nM kallistatin was shown to suppress high-glucose-induced tube formation and the expression of vascular endothelial growth factor of HRECs. Furthermore, the knockdown of kallistatin enhanced the proliferation, migration, and tube formation of HRECs. CONCLUSIONS:Our data indicated that kallistatin might be a potent inhibitory factor for PDR. The molecule plays its role by inhibiting high-glucose-induced proliferation of HRECs. The findings suggest that the upregulation of kallistatin might be an effective strategy for PDR prevention. 10.1159/000447776
    Recombinant humanized anti-vascular endothelial growth factor monoclonal antibody efficiently suppresses laser-induced choroidal neovascularization in rhesus monkeys. Ji Wan-Wan,Yu Dong-An,Yang Peng,Fang Peng,Cao Yu-Xia,Li Hu,Xie Ning,Yan Shou-Sheng European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences Neovascular age-related macular degeneration, characterized by abnormal choroidal neovascularization (CNV), is a major cause of blindness worldwide. Anti-vascular endothelial growth factor (VEGF) antibodies have demonstrated significant efficacy in improving visual acuity. TMAB001 is a new recombinant humanized rabbit anti-VEGF monoclonal antibody. It presents high activities in vitro studies. In the binding affinity assay, TMAB001 exhibited a high binding capability to VEGF with an affinity constant of 10M. In the receptor antagonist activity assay, IC of TMAB001 was 0.15μg/ml. In a cell-based assay, TMAB001 inhibited VEGF-induced HUVEC cells proliferation in a dose-dependent manner. Furthermore, in the rhesus monkey model of laser-induced CNV, results showed the growth and leakage of experimental CNV were significantly decreased with a single bilateral intravitreal injection of TMAB001, and the grade 4 lesions were complete absence in TMAB001 groups. The efficacy of TMAB001 was maintained for at least 28days. In a mice model of oxygen-induced retinopathy, the retina fluorescence leakage was reduced and the vascular morphology in retina was normalized by TMAB001 intraperitoneal administration. In conclusion, those results indicate that TMAB001 might be a potential drug candidate for wet AMD. 10.1016/j.ejps.2017.09.021
    LncRNA TDRG1-Mediated Overexpression of VEGF Aggravated Retinal Microvascular Endothelial Cell Dysfunction in Diabetic Retinopathy. Gong Qiaoyun,Dong Wenpei,Fan Ying,Chen Feng'e,Bian Xiaolan,Xu Xun,Qian Tianwei,Yu Ping Frontiers in pharmacology Purpose:Diabetic retinopathy (DR), a neurovascular disease, is one of the leading causes of blindness in working-age adults. Long noncoding RNAs (lncRNAs) have attracted attention as indicators for DR. This study aimed to characterize the role of lncRNA human testis development-related gene 1 (TDRG1) and its modulation of vascular endothelial growth factor (VEGF) in deteriorating DR. Methods:Tissue samples were obtained from patients with epiretinal membranes (EMs) or proliferative DR, and human retinal microvascular endothelial cells (HRECs) were cultured with high-glucose medium to mimic DR as the model. The expression of lncRNA TDRG1 and VEGF was determined by immunofluorescence staining, Western blotting, and RT-qPCR. Transfection of small-interfering RNA was conducted to knock down target gene expression. HREC functions were evaluated by cell viability, fluorescein isothiocyanate (FITC)-dextran extravasation, migration, and tube formation assays under different conditions. Results:LncRNA TDRG1 and VEGF were found to be co-expressed and significantly upregulated in fibrovascular membranes (FVMs) from DR patients compared to those from EM patients. In the model, hyperglycemic treatment markedly increased the expression of lncRNA TDRG1 and VEGF at the mRNA and protein levels, which promoted cell proliferation and migration, enhanced permeability, and disrupted tube formation of HRECs. However, knockdown of lncRNA TDRG1 or VEGF notably decreased the expression of VEGF and reversed the impaired functions of high-glucose-treated HRECs. Conclusions:LncRNA TDRG1 promoted microvascular cell dysfunction upregulating VEGF in the progression of DR and may serve as a potential therapeutic target in DR treatment. 10.3389/fphar.2019.01703
    ANRIL: A Regulator of VEGF in Diabetic Retinopathy. Thomas Anu Alice,Feng Biao,Chakrabarti Subrata Investigative ophthalmology & visual science Purpose:Long noncoding RNAs (lncRNAs) previously thought to be "dark matter" of the genome, play key roles in various biological processes. The lncRNA ANRIL is located at a genetic susceptibility locus for coronary artery diseases and type 2 diabetes. We examined the role of ANRIL in diabetic retinopathy, through study of its regulation of VEGF both in vitro and in vivo. Methods:Human retinal endothelial cells (HRECs) were subjected to incubation in high glucose to mimic diabetes. ANRIL expression was measured with or without small interfering RNA (siRNA) knockdown in HRECs. ANRIL knockout mice, with or without streptozotocin-induced diabetes, were also investigated. Cell and tissues were measured for VEGF mRNA and protein expression. Functional alterations in VEGF were determined through tube formation, cell proliferation, and retinal vascular permeability assays. Vascular endothelial growth factor regulation through ANRIL's interactions with polycomb repressive complex 2 (PRC2) components and p300 were studied thorough PRC2 blocker, siRNA, and RNA immunoprecipitation (RNA-IP) assays. Results:High glucose and diabetes caused ANRIL upregulation in HRECs and in the retina. Glucose-mediated elevation of ANRIL, on silencing, prevented VEGF expression. Such regulation involved ANRIL-mediated control of the PRC2 components p300 and miR200b. Direct binding of ANRIL to p300 and enhancer of zeste homolog 2 (EZH2; a PRC2 component) were elevated following exposure to high glucose levels. Conclusions:Our results demonstrate for the first time that ANRIL regulates VEGF expression and function in diabetic retinopathy. This regulation is mediated by p300, miR200b, and EZH2 of the PRC2 complex. 10.1167/iovs.16-20569
    Antiangiogenic effect of dasatinib in murine models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Seo Songyi,Suh Wonhee Molecular vision Purpose:Vascular endothelial growth factor (VEGF) is a principal mediator of pathological ocular neovascularization, which is the leading cause of blindness in various ocular diseases. As Src, a non-receptor tyrosine kinase, has been implicated as one of the major signaling molecules in VEGF-mediated neovascularization, the present study aimed to investigate whether dasatinib, a potent Src kinase inhibitor, could suppress pathological ocular neovascularization in murine models of oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). Methods:Tube formation, scratch wounding migration, and cell proliferation assays were performed to measure the inhibitory effect of dasatinib on VEGF-induced angiogenesis in human retinal microvascular endothelial cells. Murine models of OIR and laser-induced CNV were used to assess the preventive effect of an intravitreal injection of dasatinib on pathological neovascularization in the retina and choroid. Neovascularization and Src phosphorylation were evaluated with immunofluorescence staining. Results:Dasatinib efficiently inhibited VEGF-induced endothelial proliferation, wounding migration, and tube formation. In mice with OIR and laser injury-induced CNV, eyes treated with a single intravitreal injection of dasatinib exhibited significant decreases in pathological neovascularization compared with that of controls injected with vehicle. The dasatinib-treated OIR mice also showed a decrease in Src phosphorylation in the periretinal tufts. The intravitreal injection of dasatinib did not cause ocular toxicity at the treatment dose administered. Conclusions:These results demonstrated that dasatinib suppressed pathological neovascularization in the mouse retina and choroid. Therefore, dasatinib may be indicated for the treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.
    Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases. Qiu Beiying,Tan Alison,Veluchamy Amutha Barathi,Li Yong,Murray Hannah,Cheng Wei,Liu Chenghao,Busoy Joanna Marie,Chen Qi-Yin,Sistla Srivani,Hunziker Walter,Cheung Chui Ming Gemmy,Wong Tien Yin,Hong Wanjin,Luesch Hendrik,Wang Xiaomeng Investigative ophthalmology & visual science Purpose:Abnormal blood vessel formation is a defining feature of many blinding eye diseases. Targeting abnormal angiogenesis by inhibiting VEGF has revolutionized the treatment of many ocular angiogenic diseases over the last decade. However, a substantial number of patients are refractory to anti-VEGF treatment or may develop resistance over time. The objective of this study was to determine the efficacy and the mechanism of action of Apratoxin S4 in ocular angiogenesis. Methods:Retinal vascular cell proliferation, migration, and the ability to form tube-like structure were studied in vitro. Ex vivo aortic ring, choroid, and metatarsal assays were used to study Apratoxin S4's impact on vessel outgrowth in a multicellular environment. Apratoxin S4 was also tested in mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), and in a rabbit model of persistent retinal neovascularization (PRNV). Western blot and ELISA were used to determine the expression of key angiogenic regulators after Apratoxin S4 treatment. Results:Apratoxin S4 strongly inhibits retinal vascular cell activation by suppressing multiple angiogenic pathways. VEGF-activated vascular cells and angiogenic vessels are more susceptible to Apratoxin S4 treatment than quiescent vascular cells and vessels. Both intraperitoneal and intravitreal delivery of Apratoxin S4 are able to impede ocular neovascularization in vivo. Apratoxin S4 specifically attenuates pathological ocular angiogenesis and exhibits a combinatorial inhibitory effect with standard-of-care VEGF inhibitor drug (aflibercept). Conclusions:Apratoxin S4 is a potent antiangiogenic drug that inhibits the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways. 10.1167/iovs.19-26936
    Senescence-associated secretory phenotype contributes to pathological angiogenesis in retinopathy. Oubaha Malika,Miloudi Khalil,Dejda Agnieszka,Guber Vera,Mawambo Gaëlle,Germain Marie-Anne,Bourdel Guillaume,Popovic Natalija,Rezende Flavio A,Kaufman Randal J,Mallette Frédérick A,Sapieha Przemyslaw Science translational medicine Pathological angiogenesis is the hallmark of diseases such as cancer and retinopathies. Although tissue hypoxia and inflammation are recognized as central drivers of vessel growth, relatively little is known about the process that bridges the two. In a mouse model of ischemic retinopathy, we found that hypoxic regions of the retina showed only modest rates of apoptosis despite severely compromised metabolic supply. Using transcriptomic analysis and inducible loss-of-function genetics, we demonstrated that ischemic retinal cells instead engage the endoplasmic reticulum stress inositol-requiring enzyme 1α (IRE1α) pathway that, through its endoribonuclease activity, induces a state of senescence in which cells adopt a senescence-associated secretory phenotype (SASP). We also detected SASP-associated cytokines (plasminogen activator inhibitor 1, interleukin-6, interleukin-8, and vascular endothelial growth factor) in the vitreous humor of patients suffering from proliferative diabetic retinopathy. Therapeutic inhibition of the SASP through intravitreal delivery of metformin or interference with effectors of senescence (semaphorin 3A or IRE1α) in mice reduced destructive retinal neovascularization in vivo. We conclude that the SASP contributes to pathological vessel growth, with ischemic retinal cells becoming prematurely senescent and secreting inflammatory cytokines that drive paracrine senescence, exacerbate destructive angiogenesis, and hinder reparative vascular regeneration. Reversal of this process may be therapeutically beneficial. 10.1126/scitranslmed.aaf9440
    Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia. Yamaguchi Muneo,Nakao Shintaro,Arita Ryoichi,Kaizu Yoshihiro,Arima Mitsuru,Zhou Yedi,Kita Takeshi,Yoshida Shigeo,Kimura Kazuhiro,Isobe Tomoyuki,Kaneko Yoshio,Sonoda Koh-Hei,Ishibashi Tatsuro Investigative ophthalmology & visual science PURPOSE:In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. METHODS:In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS:Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 μmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 μmol/L and 6.8 to 14.8 μmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. CONCLUSIONS:Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization. 10.1167/iovs.15-17411
    FGF21 Administration Suppresses Retinal and Choroidal Neovascularization in Mice. Fu Zhongjie,Gong Yan,Liegl Raffael,Wang Zhongxiao,Liu Chi-Hsiu,Meng Steven S,Burnim Samuel B,Saba Nicholas J,Fredrick Thomas W,Morss Peyton C,Hellstrom Ann,Talukdar Saswata,Smith Lois E H Cell reports Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration: very-low-density lipoprotein-receptor-deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited tumor necrosis α (TNF-α) expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. 10.1016/j.celrep.2017.01.014
    Microglia enhanced the angiogenesis, migration and proliferation of co-cultured RMECs. Ding Xinyi,Gu Ruiping,Zhang Meng,Ren Hui,Shu Qinmeng,Xu Gezhi,Wu Haixiang BMC ophthalmology BACKGROUND:Attention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia contribute to disruption of the blood-retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs). METHODS:We activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis. RESULTS:We successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs. CONCLUSIONS:Microglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification. 10.1186/s12886-018-0886-z
    Role of ω3 polyunsaturated fatty acids in diabetic retinopathy: a morphological and metabolically cross talk among blood retina barriers damage, autoimmunity and chronic inflammation. Eynard Aldo R,Repossi Gaston Lipids in health and disease Vision disorders are one of the most serious complications of diabetes mellitus (DM) affecting the quality of life of patients and eventually cause blindness. The ocular lesions in diabetes mellitus are located mainly in the blood vessels and retina layers. Different retina lesions could be grouped under the umbrella term of diabetic retinopathies (DMRP).We propose that one of the main causes in the etiopathogenesis of the DMRP consists of a progressive loss of the selective permeability of blood retinal barriers (BRB). The loss of selective permeability of blood retinal barriers will cause a progressive autoimmune process. Prolonged autoimmune injures in the retinal territory will triggers and maintains a low-grade chronic inflammation process, microvascular alterations, glial proliferation and subsequent fibrosis and worse, progressive apoptosis of the photoreceptor neurons.Patients with long-standing DM disturbances in retinal BRBs suffer of alterations in the enzymatic pathways of polyunsaturated fatty acids (PUFAs), increase release of free radicals and pro-inflammatory molecules and subsequently incremented levels of vascular endothelial growth factor. These facts can produce retinal edema and photoreceptor apoptosis.Experimental, clinical and epidemiological evidences showing that adequate metabolic and alimentary controls and constant practices of healthy life may avoid, retard or make less severe the appearance of DMRP. Considering the high demand for PUFAs ω3 by photoreceptor complexes of the retina, it seems advisable to take fish oil supplements (2 g per day). The cellular, subcellular and molecular basis of the propositions exposed above is developed in this article.Synthesizer drawings the most relevant findings of the ultrastructural pathology, as well as the main metabolic pathways of the PUFAs involved in balance and disbalanced conditions are provided. 10.1186/s12944-019-1049-9
    Cutaneous microangiopathy in patients with type 2 diabetes: Impaired vascular endothelial growth factor expression and its correlation with neuropathy, retinopathy and nephropathy. Sugimoto Kazuhiro,Murakami Hiroshi,Deguchi Takahisa,Arimura Aiko,Daimon Makoto,Suzuki Susumu,Shimbo Takuro,Yagihashi Soroku Journal of diabetes investigation AIMS/INTRODUCTION:To examine the three-dimensional morphology and vascular endothelial growth factor (VEGF) expression of skin microvasculature in patients with type 2 diabetes in relation to neuropathy, retinopathy and nephropathy. MATERIALS AND METHODS:The present study enrolled 17 individuals with type 2 diabetes and 16 without. Skin sections were double-immunostained for type IV collagen and VEGF-A or protein gene product 9.5. Projected images from confocal microscopy served to quantify the occupancy rate of subepidermal type IV collagen-immunoreactive microvascular basement membrane area (OR-T4MBM), subepidermal VEGF-A-immunoreactive area and the VEGF/T4MBM ratio, as well as the protein gene product 9.5-immunoreactive intraepidermal nerve fiber density. Reduced intraepidermal nerve fiber density was applied for the diagnosis of neuropathy, fundic ophthalmoscopy and fluorescein angiography for retinopathy, and microalbuminuria or persistent proteinuria for nephropathy. RESULTS:A total of 12 patients with diabetes had neuropathy, 10 had retinopathy and eight had nephropathy. Regardless of the presence or absence of neuropathy, retinopathy or nephropathy, OR-T4MBM was significantly increased in patients with diabetes compared with individuals without diabetes. In contrast, VEGF/T4MBM ratio was significantly decreased in those with neuropathy and retinopathy, as well as in those with and without nephropathy, whereas a trend toward a decreased VEGF/T4MBM ratio was seen in patients without retinopathy, as compared with individuals without diabetes. CONCLUSIONS:The present study is the first report to show that cutaneous microangiopathy, as indicated by subepidermal microvascular proliferation and impaired VEGF expression, appears to occur before the development of overt clinical neuropathy, retinopathy or nephropathy in patients with type 2 diabetes. 10.1111/jdi.13020
    Lonicerae Japonicae Flos attenuates diabetic retinopathy by inhibiting retinal angiogenesis. Zhou Lingyu,Zhang Tianyu,Lu Bin,Yu Zengyang,Mei Xiyu,Abulizi Palida,Ji Lili Journal of ethnopharmacology ETHNOPHARMACOLOGICAL RELEVANCE:Lonicerae Japonicae Flos (Jin-Yin-Hua) is a well-known traditional Chinese medicine used for clearing away heat and toxic material. AIM OF THE STUDY:This study aims to observe the attenuation of aqueous extract of Lonicerae Japonicae Flos (FL) against streptozotocin (STZ)-induced diabetic retinopathy (DR) and its engaged mechanism. MATERIALS AND METHODS:STZ-induced proliferative DR (PDR) for 5 month in C57BL/6 mice was used in this study. Retinal vessels were observed by immunofluorescence staining with cluster of differentiation 31 (CD31) and histopathological evaluation. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum vascular endothelial growth factor (VEGF) content. Cell proliferation was detected by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay in choroid-retinal endothelial RF/6A cells. VEGF-induced tube formation in RF/6A cells was observed. The contents of chlorogenic acid (CGA), caffeic acid (CA), and luteolin in FL were detected by high-performance liquid chromatography (HPLC). RESULTS:Histopathological evaluation demonstrated that retinal vessels were increased in STZ-induced PDR mice, whereas FL decreased such increase. The results of CD31 staining also showed that FL decreased the increased number of retinal vessels in STZ-induced PDR mice. In addition, FL reduced the increased serum VEGF content in STZ-induced PDR mice. FL reduced VEGF-induced RF/6A cell proliferation in the concentration-dependent manner, but had no obvious effect on RF/6A cell viability without VEGF stimulation. VEGF-induced tube formation in RF/6A cells was inhibited by different concentrations of FL. CGA, CA and luteolin all inhibited VEGF-induced tube formation in RF/6A cells, and the lowest effective concentration of CGA and CA was both 0.625μM, but of luteolin was 5μM. Furthermore, the results of HPLC demonstrated that the amount of CGA was the highest in FL. CONCLUSIONS:FL ameliorates STZ-induced PDR by inhibiting retinal angiogenesis. Phenolic acid CGA is the main compound contributing to the inhibition of FL on retinal angiogenesis. 10.1016/j.jep.2016.05.039
    Imatinib Ameliorated Retinal Neovascularization by Suppressing PDGFR-α and PDGFR-β. Zhou Lingli,Sun Xiaowei,Huang Zijing,Zhou Tian,Zhu Xiaowei,Liu Yan,Wang Jing,Cheng Bing,Li Mei,He Chang,Liu Xialin Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology BACKGROUND/AIMS:Platelet-derived growth factors (PDGFs) have emerged as pivotal in pathological angiogenesis, which is a hallmark of various tumors and retinal diseases. Here we evaluated the anti-angiogenic effect of imatinib, an inhibitor of PDGF receptors α and β (PDGFR-α and -β), in retinal neovascularization using an oxygen-induced retinopathy (OIR) model. METHODS:The OIR model was established and given imatinib or vehicle treatments daily from P12 to P16. At the peak of angiogenesis at P17, the neovascularization area was quantified on retinal whole-mounts with isolectin B4 staining. Immunofluorescence staining and western blots were used to determine the effect of imatinib on different vascular cells and the pathway molecules involved. RESULTS:Imatinib effectively suppressed pathological angiogenesis in OIR mice and reduced the number of all three types of vascular cells, including endothelial cells, pericytes, and smooth muscle cells. Moreover, the expression and activation of PDGFR-α and -β were inhibited by imatinib. The imatinib-treated OIR mice presented with reduced expression of other potent pro-angiogenic factors such as VEGF and FGF2. No obvious retinal or systemic side effects were observed in the imatinib treatment group. CONCLUSIONS:Imatinib appears to be safe and effective in suppressing retinal neovascularization. Targeting PDGFs/PDGFRs may also be important for anti-angiogenic treatment and offer a viable alternative treatment for retinal angiogenic diseases. 10.1159/000491726
    YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells. Zhu Manhui,Liu Xiaojuan,Wang Ying,Chen Lili,Wang Li,Qin Xiao,Xu Jiaowen,Li Lele,Tu Yuanyuan,Zhou Taohu,Sang Aimin,Song E Experimental cell research Endothelial dysfunction is a main feature of retinal neovascular diseases which are the leading cause of blindness in developed countries. Yes-associated protein (YAP) and signal transducer and activator of transcription factor 3 (STAT3) participate in angiogenesis via vascular endothelial growth factor (VEGF) signaling. Additionally, YAP can bind STAT3 in endothelial cells. In the study, dimethyloxalylglycine (DMOG) stimulated human retinal microvascular endothelial cells (HRMECs) was used as retinal endothelial hypoxia model. The proliferation of HRMECs, as well as t-YAP, p-STAT3 (Tyr705) increased, while p-YAP (Ser127), p-YAP (Ser397) decreased following hypoxia. Meanwhile, YAP and STAT3 translocated to the nucleus. YAP knockdown inhibited the proliferation, migration and tube formation of HRMECs. YAP overexpression up-regulated phosphorylation of STAT3. The YAP overexpression-induced HRMECs proliferation, migration and tube formation were reversed by S3I-201, a selective STAT3 inhibitor. YAP interacted with STAT3 to promote STAT3 nuclear translocation. Additionally, YAP and STAT3 promoted the transcription of VEGF synergistically. Finally, inhibition of YAP alleviated retinal pathological neovascularization in mouse oxygen-induced retinopathy (OIR) model. In summary, activated YAP interacted with STAT3 to promote the activation and nuclear translocation of STAT3, hence boosted the proliferation, migration and tube formation of HRMECs via VEGF signaling following hypoxia. The data will further elucidate the mechanisms of retinal neovascular diseases. 10.1016/j.yexcr.2018.10.007
    PF4 antagonizes retinal neovascularization via inhibiting PRAS40 phosphorylation in a mouse model of oxygen-induced retinopathy. Cai Siwei,Yang Qianhui,Cao Yunshan,Li Yahong,Liu Juping,Wang Jiantao,Zhang Xiaomin,Liu Liren,Li Xiaorong,Zhang Yan Biochimica et biophysica acta. Molecular basis of disease Retinal neovascularization (RNV) is a common pathology of blinding proliferative retinopathies. The current treatments to RNV, however, are hindered by limited efficacy, side effects, and drug resistance. A naturally-occurring cytokine in retina that is amicable to immune system and possesses robust anti-neovascular function would facilitate to overcome the hurdles. In this study, retinas from a mouse model of oxygen-induced retinopathy (OIR) underwent a protein array to screen the naturally-occurring cytokines that may antagonize RNV. Among the 62 angiogenesis-associated cytokines, platelet factor 4 (Pf4) stood out with the most prominent upregulation and statistical significance. Moreover, an intravitreal injection of mouse Pf4 demonstrated dramatic anti-vaso-obliteration and anti-neovascularization effects dose dependently in the OIR model; whereas human PF4 inhibited the proliferation, migration, and tubulogenesis of monkey retinal vascular endothelial cells treated with VEGF and TNF-α. These previously undescribed angiostatic effects of PF4 in OIR retinas and retinal vascular endothelial cells support translation of this naturally-occurring chemokine into a therapeutic modality to RNV supplementary to the anti-VEGFs. Mechanistically, a phosphorylation array and western blots indicated that downregulation of proline-rich Akt substrate of 40 kDa (Pras40) and its phosphorylation were necessary for Pf4's anti-neovascular effects in the OIR retinas. Indeed, overexpression of the wildtype Pras40 and the mutant version with deficient phosphorylation abolished and mimicked the Pf4's angiostatic effects in the OIR retinas, respectively. The similar effects were also observed in vitro. This study, for the first time, links PF4's anti-RNV function to an intracellular signaling molecule PRAS40 and its phosphorylation. 10.1016/j.bbadis.2019.165604
    Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis. Liu Zhiping,Yan Siyuan,Wang Jiaojiao,Xu Yiming,Wang Yong,Zhang Shuya,Xu Xizhen,Yang Qiuhua,Zeng Xianqiu,Zhou Yaqi,Gu Xuejiao,Lu Sarah,Fu Zhongjie,Fulton David J,Weintraub Neal L,Caldwell Ruth B,Zhang Wenbo,Wu Chaodong,Liu Xiao-Ling,Chen Jiang-Fan,Ahmad Aftab,Kaddour-Djebbar Ismail,Al-Shabrawey Mohamed,Li Qinkai,Jiang Xuejun,Sun Ye,Sodhi Akrit,Smith Lois,Hong Mei,Huo Yuqing Nature communications Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment. 10.1038/s41467-017-00551-2
    Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling. Liu Yuan,Chen Zhijun,Cheng Haixia,Chen Juan,Qian Jing Oncotarget Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases. 10.18632/oncotarget.13518
    The Effect of Interleukin 38 on Angiogenesis in a Model of Oxygen-induced Retinopathy. Zhang Jing,Zhao Ruijuan,Chen Jianping,Jin Jiayi,Yu Ying,Tian Yunzhe,Li Weihua,Wang Wencong,Zhou Hongyan,Bo Su Shao Scientific reports Interleukin 38 (IL-38) is a novel identified cytokine of IL-1 family in which some members are important in inflammation and angiogenesis. However, the role of IL-38 in regulating angiogenesis is unknown. The aim of the present study is to explore the effect of IL-38 on angiogenesis. Oxygen-induced retinopathy (OIR) of C57BL/6 J mice was induced by exposure of hyperoxia (75% oxygen) from postnatal day 7 (P7) to P12 and then returned to room air. The mice were injected with IL-38. At P17, neovascular region (tufts) and avascular area of the retinas were analyzed. The data showed that administration of IL-38 in vivo inhibited retinal angiogenesis significantly. Furthermore, the addition of IL-38 to the cell cultures attenuated the proliferation, scratch wound healing and tube formation of vascular endothelial cells induced by VEGF significantly. Our findings suggest that IL-38 is an antiangiogenic cytokine in pathophysiological settings and may have therapeutic potential for angiogenesis related diseases. 10.1038/s41598-017-03079-z
    Chlorogenic acid attenuates diabetic retinopathy by reducing VEGF expression and inhibiting VEGF-mediated retinal neoangiogenesis. Mei Xiyu,Zhou Lingyu,Zhang Tianyu,Lu Bin,Sheng Yuchen,Ji Lili Vascular pharmacology Diabetic retinopathy (DR) is one of the most common and serious complications of diabetes mellitus (DM). This study aims to investigate the amelioration of chlorogenic acid (CGA) on proliferative DR (PDR) via focusing on inhibiting retinal neoangiogenesis. CGA reduced the increased cell proliferation, migration and tube formation induced by vascular endothelial growth factor (VEGF) in human retinal endothelial cells (HREC) and choroid-retinal endothelial RF/6A cells. CGA abrogated VEGF-induced the phosphorylation of VEGFR2 and its downstream mitogen-activated extracellular regulated kinase (MEK1/2), extracellular regulated protein kinase (ERK1/2) and p38 kinase. CGA reduced high glucose (HG)-induced the activation of microglia BV-2 cells. CGA also reduced HG-induced the increased VEGF expression and hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus in BV2 cells. Retinal immunofluorescence staining with cluster of differentiation 31 (CD31) and retinal histopathological observation both demonstrated that CGA (1, 10mg/kg) decreased the increased retinal vessels in streptozotocin (STZ)-induced hyperglycemic mice. CGA reduced the elevated serum VEGF level and microglia activation in STZ-induced hyperglycemic mice. In conclusion, CGA inhibits retinal neoangiogenesis during the process of DR by abrogating HG-induced HIF-1α-mediated paracrine VEGF expression in microglia cells and inhibiting VEGF-induced angiogenesis in retinal endothelial cells. 10.1016/j.vph.2017.11.002
    New Lipid Mediators in Retinal Angiogenesis and Retinopathy. Fleming Ingrid Frontiers in pharmacology Retinal diseases associated with vascular destabilization and the inappropriate proliferation of retinal endothelial cells have major consequences on the retinal vascular network. In extreme cases, the development of hypoxia, the upregulation of growth factors, and the hyper-proliferation of unstable capillaries can result in bleeding and vision loss. While anti-vascular endothelial growth factor therapy and laser retinal photocoagulation can be used to treat the symptoms of late stage disease, there is currently no treatment available that can prevent disease progression. Cytochrome P450 enzymes metabolize endogenous substrates (polyunsaturated fatty acids) to bioactive fatty acid epoxides that demonstrate biological activity with generally protective/anti-inflammatory and insulin-sensitizing effects. These epoxides are further metabolized by the soluble epoxide hydrolase (sEH) to fatty acid diols, high concentrations of which have vascular destabilizing effects. Recent studies have identified increased sEH expression and activity and the subsequent generation of the docosahexaenoic acid-derived diol; 19,20-dihydroxydocosapentaenoic acid, as playing a major role in the development of diabetic retinopathy. This review summarizes current understanding of the roles of cytochrome P450 enzyme and sEH-derived PUFA mediators in retinal disease. 10.3389/fphar.2019.00739
    Effect of intravitreal and intraperitoneal cyanidin-3-glucoside injection in oxygen-induced retinopathy mouse model. Ercan Zeynep E,Haberal Nihan,Helvacıoglu Fatma,Dağdeviren Atilla,Yılmaz Gürsel Indian journal of ophthalmology Purpose:To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model. Methods:In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/μL), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling. Results:The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group. Conclusion:C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations. 10.4103/ijo.IJO_166_18
    CCN1-Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis. Lee Sangmi,Ahad Afruja,Luu Michele,Moon Sohyun,Caesar JoyAnn,Cardoso Wellington V,Grant Maria B,Chaqour Brahim Molecular and cellular biology Cellular communication network factor 1 (CCN1) is a dynamically expressed, matricellular protein required for vascular development and tissue repair. The gene is a presumed target of Yes-associated protein (YAP), a transcriptional coactivator that regulates cell growth and organ size. Herein, we demonstrate that the promoter is indeed a direct genomic target of YAP in endothelial cells (ECs) of new blood vessel sprouts and that deficiency in mice downregulates and alters cytoskeletal and mitogenic gene expression. Interestingly, overexpression in cultured ECs inactivates YAP in a negative feedback and causes its nuclear exclusion. Accordingly, EC-specific deletion of the gene in mice mimics a gain-of-function phenotype, characterized by EC hyperproliferation and blood vessel enlargement. CCN1 brings about its effect by providing cells with a soft compliant matrix that creates YAP-repressive cytoskeletal states. Concordantly, pharmacological inhibition of cell stiffness recapitulates the deletion vascular phenotype. Furthermore, adeno-associated virus-mediated expression of reversed the pathology of YAP hyperactivation and the subsequent aberrant growth of blood vessels in mice with ischemic retinopathy. Our studies unravel a new paradigm of functional interaction between CCN1 and YAP and underscore the significance of their interplay in the pathogenesis of neovascular diseases. 10.1128/MCB.00107-19
    Overexpressing kringle 1 domain of hepatocyte growth factor with adeno-associated virus inhibits the pathological retinal neovascularization in an oxygen-induced retinopathy mouse model. Sun Peng,Liu Zheli Biochemical and biophysical research communications Current clinical treatments for ocular neovascularization are characterized by high possibility of damaging healthy tissues and high recurrence rates. It is necessary to develop new treatment methods to control neovascularization with a stable and effective effect. Kringle1 domain of hepatocyte growth factor (HGFK1) has anti-angiogenesis activity. Here, we established oxygen-induced retinopathy (OIR) model to study if using adeno-associated virus (AAV) as a delivery system to overexpression HGFK1 in retinal cells could benefit retinal neovascularization. We show that, overexpressed exogenous gene was mainly expressed in the inner and outer nuclear layer of the retina. Compared with control mice, the mice pretreated with rAAV-HGFK1 at P3 showed relatively normal vascular branches examined by fluorescence fundus angiography. Subsequent H&E staining and immunohistochemical staining of CD31 of the eye tissue sections showed that the mice received rAAV-HGFK1 had a relatively normal distribution of vascular endothelial cells. Additionally, immunohistochemical staining indicated a lower expression of VEGF in the eye tissues of rAAV-HGFK1 treated OIR mice. Further in vitro studies showed that HGFK1 could inhibit the proliferation but promote the apoptosis of bovine retinal microvascular endothelial cells (BRECs) under the presence of VEGF. Moreover, HGFK1 could inhibit VEGF induced ERK activation but promote p38 activation in BRECs. Therefore, we propose that intravitreal injection of rAAV-HGFK1 might be used to improve the retinal neovascularization and HGFK1 may function through regulating VEGF signaling pathway to inhibit neovascularization. 10.1016/j.bbrc.2018.11.111
    Effects of microRNA-133b on retinal vascular endothelial cell proliferation and apoptosis through angiotensinogen-mediated angiotensin II- extracellular signal-regulated kinase 1/2 signalling pathway in rats with diabetic retinopathy. Liu Tao-Tao,Hao Qian,Zhang Yan,Li Zhao-Hui,Cui Zhi-Hua,Yang Wei Acta ophthalmologica PURPOSE:This study aimed to explore the effects of microRNA-133b (miR-133b) on diabetic retinopathy (DR) by targeting angiotensinogen (AGT) through the angiotensin-II (AngII) extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway in rats. METHODS:The DR rat model was established using retinal tissues of DR rats and normal rats. Immunohistochemistry was performed to detect the protein expressions of AGT and CD34 in retinal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect miR-133b expression, AGT, AngII, ERK1/2 mRNA, and protein expressions in tissues and cells after transfection. Retinal vascular endothelial cells were cultured and divided into normal, blank, miR-133b mimics, miR-133b mimics negative control (NC), miR-133b inhibitors, miR-133b inhibitors NC, siRNA NC, siRNA-AGT, and miR-133b inhibitors + siRNA-AGT groups. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to detect cell proliferation. Cell cycle and apoptosis were evaluated using flow cytometry. RESULTS:Compared with normal rats, AGT and CD34 were expressed more frequently in DR rats. MicroRNA (miR)-133b expression was downregulated but AGT, AngII, ERK1 and ERK2 mRNA expressions were upregulated in retinal tissues of DR rats. When compared to the normal group, all other groups displayed decreased cell proliferation, increased cell number in G0/G1, decreased cell number in S stage, increased cell apoptosis rate and declined miR-133b expression. As well, significant increased expressions of AGT and the AngII-ERK1/2 pathway-related proteins were observed in retinal vascular endothelial cells in all groups except the normal group. The miR-133b mimics and siRNA-AGT groups had increased cell proliferation, decreased cell number in the G0/G1 phase, increased cell number in S stage, decreased cell apoptosis rate and decreased expressions of AGT and AngII-ERK1/2 pathway-related proteins than the miR-133b inhibitors + siRNA-AGT group. The miR-133b inhibitors group exhibited opposite trends compared with the miR-133b mimics and siRNA-AGT groups. CONCLUSION:The study provides data to suggest that miR-133b induces proliferation and inhibits apoptosis of retinal vascular endothelial cells by targeting AGT through the AngII-ERK1/2 signalling pathway in DR rats. 10.1111/aos.13715
    Downregulation of circRNA DMNT3B contributes to diabetic retinal vascular dysfunction through targeting miR-20b-5p and BAMBI. Zhu Ke,Hu Xin,Chen Han,Li Fang,Yin Ning,Liu Ai-Lin,Shan Kun,Qin Yao-Wu,Huang Xin,Chang Qing,Xu Ge-Zhi,Wang Zhongfeng EBioMedicine BACKGROUND:Diabetic retinopathy, a vascular complication of diabetes mellitus, is the leading cause of visual impairment and blindness. circRNAs act as competing endogenous RNA, sponging target miRNA and thus influencing mRNA expression in vascular diseases. We investigated whether and how circDNMT3B is involved in retinal vascular dysfunction under diabetic conditions. METHODS:qRT-PCR was performed to detect expression of circDNMT3B, miR-20b-5p, and BAMBI in retinal microvascular endothelial cells under diabetic conditions. Western blot, Cell Counting Kit-8, Transwell, Matrigel tube formation, and retinal trypsin digestion assays were conducted to explore the roles of circDNMT3B/miR-20b-5p/BAMBI in retinal vascular dysfunction. Bioinformatics analysis and luciferase reporter, siRNA, and overexpression assays were used to reveal the mechanisms of the circDNMT3B/miR-20b-5p/BAMBI interaction. Electroretinograms were used to evaluate visual function. FINDINGS:Upregulation of miR-20b-5p under diabetic conditions promoted proliferation, migration, and tube formation of human retinal microvascular endothelial cells (HRMECs), which was mediated by downregulated BAMBI. Under diabetic conditions, circDNMT3B, which acts as a sponge of miR-20b-5p, is downregulated. circDNMT3B overexpression reduced retinal acellular capillary number and alleviated visual damage in diabetic rats. Changes in expression of circDNMT3B and miR-20b-5p were confirmed in the proliferative fibrovascular membranes of patients with diabetic retinopathy. INTERPRETATION:Downregulation of circDNMT3B contributes to vascular dysfunction in diabetic retinas through regulating miR-20b-5p and BAMBI, providing a potential treatment strategy for diabetic retinopathy. FUNDING:National Natural Science Foundation of China, National Key Basic Research Program of China, Shanghai Municipal Science and Technology Major Project, and ZJLab. 10.1016/j.ebiom.2019.10.004
    Role of TLR4-MAP4K4 signaling pathway in models of oxygen-induced retinopathy. Chen Wenwen,Zhang Juan,Zhang Peijun,Hu Fangyuan,Jiang Tingting,Gu Junxiang,Chang Qing FASEB journal : official publication of the Federation of American Societies for Experimental Biology Retinopathy of prematurity is a vision-threatening condition, and therapies based on antagonizing VEGF may elicit serious side effects in premature infants. Mechanisms of retinal angiogenesis, particularly the signaling pathways independent of VEGF, remain elusive. The goals of our study were to explore TLR4-mediated signaling pathways in human retinal microvascular endothelial cells (HRMECs) and to examine the effects of TLR4 antagonists in models of oxygen-induced retinopathy (OIR). Our results show that intravitreal injection of the TLR4 antagonist TAK-242 reduced areas of nonperfusion, inhibited aberrant angiogenesis, and improved vascular density in the retina of OIR mice. The effects were further potentiated by the anti-VEGF antibody ranibizumab. In cultured HRMECs, the TLR4 agonist LPS up-regulated TLR4/MAPKK kinase kinase 4 (MAP4K4) signaling, and promoted cell proliferation and migration, and reduced barrier functions of the cells. Down-regulation of MAP4K4 in HRMECs abolished the proangiogenic effects by LPS. Our data suggest that the TLR4-MAP4K4 pathway can regulate retinal neovascularization via mechanisms independent of VEGF.-Chen, W., Zhang, J., Zhang, P., Hu, F., Jiang, T., Gu, J., Chang, Q. Role of TLR4-MAP4K4 signaling pathway in models of oxygen-induced retinopathy. 10.1096/fj.201801086RR
    Sphingosine Kinase 2 Modulates Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy. Eresch Jeanette,Stumpf Martin,Koch Alexander,Vutukuri Rajkumar,Ferreirós Nerea,Schreiber Yannick,Schröder Katrin,Devraj Kavi,Popp Rüdiger,Huwiler Andrea,Hattenbach Lars-Olof,Pfeilschifter Josef,Pfeilschifter Waltraud Investigative ophthalmology & visual science Purpose:Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR). Methods:Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR. Results:tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression. Conclusions:Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies. 10.1167/iovs.17-22544
    Association of 150-kDa oxygen-regulated protein with vascular endothelial growth factor in proliferative diabetic retinopathy. Abu El-Asrar Ahmed M,Ahmad Ajmal,Alam Kaiser,Bittoun Emilie,Siddiquei Mohammad Mairaj,Mohammad Ghulam,Mousa Ahmed,De Hertogh Gert,Opdenakker Ghislain Acta ophthalmologica PURPOSE:150-kDa oxygen-regulated protein (ORP150), a member of heat-shock protein family located in endoplasmic reticulum (ER), has a critical role in secretion of vascular endothelial growth factor (VEGF). We investigated expression levels of ORP150 and correlated these levels with VEGF and total vitreous antioxidant capacity (TAC) in proliferative diabetic retinopathy (PDR). We also examined expression of ORP150 in retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMEC). METHODS:Vitreous samples from 40 PDR and 20 non-diabetic patients, epiretinal membranes from 14 patients with PDR, retinas of rats and HRMEC were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. RESULTS:We showed a significant increase in expression of VEGF and ORP150 in vitreous samples from PDR patients compared with controls (p < 0.0001 for both comparisons). Total vitreous antioxidant capacity (TAC) levels were significantly lower in patients with PDR than those in controls (p < 0.0001). Vascular endothelial growth factor (VEGF) and ORP150 levels in PDR with active neovascularization were significantly higher than that in inactive PDR (p = 0.016; p = 0.011, respectively). A significant positive correlation was observed between levels of ORP150 and levels of VEGF (r = 0.42; p = 0.001). In epiretinal membranes, ORP150 was expressed in vascular endothelial cells and stromal cells. We also demonstrated colocalization of the nuclear cell proliferation marker Ki67 and ORP150 in endothelial cells of pathologic new blood vessels. 150-kDa oxygen-regulated protein (ORP150) levels were significantly increased in rat retinas after induction of diabetes. Vascular endothelial growth factor (VEGF) and the pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) induced upregulation of ORP150 in HRMEC. CONCLUSION:These results suggest a role for ORP150 in PDR angiogenesis. 10.1111/aos.13600
    Knockout of insulin and IGF-1 receptors on vascular endothelial cells protects against retinal neovascularization. Kondo Tatsuya,Vicent David,Suzuma Kiyoshi,Yanagisawa Masashi,King George L,Holzenberger Martin,Kahn C Ronald The Journal of clinical investigation Both insulin and IGF-1 have been implicated in control of retinal endothelial cell growth, neovascularization, and diabetic retinopathy. To precisely define the role of insulin and IGF-1 signaling in endothelium in these processes, we have used the oxygen-induced retinopathy model to study mice with a vascular endothelial cell-specific knockout of the insulin receptor (VENIRKO) or IGF-1 receptor (VENIFARKO). Following relative hypoxia, VENIRKO mice show a 57% decrease in retinal neovascularization as compared with controls. This is associated with a blunted rise in VEGF, eNOS, and endothelin-1. By contrast, VENIFARKO mice show only a 34% reduction in neovascularization and a very modest reduction in mediator generation. These data indicate that both insulin and IGF-1 signaling in endothelium play a role in retinal neovascularization through the expression of vascular mediators, with the effect of insulin being most important in this process. 10.1172/JCI17455
    Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1. Joyal Jean-Sébastien,Sun Ye,Gantner Marin L,Shao Zhuo,Evans Lucy P,Saba Nicholas,Fredrick Thomas,Burnim Samuel,Kim Jin Sung,Patel Gauri,Juan Aimee M,Hurst Christian G,Hatton Colman J,Cui Zhenghao,Pierce Kerry A,Bherer Patrick,Aguilar Edith,Powner Michael B,Vevis Kristis,Boisvert Michel,Fu Zhongjie,Levy Emile,Fruttiger Marcus,Packard Alan,Rezende Flavio A,Maranda Bruno,Sapieha Przemyslaw,Chen Jing,Friedlander Martin,Clish Clary B,Smith Lois E H Nature medicine Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy-consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr(-/-) mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr(-/-) photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr(-/-) retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases. 10.1038/nm.4059
    Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization. Rama Nicolas,Dubrac Alexandre,Mathivet Thomas,Ní Chárthaigh Róisín-Ana,Genet Gael,Cristofaro Brunella,Pibouin-Fragner Laurence,Ma Le,Eichmann Anne,Chédotal Alain Nature medicine Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease. 10.1038/nm.3849