Inhibition Of Expression Of VEGF In Retinal Pigment Epithelium Cells Under Hypoxia With Specific SiRNA Targeting HIF-1_α

Background A great number of study has demonstrated that VEGF is a key factor for neovascularization in retinal diseases. VEGF is a mitogen of endothelial cells, acts as hyperpermeability factor and activates the proliferation of vascular endothelial cells. VEGF is upregulated by hypoxia and is a major stimulatory factor for retinal neovascularization in ischemic retinopathies.Age related macular degeneration (AMD) is the leading cause of visual impairment in people over 50 years old. There are two forms of macular degeneration: the atrophic (dry) type and the exudative (wet) type. Although only 10% of patients with AMD have exudative type, it accounts for 90% of severe vision loss from the disease. Choroidal neovascularization (CNV) in exudative AMD is responsible for loss of central vision in the majority of patients. The exact causes of exudative AMD are still unknown.Although the effect of hypoxia on the development of CNV hasnot been identified, the expression of VEGF has been reported increasing significantly in RPE cells of exudative AMD. In an animal model, CNV was induced by subretinal injection of VEGF. A transgenic murine model of AMD has been established successfully, in which the overexpression of VEGF by RPE cells induces CNV.The up-regulation of VEGF expression in response to hypoxia occurs through hypoxia-inducible factor 1 (HIF-1), a crucial transactivator of the VEGF gene. HIF-1 is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis. HIF-1 consists of a B subunit and a a subunit, HIF-1B expresses constitutively under normoxia or hypoxia .The stability and activity of HIF-1a are regulated by hypoxia. Under normoxia, the HIF-1B subunit is rapidly degraded via the ubiquitin-proteasome pathway. On the contrary, in the hypoxia condition, HIF-1 a becomes stable and interacts with HIF-1B to form HIF-1, to modulate its transcriptional activity. Therefore, HIF-1 a is the active center of HIF-1. Once activated by hypoxia, HIF-1 binds to the consensus HIF-1 DN A binding site (HBS) which present in the hypoxia-response elements (HREs) of many oxygen-regulated genes, such as VEGF. It's presumed that blocking of HIF-1a can inhibit angiogenesis. Based on these findings, HIF-1 a can be a prime target for gene therapy to inhibit intraocular neovascularization.In recent years, the phenomenon of RNA interference (RNAi) in cells was found. RNAi is a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Nowadays, RNAi has been a focus of RNA molecular therapy. Purpose (1) To evaluate the relationship between ischemia and hypoxia of chorioid with CNV in eyes with AMD. (2) To investigate the expression of HIF-1 a and VEGF in human RPE cells underhypoxia. (3) To evaluate the effect of hypoxia on proliferation of human RPE cells and the effect of PKC signaling pathway on VEGF expression in human RPE cells under hypoxia. (4) Construction of HIF-1a siRNA vector and to examine the effectiveness on the specific inhibition of HIF-1aand VEGF expression in transfected RPE cells by RNAi.Methods (1) A total of 12 cases (24 eyes) were studied retrospectively, who had atrophic AMD at the first consultation, and of which 14 eyes developed exudative AMD during follow-up. Their choroidal circulation reflected on fundus fluorescence angiography(FFA) and confocal indocyanine green angiography (ICGA) before and after CNV were analysed. (2) 100uM CoCl2 were added to cell culture medium, which mimics hypoxia. The passage human RPE cells were exposed to CoCl2 for 0.5, 1, 2, 4, 8, 12 and 24h differently, the RPE cells cultured under normoxia as control group. The expression of HEF-la mRNA and protein in RPE cells was detected by Western blot , RT-PCR assay and immunofluorescence staining. The passage human RPE cells were exposed to CoCl2 for 0.5, 1, 2, 4, 8, 12 and 24h differently. The expression of VEGF protein in RPE cells was measured by immunocytochemical staining. The concentration of VEGF protein secreted by RPE cells in culture medi...