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Myeloid-Derived Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor Are Dispensable for Ocular Neovascularization--Brief Report. Liyanage Sidath E,Fantin Alessandro,Villacampa Pilar,Lange Clemens A,Denti Laura,Cristante Enrico,Smith Alexander J,Ali Robin R,Luhmann Ulrich F,Bainbridge James W,Ruhrberg Christiana Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined. APPROACH AND RESULTS:We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy. CONCLUSIONS:The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies. 10.1161/ATVBAHA.115.306681
Smooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemia. Dou Yong-Qing,Kong Peng,Li Chang-Lin,Sun Hong-Xing,Li Wei-Wei,Yu Yuan,Nie Lei,Zhao Li-Li,Miao Sui-Bing,Li Xiao-Kun,Dong Chen,Zhang Jin-Wen,Liu Yang,Huo Xiao-Xia,Chi Kui,Gao Xiang,Zhang Ning,Weng Lin,Yang Hongyuan,Zhang Fan,Han Mei Theranostics Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (-Tg) and knockout (KO) mice. Angiogenesis was assessed in by quantification of the total number of capillaries, wound healing and matrigel plug assays, and ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and hybridization assays. The blood flow recovery was significantly attenuated in -Tg mice, and markedly improved in -Tg mice treated with SIRT1 inhibitor EX527 and in -KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of Tg mice compared with -KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of -Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with -KO and WT VSMCs. Mechanistically, -Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in Tg mice. : Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases. 10.7150/thno.39320
Upregulating Hif-1α by Hydrogel Nanofibrous Scaffolds for Rapidly Recruiting Angiogenesis Relative Cells in Diabetic Wound. Chen Hao,Jia Peng,Kang Hui,Zhang Hongbo,Liu Yi,Yang Peilang,Yan Yufei,Zuo Guilai,Guo Lei,Jiang Min,Qi Jin,Liu Yuanyuan,Cui Wenguo,Santos Hélder A,Deng Lianfu Advanced healthcare materials Nonhealing chronic wounds on foot are one of the most dreaded complications of diabetes, and biomedical scaffolds remain an attractive option for repairing or regenerating tissues. Accelerating angiogenesis in the early stage after injury is critical to wound healing process; however, the scaffolds accelerate the angiogenesis in the beginning but with the acceleration of vessel network formation the scaffold network hinders the process. In this study, the water soluble drugs-loaded hydrogel nanofibrous scaffolds are designed for rapidly recruiting angiogenesis relative cells and promoting wound healing. The sustained release profile of desferrioxamine (DFO), which continues for about 72 h, leads to significantly increase of neovascularization. The majority of the scaffold is degraded in 14 d, leaving enough space for cell proliferation and vessel formation. The in vitro results show that the scaffolds upregulate the expression of Hif-1α and vascular endothelial growth factor, and enhance the interaction between fibroblasts and endothelial cells. The in vivo studies show a higher expression of angiogenesis related cytokines. This study demonstrates that the DFO released from hydrogel nanofibrous scaffolds of quick degradation can interfere with the required prolyl-hydroxylases cofactors by acting as Fe(2+) chelator and upregulate the expression of Hif-1α, leading to a significant increase of the neovascularization. 10.1002/adhm.201501018
LncRNA GAS5 activates the HIF1A/VEGF pathway by binding to TAF15 to promote wound healing in diabetic foot ulcers. Laboratory investigation; a journal of technical methods and pathology A diabetic foot ulcer (DFU) is one of the most devastating complications of diabetes. It has been reported that lncRNA GAS5 plays a vital role in wound healing in DFUs. However, the specific mechanism remains unclear. In this research, we aimed to investigate the role of GAS5 in wound healing in DFUs as well as the underlying mechanism. qPCR or western blotting was performed to measure the expression levels of GAS5, HIF1A, VEGF and TAF15. CCK-8 or EdU assays, flow cytometry, wound healing assays and tube formation assays were carried out to assess the proliferation, apoptosis, wound healing and in vitro angiogenesis of HUVECs, respectively. RNA pull-down and RIP assays were performed to verify the interaction between GAS5 and TAF15. ChIP and luciferase assays were conducted to verify the binding of TAF15 to the HIF1A promoter. In the DFU mouse model, H&E and Masson staining were used to determine epidermal and dermal thickness and collagen formation. GAS5 and HIF1A were downregulated in the skin tissues of DFU patients, and GAS5 overexpression promoted cell proliferation, wound healing and tubule formation in HG-treated HUVECs. In addition, GAS5 facilitated HIF1A expression by interacting with TAF15. Rescue assays demonstrated that the suppression of HIF1A/VEGF pathway activation partially reversed the functional roles of GAS5 in HUVECs. Furthermore, GAS5 accelerated wound healing by activating the HIF1A/VEGF pathway in mice with DFUs. GAS5 activates the HIF1A/VEGF pathway by binding to TAF15, resulting in accelerated wound healing in DFUs. Our findings may provide a theoretical basis for the clinical treatment of DFUs. 10.1038/s41374-021-00598-2
High glucose and/or high insulin affects HIF-1 signaling by regulating AIP1 in human umbilical vein endothelial cells. Li Shuang,Li Qin,Yu Wenlin,Xiao Qiang Diabetes research and clinical practice OBJECTIVE:The objective of this study was to explore the effects of high glucose/high insulin on AIP1 expression in HUVECs and the possible regulation of HIF-1α signaling by AIP1. METHODS:We investigated the expression of AIP1 and HIF-1α signaling in HUVECs at the levels of mRNA and protein following exposure to 30 mmol/L glucose (high glucose), 1 nmol/L insulin (high insulin), and the combination of the two (high glucose/high insulin). We detected changes in HIF-1α and VEGF expression with AIP1 siRNA interference by real-time PCR and western blotting. The CCK8 cell proliferation assay, the scratch/wound-healing assay, and flow cytometry were used to assess cell proliferation, migration and apoptosis, respectively. Matrigel was used to perform a tubule formation assay. RESULTS:Compared with 5.5 mmol/L glucose alone (control), high glucose, high insulin, and the combination of high glucose+high insulin increased AIP1 expression at 24 h at the mRNA and protein levels. High glucose, high insulin, and high glucose+high insulin decreased HIF-1α expression at the mRNA and protein levels. AIP1 knockdown significantly increased HIF-1α and VEGF expression at both the mRNA and protein levels in HUVECs under high glucose conditions. In the presence of high insulin, the effect of high glucose on target gene expression was altered. The downregulation of AIP1 promoted cell proliferation, migration, and tubule formation, and it decreased apoptosis. CONCLUSIONS:High glucose increases AIP1 expression and decreases the expression of HIF-1α and downstream molecules. Decreased HIF-1α signaling may be regulated by increased AIP1 under high glucose. 10.1016/j.diabres.2015.05.005
Genipin ameliorates diabetic retinopathy via the HIF-1α and AGEs-RAGE pathways. Phytomedicine : international journal of phytotherapy and phytopharmacology BACKGROUND:Traditional Chinese medicine (TCM) is useful in disease treatment and prevention. Genipin is an active TCM compound used to treat diabetic retinopathy (DR). In this study, a network pharmacology (NP)-based approach was employed to investigate the therapeutic mechanisms underlying genipin administration in DR. METHODS:The potential targets of DR were identified using the gene expression omnibus (GEO) database. TCM database screening and NP were used to predict the potential active targets and pathways of genipin in DR. Cell viability was tested in vitro to determine the effects of different doses of glucose and genipin on Human Retinal Microvascular Endothelial Cells (hRMECs). CCK-8, CCK-F, colony formation, CellTiter-Lum, Annexin V-FITC, wound healing, Transwell, tube-forming, reactive oxygen species (ROS), and other assay kits were used to detect the effects of genipin on hRMECs during high levels of glucose. In vivo, a streptozotocin (STZ)-mouse intraocular genipin injection (IOI.) model was used to explore the effects of genipin on diabetes-induced retinal dysfunction. Western blotting was performed to identify the cytokines involved in proliferation, apoptosis, angiogenesis, ROS, and inflammation. The protein expression of the AKT/ PI3K/ HIF-1α and AGEs/ RAGE pathways was also examined. RESULTS:Approximately 14 types of TCM, and nearly 300 active ingredients, including genipin, were identified. The NP approach successfully identified the HIF-1α and AGEs-RAGE pathways, with the EGR1 and UCP2 genes, as key targets of genipin in DR. In the in vitro and in vivo models, we discovered that high glucose increased cell proliferation, apoptosis, angiogenesis, ROS, and inflammation. However, genipin application regulated cell proliferation and apoptosis, inhibited angiogenesis, and reduced ROS and inflammation in the HRMECs exposed to high glucose. Furthermore, the retinal thickness in the genipin-treated group was lower than that in the untreated group. AKT/ PI3K/ HIF-1α and AGEs/ RAGE signaling was increased by high glucose levels; however, genipin treatment decreased AKT/ PI3K and AGEs/ RAGE pathway expressions. Genipin also increased HIF-1α phosphorylation, oxidative phosphorylation of ATP synthesis, lipid peroxidation, and the upregulation of oxidoreductase. Genipin was found to protect HG-induced hRMECs and the retina of STZ-mice, based on; 1 the inhibition of UCP2 and Glut1 decreased intracellular glucose, and glycosylation; 2 the increased presence of HIF-1α, which increased oxidative phosphorylation and decreased substrate phosphorylation; 3 the increase in oxidative phosphorylation from ATP synthesis increased lipid peroxidation and oxidoreductase activity, and; 4 the parallel effect of phosphorylation and glycosylation on vascular endothelial growth factor (VEGF), MMP9, and Scg3. CONCLUSION:Based on NP, we demonstrated the potential targets and pathways of genipin in the treatment of DR and confirmed its effective molecular mechanism in vitro and in vivo. Genipin protects cells and tissues from high glucose levels by regulating phosphorylation and glycosylation. The activation of the HIF-1α pathway can also be used to treat DR. Our study provides new insights into the key genes and pathways associated with the prognosis and pathogenesis of DR. 10.1016/j.phymed.2024.155596