| Background:Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Choroidal neovascularization (CNV), the main pathological change in exudative AMD, is the primary cause for vision loss in patients with AMD. Hypoxia may play an important role in the development of CNV as hypoxia markedly increases VEGF-A secretion by RPE cells and CNV may be initiated by these accumulations of high amounts of VEGF-A. There are two families of VEGF-A isoforms formed by alternative splicing, the angiogenic VEGF-A family (VEGFXXX), known to contribute to ocular neovascularization, and the anti-angiogenic VEGF-A family (VEGFxxxb), which is found in normal ocular tissues but downregulated in diabetic retinopathy in humans. VEG165b, the most widely studied VEGFxxxb isoform, inhibits corneal and retinal neovascularization in animal models, howerver shows no inhibiton of physiological angiogenesis. Since hypoxia induced angiogenesis in the choroid lead to CNV predominantly through a VEGF-A mediated mechanism, the effect of VEGF165sb on hypoxia dependent angiogenesis in CNV in vitro is determined.Objective:To determine whether VEGF165b could inhibit CNV in vitro and be therapeutically valuable in the treatment of CNV, human RPE cell line ARPE-19 cells were studied to mimc for hypoxia. The investigation aims to provide experimental basis for the VEGF165b treatment of CNV.Methods:1. Hypoxia was induced by addition of 150μM CoCl2 to ARPE-19 cells and verified by HIF-la expression. Apoptotic morphological changes of ARPE-19 cells were examined by light microscopy and stained with Hoechst 33258. Cell viability of ARPE-19 cells was determined by MTT methods.2. In normoxia and hypoxia ARPE-19 cells, the mRNA expression of VEGFxxxb was studied by RT-PCR and the mRNA level of VEGF165b was measured using RQ-PCR. The VEGFxxxb protein expression was measured using ELISA and Western blot with a VEGFxxxb-specific antibody.3. Two coculture models were used to observe the effects of VEGF165b on the proliferation and migration of choroidal endothelial cell line RF/6A cells. The wound healing activity and capillary tube formation of RF/6A cells cultured in conditioned media of ARPE-19 cells was also assessed by wound healing assay and tube fonnation assay.4. To determine whether VEGF165b protects ARPE-19 cells from hypoxia, cell viability of ARPE-19 cells was determined by MTT methods.The effect of VEGF165b on HIF-1αexpression was demonstrated with the use of Western blot analysis, immunofluorescent staining and verified by Western blot on isolated nuclear protein extracts and cytosolic fraction.Results:1. After a 24 h treatment with COCl2, alteration in the structure, size and shape of ARPE-19 cells were detected. An impairment of the plasma membrane was observed after 72 h treatment with COCl2 and chromatins of condensed appearance as well as nuclear fragmentation were observed after a 96 h. The apoptotic index increased in a time-dependent manner treated by COCl2. Cell viability was 60.33% after 24 h, and decrease to 29.65% after 48 h. HIF-la was not present in 0 h but it was present after 3 h treatment with COCl2. It can be noted that the HIF-1αlevel increased with COCl2 treated time going on. The maximum increase in HIF-la occurred after 24 h and then decreased after 48 h.2. VEGFxxxb mRNA and protein isoforms were more detectable in normoxia than in hypoxia ARPE-19 cells. The expression of VEGF165 mRNA in normoxia was increased than that of in hypoxia but the expression of VEGF165b mRNA in normoxia was decreased than that of in hypoxia. The concentration of VEGFxxxb in normoxia was (166.82±2.55) pg/ml and decreased to (125.35±2.10) pg/ml in hypoxia after 24 h, whereas the concentration of pan-VEGF-A in normoxia was (294.27±11.97) pg/ml and raised (582.26±12.98) pg/ml in hypoxia after 24 h.3. Compared to group without VEGF165b treatment, the number of proliferated and transmigrated RF/6A cells significantly decreased when RF/6A cells were cultured in coculture assays. Conditioned media from ARPE-19 cells grown in hypoxia after VEGF165b treatment resulted in significantly less RF/6A wound gap and tube formation than conditioned medium from hypoxic ARPE-19 cells without any VEGF165b treatment. 4. VEGF165b increased the cell viability of ARPE-19 cells after 48 h treatment with hypoxia. The expression of HIF-1αprotein could be inhibited by 40ng/ml VEGF165b and in a concentration-dependent manner. HIF-la downregulation induced by VEGF165b was prevented by treatment with proteasomal inhibitor MG132. Confocal microscopy and Western blot on isolated nuclear protein extracts and cytosolic fraction confirmed HIF-1αis mainly localized into the nucleus and VEGF165b induces HIF-1αproteasomal degradation.Conclusion:1. Addition of 150μM CoCl2 to ARPE-19 cells may use for studying to mimic for hypoxia. The 24 h treatment with CoCl2 was chosen because it induced HIF-1αexpression without sharply decreasing the Cell viability.2. VEGFxxxb isoforms are the predominant isoform in normoxia ARPE-19 cells. In hypoxia ARPE-19 cells, however, the expression of VEGFxxx is significantly greater than that of VEGFxxxb.3. VEGFxxxb inhibited proliferation, migration and tube formation of RF/6A cells cultured in coculture modals and conditioned media from ARPE-19 cells.4. The survival effects of VEGF165b could protect ARPE-19 cells from hypoxia damage. The expression of HIF-1αprotein in hypoxia ARPE-19 cells was downregulated by VEGF165b which induced HIF-1αproteasomal degradation. |
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