The Effect Of CXCR7 / CXCR4 / SDF-1 Axis And VEGF On Chorioretinal Vascular Endothelial Cells

Choroidal neovascularization (CNV) is an important cause of visual imparement in age-related macular degeneration (AMD), pathological myopia, central exudative chorioretinopathy (CEC), ocular histoplasmosis syndrome (OHS), ocular tumor and trauma. CNV frequently occurs at macula and often leads to repeated hemorrhage, exudation, and consequent fibrosis. It severly impairs central vision. Until now, CNV-related diseases are the key and difficult point in ophthalmology.CNV is regulated by many factors and its specific mechanisms were not fully elucidated. This study investigated the functions of several CNV related factors such as stromal cell-derived factor-1(SDF-1), chemokine receptor CXCR4, the recently identified alternate receptor for SDF-1—CXCR7, and vascular endothelial growth factor.Part1:In vitro study of CXCR7/CXCR4/SDF-1on choroidal neovascularization Purpose and background:Chemokine receptor CXCR4has long been regarded as the only receptor for stromal cell-derived factor-1(SDF-1), and their binding could mediate a broad range of biological and pathological processes. CXCR7is a recently identified receptor for SDF-1. This study aims to investigate whether the CXCR7/CXCR4/SDF-1axis is involved in choroidal neovascularization (CNV) in a choroid retinal endothelial cell hypoxic model.Methods:Choroid retinal endothelial cell (RF/6A) was incubated in a hypoxic chamber flushed with a gas mixture of1%O2-94%N2-5%C02to establish an in vitro hypoxia model. CXCR4-siRNA and/or CXCR7-siRNA were transfected into RF/6A cell by lipofectamine2000. Real-time PCR and Western blot analysis were carried out to investigate the mRNA and protein level of CXCR4and CXCR7in normal control group, hypoxia group, CXCR4-siRNA transfection group, CXCR7-siRNA transfection group, and CXCR4-siRNA combined CXCR7-siRNA transfection group. CCK-8cell proliferation analysis, Transwell migration analysis, Annexin V-FITC and PI staining, and Matrigel tube formation assay were implemented to test the proliferation, migration, apoptosis and tube formation ability of RF/6Acell line. Results:CXCR4and CXCR7were up-regulated in mRNA and protein levels under hypoxia. Specific siRNA transfection could down-regulate CXCR4and CXCR7expression levels. Chemokine receptor CXCR4, but not CXCR7, was involved in SDF-1-induced RF/6A cell proliferation and migration, whereas CXCR7was exclusively involved in RF/6A apoptosis. In addition, CXCR4and CXCR7acted together in regulating RF/6Atube formation.Conclusions:CXCR7/CXCR4/SDF-laxis is involved in RF/6A cell proliferation, migration, apoptosis and tube formation, moreover, CXCR4and CXCR7played in a different but orchestrated manner.Part2:Effects and mechanism of vascular endothelial growth factor Purpose and background:Vascular endothelial growth factor is a potent angiogenic factor and is involve in various pathological process. This study aims to investigate the effects and mechanism of VEGF on choroid retinal endothelial cells under hypoxia.Methods:Choroid retinal endothelial cells (RF/6A) were pretreated with MEK inhibitor or PI3K inhibitor respectively, and then incubated in a hypoxia chamber flushed with a gas mixture of1%O2-5%CO2-94%N2. Real-time PCR and western blot analysis were carried out to explore VEGF expression on mRNA and protein levels under hypoxia, exogenous recombinant VEGF, and different signal pathway inhibitors, respectively. CCK-8analysis and tube formation assay were tested. Mean while, the PI3K/Akt and MEK/ERK pathways in this process were also investigated. Results:VEGF, p-ERK, and p-Akt were up-regulated in RF/6A cells under hypoxic conditions. MEK inhibitor (PD98059) and PI3K inhibitor (LY294002) decreased ERK and Akt activity respectively, and reduced VEGF expression. VEGF-induced RF/6A proliferation and tube formation requires MEK/ERK and PI3K/Akt signaling, and both of the two pathways were needed in regulating VEGF expression.Conclusions:These suggest that VEGF plays an important role in RF/6A proliferation and tube formation, and MEK/ERK and PI3K/Akt pathway may be responsible for this process.