| Background Choroidal neovascularization (CNV) is common in many ocular diseases, such as age-related macular degeneration (AMD), chorioretinitis, ocular histoplasmosis syndrome (OHS), high myopia, and so on. CNV often implicated the macular, leading to bleeding, exudation and scar forming. It is one of the causes of blindness. However the exact causes of CNV are still unknown.VEGF is upregulated by hypoxia and is a major stimulatory factor for neovascularization. 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. All these showed the important role of VEGF in the formation of CNV.The transform of choroid circulation and the incrassation of Bruch's membrane in the CNV patients could lead to hypoxia condition. 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 β subunit and a asubunit, HIF-1β expresses constitutively under normoxia or hypoxia. The stability and activity of HIF-1α are regulated by hypoxia. Under normoxia, the HIF-la subunit is rapidly degraded via the ubiquitin-proteasome pathway. On the contrary, in the hypoxia condition, HIF-la becomes stable and interacts with HIF-lp to form HIF-1, to modulate its transcriptional activity. Therefore, HIF-1α is the active center of HIF-1. Once activated by hypoxia, HIF-1 binds to the consensus HIF-1 DNA binding site (HBS) which present in the hypoxia-response elements (HREs) of many oxygen-regulated genes, such as VEGF.Calcium ion, as a secondary messenger, plays an important role in the intercellur signal transduction, secretion, cell proliferation and differentiation. Studies showed a significant increase of [Ca2+]i in the endothelial cells during 2h hypoxia. Such increased intracellular calcium concentrations could lead to the expression of HIF-1α induced by hypoxia.Objective Cobalt Chloride(CoCl2) is used to establish a hypoxia model in cultured human RPE cells. We investigate the influence of hypoxia on the activities of mitochondrial enzyme in RPE cells and evaluate the effect of hypoxia on proliferation of RPE cells.We investigate the effect of Calcium ion on HIF-la activation and VEGF expression in human RPE cells after hypoxia.Methods (1) The hypoxia model was set up using CoCl2. The human RPE cells were exposed to 200μM CoCl2 for 0.5, 1, 2, 4, 8, 16 and 24h differently. The RPE cells cultured under normoxia as control group. The mitochondrial enzyme activity was determined with histochemistry technique. (2) The human RPE cells were exposed to 200μM CoCl2 for 1, 2, 3, 4 and 5d differently. The effect of hypoxia on the proliferation of cultured RPE cells was examined by 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. (3) The human RPE cells were exposed to 200μM CoCl2 for 15min, 30min, 1h and 2h differently. The cytoplasmic free Ca2+ concentration([Ca2+]i) of RPE cellswere detected by laser scanning confocal microscope (LSCM). (4) The human RPE cells were exposed to 200μM CoCl2 for 0.5, 1, 2, 4, 8, 16 and 24h differently. The expression of HIF-1μ mRNA and protein in RPE cells was detected by in suit hybridization (ISH), immunocytochemical staining and immunofluorescence staining. The concentration of VEGF protein secreted by RPE cells in culture medium was measured by enzyme-linked immunosorbent assy (ELISA) kits. (5) The [Ca2+]i of RPE cells, the expression of HIF-la mRNA and protein, the concentration of VEGF protein in culture medium was investigated after 5μM A23187 (the calcium ionophore) and 10μM BAPTA-AM (the intracellular calcium chelator) were added to the culture medium of cultured RPE cells under hypoxia for 0.5, 1,2,4, 8, 16 and 24h.Results (1) The mitochondrial succinate dehydrogenase and cytochrome oxidase activity of RPE cells in the hypoxia group showed a significant suppression compared with the normal group(P<0.05). (2) MTT test showed the cellular proliferation of RPE cells in the hypoxia group was increased(P<0.05). (3) The level of [Ca2+]i in RPE cells increased rapidly after exposed to hypoxia or A23187. While BAPTA-AM could restrain the [Ca2+]i increase in hypoxic RPE cells . (4) It was showed that the HIF-la mRNA located in the nuclea at normoxia by in suit hybridization. For 1h of hypoxia the HIF-la mRNA moved to the cytoplasm. Immunocytochemical staining and immunofluorescence staining realed that the HIF-la protein located in cytoplasm at 0.5h of hypoxia. For lh of hypoxia the accumulation of HIF-la in the nuclea increased. The concentration of VEGF protein in culture medium increased significantly (P<0.05) after hypoxia. (5) Comparing with control group, the expression of HIF-1α and the concentration of VEGF secreted by RPE cells under hypoxia increased after the administration of A23187 in culture medium (P<0.05) and decreased after the treatment of BAPTA-AM (P<0.05). |
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