| Background Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age related macular degeneration (AMD). The pathogenesis of CNV is clearly multifactorial, but not completely understood. Generally, neovascularization incorporates two forms of new blood vessel growth: angiogenesis and vasculogenesis. Angiogenesis were generally promoted by growth factors, and most cellular components of the new vessel complex (endothelial cells, smooth muscle cells, and pericytes) were derived from cells resident within adfacent pre-existing blood vessels. However, more and more studies in animal models suggest that circulating bone marrow-derived endothelial precursor cells (EPCs) may be participate in pathophysiological noevascularization, including retinal and choroidal neovascularization.As a member of the CXC chemokine subfamily, stromal cell derived factor 1 (SDF-1) is implicated for CXCR4-positive EPCs recruitment from the bone marrow to the target tissue. CXCR4, a seven transmembrane spanning G protein coupled receptor, is the only known receptor for SDF-1 and is expressed on various endothelial cells and EPCs. It has been reported that SDF-1 acts as an angiogenic agent in several in vivo and in vitro model systems. Mice deficient in either SDF-1 or CXCR4 had defective formation of large vessels supplying the gastrointestinal tract. In proliferative retinopathy and CNV model, SDF-1/CXCR4 may have a role in orienting EPC to reach sites of neovascularization. Besides, CXCR4 antagonists were effective supppressors of several types of ocular NV, such as ischemia-induced retinal NV and CNV. Clinically, in aged control and AMD choroid, SDF-1 and CXCR4 were most prominent in retinal pigment epithelial (RPE) cells and choroidal stroma.Some studies showed that SDF-1 was expressed in ischemic tissues under the control of HIF-1α. In models of wound healing and several models of tissue hypoxia, SDF-1 mediated homing of endothelial progenitor cells to blood vessel walls in the ischemic tissue. It was concluded that recruitment of CXCR4 positive progenitor cells to regenerating tissues were mediated by hypoxic gradients via HIF-1α-induced expression of SDF-1. HIF-1αinduced SDF-1 expression facilitated adhesion of progenitor cells to ischemic endothelium and induced their migration in transwell assays, suggesting a specific role in homing of circulating progenitor cells to ischemic vascular beds. In this study, we will investigate the potential role of hypoxia induced SDF-1 expression in formation of CNV.Objectives It would be helpful for our understanding of CNV to study the effect of SDF-1 on CNV formation. In this study, we investigated the expression of HIF-1αand SDF-1 in CNV models; then, we studied the SDF-1 expression in cultured RPE treated with CoCl2 and HIF-1siRNA. Then, we used C57BL/6J-GFP chimeric mice to investigate the SDF-1 induced bone marrow derived-EPCs to CNV. Finally, We established an in vitro 3-dimensional (3D) matrix system to study the neovascularization under different SDF-1 levels and microenviroments.Methods 1) The C57BL/6J mice underwent laser rupture of Bruch's membrane to induce CNV and were killed at 1, 4, 8, 16, 24 hours and 1, 3, 7, 14, 28 days after laser injury. Immunofluorescence analysis were processed the expression of HIF-1αand SDF-1. Cultured human RPE cells were exposed to hypoxia (CoCl2, 0.2mM) for 0, 1, 2, 4, 8, 16, 24 and 48 hours, HIF-1αand SDF-1 was detected with Western Blot, RT-PCR and ELISA respetively. HIF-1αshRNA was used to knock down the HIF-1αgene in hRPE, the expression of HIF-1αand SDF-1 under hypoxia was detected. 2) Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells from GFP+/+ transgenic mice to adult C57BL/6J mice and established CNV model after 1 month when confirmed the successful bone marrow transplantation. The chimeric mice were killed at 1, 3, 7, 14 and 28 days after laser injury. The eyes were enucleated and frozen section. SDF-1, CXCR4, CD34, CD31 and c-kit were detected by immunofluorescence. The recruitment of BMCs to CNV is measured by choroidal flatmount. 3) 3D fibrin matrix system was prepared in vitro with human fibrinogen, thrombin and aprotinin. Cultured human BMCs (marked with DiO) and RF/6A cells were seed in the gel, cocultured with different conditional media. The tubular structure and BMCs participating in neovascularization were investigated with a phase contrast microscope. Gels were fixed in 4% buffered formalin and processed for paraffin embedding. The embedded matrixes were cut (5μm) perpendicularly to the surface of the matrix sheet. The expression of CD34 and CD31 on BMCs was examined by immunofluorescence.Results Immunofluorescence showed that the expression of HIF-1αand SDF-1 in CNV were near RPE layer, which were gradually higher and reached the peak at 24 hour and 3d respectively after laser injury. RT-PCR and Western Blot showed that the hypoxic stimulus induced the expression of HIF-1αand SDF-1 in cultured hRPE, and reached the peak at 4th hour, while shHIF-1αhad knockdown the expression of HIF-1αand down-regulated the SDF-1 level. Choroidal flatmount showed that at the early stage of CNV, BMCs gathered around RPE layer near the laser lesion; the GFP-labeled cells were morphologically green fluorescent round spots. During CNV formation, CXCR4 marked BMCs in CNV lesion were differentiated into endothelial cells (CD31+) and participated in vessel formation. When CNV came into being, there were amount of BMCs derived endothelial cells participating in the neovasculature network. Immunofluorescence showed that most BMCs gathered in CNV were CXCR4 positive cells, and some acquired phenotypic markers of EPCs (CD34 and c-kit) and ECs (CD31). In the RF/6A-BMCs-cocultured 3D fibrin matrix system, the DiO labled BMCs were particitated in the tubular networks. There were more CD31 positive BMCs (Dio labeled) in SDF-1 constitutive-expression, SDF-1 and hypoxia group.Conclusion During CNV formation, the hypoxia induced higher expression of HIF-1αin RPE layer, which up-regulated the SDF-1 expression. SDF-1 and its receptor CXCR4 were critical mediators for ischemia-specific recruitment to CNV. Also did SDF-1 can induce BMCs differentiate into endothelial cells to participate in the neovascularization.
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