Up-regulated basigin-2 in microglia induced by hypoxia promotes retinal angiogenesis.
Yin Jie,Xu Wen-Qin,Ye Ming-Xiang,Zhang Yong,Wang Hai-Yan,Zhang Jian,Li Yu,Wang Yu-Sheng
Journal of cellular and molecular medicine
Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well-characterized mouse model of oxygen-induced retinopathy, which recapitulated hypoxia-induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up-regulation of PI3K-AKT and IGF-1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro-angiogenic role, providing new insights into microglia-promoting retinal angiogenesis.
10.1111/jcmm.13256
Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers.
eLife
Beta-catenin (i.e., canonical Wnt) signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling.
10.7554/eLife.45542
In vitro and ex vivo retina angiogenesis assays.
Rezzola Sara,Belleri Mirella,Gariano Giuseppina,Ribatti Domenico,Costagliola Ciro,Semeraro Francesco,Presta Marco
Angiogenesis
Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti-angiogenic therapeutics.
10.1007/s10456-013-9398-x
A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P.
Adini Avner,Adini Irit,Chi Zai-Long,Derda Ratmir,Birsner Amy E,Matthews Benjamin D,D'Amato Robert J
Angiogenesis
Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.
10.1007/s10456-017-9556-7
PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.
Angiogenesis
VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.
10.1007/s10456-018-9634-5
Consensus guidelines for the use and interpretation of angiogenesis assays.
Angiogenesis
The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
10.1007/s10456-018-9613-x