Impact of angiogenic activation and inhibition on miRNA profiles of human retinal endothelial cells.

BACKGROUND: Human retinal microvascular endothelial cells (HRMVECs) are involved in the pathogenesis of retinopathy of prematurity. In this study, the microRNA (miRNA) expression profiles of HRMVECs were investigated under resting conditions, angiogenic stimulation (VEGF treatment) and anti-VEGF treatment. MATERIALS AND METHODS: The miRNA profiles of HRMVECs under resting and angiogenic conditions (VEGF treatment), as well as after addition of aflibercept, bevacizumab or ranibizumab were evaluated by analyzing the transcriptome of small non-coding RNAs. Differentially expressed miRNAs were validated using qPCR and classified using Gene Ontology enrichment analysis. RESULTS: Ten miRNAs were found to be significantly changed more than 2-fold. Seven of these miRNAs were changed between resting conditions and angiogenic stimulation. Four of these miRNAs (miR-139-5p/-3p and miR-335-5p/-3p) were validated by qPCR in independent experiments and were found to be associated with angiogenesis and cell migration in Gene Ontology analysis. In addition, analysis of the most abundant miRNAs in the HRMVEC miRNome (representing at least 1% of the miRNome) was conducted and identified miR-21-5p, miR-29a-3p, miR-100-5p and miR-126-5p/-3p to be differently expressed by at least 15% between resting conditions and angiogenic conditions. These miRNAs were found to be associated with apoptotic signaling, regulation of kinase activity, intracellular signal transduction, cell surface receptor signaling and positive regulation of cell differentiation in Gene Ontology analysis. No differentially regulated miRNAs between angiogenic stimulation and angiogenic stimulation plus anti-VEGF treatment were identified. CONCLUSION: In this study we characterized the miRNA profile of HRMVECs under resting, angiogenic and anti-angiogenic conditions and identified several miRNAs of potential pathophysiologic importance for angioproliferative retinal diseases. Our results have implications for possible miRNA-targeted angiomodulatory approaches in diseases like diabetic retinopathy or retinopathy of prematurity.

Semaphorin 3F Modulates Corneal Lymphangiogenesis and Promotes Corneal Graft Survival.

Purpose: Corneal vascularization significantly increases the risk for graft rejection after keratoplasty. Semaphorin 3F (Sema3F) is a known modulator of physiologic avascularity in the outer retina. The aim of this study was to investigate whether Sema3F is involved in maintaining corneal avascularity and can reduce the risk for corneal graft rejection. Methods: Corneal Sema3F expression was investigated using immunohistochemistry and qPCR in human and murine tissue. Pathologic invasion of blood and lymph vessels into corneal tissue was analyzed in the murine corneal suture and high-risk keratoplasty model. The anti-lymphangiogenic effects of Sema3F were further investigated using an in vitro spheroidal sprouting model with supernatant from isolated primary human corneal epithelial cells (hCECs). Results: Sema3F is constitutively expressed in human and murine corneal epithelium. In the corneal suture model, lymphangiogenesis was significantly suppressed by topical Sema3F treatment (P = 0.0003). In the murine high-risk keratoplasty model, pretreatment by topical Sema3F in the inflammation phase significantly promoted subsequent graft survival (P = 0.0006). In this model, both lymph- and blood angiogenesis were reduced (P < 0.05). In vitro, hCEC supernatant had a direct anti-lymphangiogenic effect on human lymphatic endothelial cells (P < 0.01). This effect was completely abolished by addition of anti-Sema3F antibodies. Conclusions: Sema3F is a novel mediator of corneal avascularity with potent anti-lymphangiogenic properties. Topical treatment with Sema3F eye drops may help to limit corneal vascularization and improve outcomes in high-risk keratoplasty patients.

Sema3f Protects Against Subretinal Neovascularization In Vivo.

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr-/-) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr-/- model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.