| BackgroundAge-related macular degeneration (AMD) is the leading cause of visual loss in persons more than 50 years of age. The exudative form of the disease is, characterized by choroidal neovascularization (CNV), in which newly formed vessels from the underlying choroid grow beneath the retinal pigment epithelium (RPE) and the neuroretina. CNV may cause acute or subacute blindness because of bleeding or scar formation. Although the morphology of angiogenesis in CNV secondary to AMD has been described in detail, the pathogenesis is still poorly understood.The deposition of drusen, the basal liner deposit, and the degenerative pigmentary changes have all been shown to be closely associated with the increased risk of CNV. The relative hypoxia caused by the disturbed balance between the limited blood supply in the macula and the high oxygen demand by the photoreceptors may also contribute to the formation of CNV by up-regulating the expression of growth factors, such as VEGF. Therefore, hypoxia was considered to play an important role and VEGF is a major pathogenic factor in the development of CNV. Several lines of evidence implicate increased levels of VEGF in retinal pigment epithelium (RPE) from patients with AMD. In cultured RPE cells, the VEGF expression was significantly up-regulated at both the mRNA and protein levels after exposure to hypoxia. As one of RTKs families, Ephrin/Eph receptor family was paid more attentions in angiogenesis by researchers. It was said that this family took part in the development of CNV, too.At present, several novel therapies for CNV have emerged based on antagonism of VEGF or the VEGF receptor, such as intravitreal administration of pegaptanib (Macugen) or ranibizumab (Lucentis), bevacizumab (avastin) and a systemically delivered, modified VEGF receptor (VEGF-Trap). However, a potential drawback of these therapies is that only one of multiple potentially important angiogenic factors is targeted. Other therapies, such as thermal laser, photodynamic therapy (PDT) or intravitreal triamcinolone, are believed to exert their effects partly through the down-regulation of multiple angiogenic factors. These therapeutic methods, however, all have significant side effects.Recently, hypoxia-inducible factor 1 (HIF-1), which is a transcription factor that regulates genes such as VEGF and erythropoietin (EPO) involved in the response to hypoxia, has been proposed as a novel therapeutic target. HIF-1 is a heterodimer composed of HIF-1αand HIF-1βsubunits. HIF-1βis constitutively expressed, while HIF-1αis induced by hypoxia. HIF-1 transactivates a repertoire of genes, including VEGF, which mediate angiogenesis, cell proliferation/survival, and glucose/iron metabolism to hypoxia. At the same time, a study indicated that hypoxia up-regulates not only HIF-1αand VEGF expression, but also Ephs and ephrins of both A and B subclasses in the mouse skin. In addition, in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins was abrogated by siRNA-mediated down-regulation of HIF-1α. These novel findings show that HIF-1αis also a key factor for expression of Ephrin/Eph.Therefore, we hypothesized that silencing the HIF-1αin RPE cells could inhibited angiogenesis. In the present study, we used coculture systems to investigate the effect of RPE cells knocked down HIF-1αon the proliferation, migration and tube formation of CECs.Purpose1. To establish a rapid and convenient method for purification and primary culture of bovine CEC in vitro and provide an in vitro model for CNV diseases.2. To observe expressions of VEGFR2, EphrinB2/EphB4 in CECs and observe VEGF/VEGFR2, EphrinB2/EphB4 expressions in RPE cells under hypoxia.3. To construct and select the most efficient short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1α) shRNA vector and observe the expressions of corresponding ligands and receptors after RPE cells were transfected by pshHIF-1α.4. To observe the effects of RPE cells transfected by pshHIF-1αon the proliferation, migration and tube formation of CECs, respectively.Methods1. Bovine choroidal microvascular vessels were isolated from choroidal tissues by microdissection and digested with 1 g?L-1 collagenase. CECs were purified selectively by immunomagnetic beads and were cultured with endothelial culture medium. The characteristics of CECs were observed by light and electron microscopy and were identified by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis. CECs grew as tubes in a collagen gel.2. RPE cells were cultured under chemical hypoxia. Immunofluorescence was used to test the EphrinB2/EphB4 expression on RPE cells. Real-time RT-PCR and Western blot were used to observe the effect of hypoxia on expression of EphrinB2/EphB4 on RPE cells. ELISA measured the secreted VEGF in supernatant of RPE cells.3. Three RNAi vectors targeting HIF-1αwere constructed and Real-time PCR selected the most efficient shRNA. Real-time RT-PCR and Western blot were used to observe the effect of hypoxia on expression of HIF-1α, VEGF and EphrinB2/EphB4 in RPE cells transfected by pshHIF-1α. ELISA was used to measure the secreted VEGF in supernatant of the RPE cells.4. Three kinds of coculture models were used to observe the effects of RPE cells transfected by pshHIF-1αon the proliferation, migration and tube formation of CECs, respectively.Results1. 95% of the cultured cells were CECs affirmed by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis. The cultured CECs were slender and spindle-shaped. After confluence, the cells had cobblestone appearance. Weibel-Palade body was found near nuclear membrane by electron microscopy. The staining of Von Willebrand factor was positive and the cells can phagocytose the Dil-Ac-LDL.2. Human RPE cells could express EphrinB2/EphB4 and chemical hypoxia could up-regulate EphB4 receptor expression at mRNA and protein levels. At mRNA level, EphB4 receptor expression increased 2.3 fold and its protein expression reach peak value at 6 h. Correspondingly, the EphrinB2 ligand expression was down-regulated under hypoxia. Bovine CECs could express EphrinB2/EphB4.3. RPE cells were cultured under hypoxia and RNAi technique was used to knock down the HIF-1αgene by pshHIF-1αin RPE cells. mRNA and protein expression of HIF-1αand VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. ELISA measured the secreted VEGF in supernatant of the three experiment groups.4. Proliferation, migration and tube formation of CECs were significantly inhibited by the knocked down RPE cells compared with the control in the coculture system. Proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on day 3, 4 and 5, respectively. Migration reduced by 49.6% at 5 h and tube formation decreased by 40.4% at 48 h.Conclusions1. Bovine CECs can be successfully isolated and purified with this modified method by which enough CEC can be got easily for CNV study;2. Human RPE cells could express EphrinB2/EphB4, which were related with HIF-1αexpression. It suggested the role of EphrinB2/EphB4 in CNV.3. The most efficient pshHIF-1αwas selected and was used for transfection, which could inhibit expression of HIF-1αand VEGF. Expression of EphB4 receptor was related with HIF-1α.4. RNAi of HIF-1αin RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1α. 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