The aim of the present study was to examine the mechanisms through which fenofibrate inhibits the ability of human retinal pigment epithelial cells (RPE cells) exposed to hypoxia to stimulate the proliferation and migration of human umbilical vein endothelial cells (HUVECs). For this purpose, RPE cells and HUVECs were divided into the following groups: RPE-normoxia, RPE + fenofibrate, RPE-hypoxia, RPE hypoxia + fenofibrate; HUVECs normal culture and HUVECs + RPE-hypoxia culture supernatant. RPE cell hypoxia was induced by cobalt(II) chloride (CoCl2). A superoxide anion probe was used to measure the production of superoxide anion, which is indicative of hypoxic conditions. Cell proliferation was assessed by MTT assay, and the expression of vascular endothelial growth factor C (VEGFC) and vascular endothelial growth factor receptor-3 (VEGFR-3) in the RPE cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The migration ability of the HUVECs was determined by scratch-wound assay, and the angiogenic ability of the HUVECs was examined by measuring cell lumen formation. The mRNA and protein expression levels of VEGFC and VEGFR-3 in the RPE cells were measured by RT-qPCR and western blot analysis, respectively. Our results revealed that fenofibrate inhibited the increase in the expression and release of VEGFC and VEGFR-3 into the RPE cell culture supernatant induced by exposure to hypoxia. The culture of HUVECs in medium supernatant of RPE cells epxosed to hypoxia enhanced the viability and migration ability of the HUVECs and promoted lumen formation; these effects were inhibited by fenofibrate. In conclusion, our data demonstrated that the exposure of RPE cells to hypoxia induced the expression and release of VEGFC and VEGFR-3 into the cell culture supernatant. The culture of HUVECs in conditioned medium from RPE cells exposed to hypoxia increased VEGFC and VEGFR-3 expression, and promoted the proliferation and migration of the HUVECs, as well as capillary tube formation, suggesting that RPE cells play an important role in the formation of choroidal neovascularization resulting from hypoxia. Fenofibrate inhibited the upregulation of VEGFC and VEGFR-3 in the RPE cells exposed to hypoxia, and thus reduced the ability of HUVECs to form new blood vessels.
calbindin-d28K (calb1) may protect human lens epithelial cells (HLEcs) from apoptosis, which is a process resulting in individual cell death. The protective effects of calb1 may be attributed to buffering high concentrations of ca 2+. The present study investigated the mechanisms through which calb1 protects SRA01/04 cells (a human lens epithelial cell line) against apoptosis induced by ultraviolet B (UVB) exposure. cells transfected with a lentivirus overexpressing calb1 and control cells were treated with 40 µW/cm 2 irradiation for 15 min and then cultured for 24 h. The changes in intracellular ca 2+ were detected by colorimetry, and the protein expression levels of Bad, Bcl-2 and caspase-12 were measured by western blot analysis. The intracellular ca 2+ concentration of control HLECs increased significantly following UVB irradiation, whereas in calb1-overexpressing cells, the ca 2+ levels remained steady. In the control cells, the expression of Bad and caspase-12 was upregulated, and that of Bcl-2 was downregulated. Notably, during UVB radiation-induced apoptosis, the overexpression of calb1 inhibited cell death, resulting in the decreased expression of Bad and caspase-12, and in the upregulated expression of Bcl-2. These results suggested that calb1 inhibited the upregulation of genes involved in apoptosis. The siRNA-mediated knockdown of calb1 resulted in increased rates of UVB radiation-induced apoptosis, the increased expression of Bad and caspase-12, and the decreased expression of Bcl-2, further demonstrating that calb1 may mediate UVB radiation-mediated apoptosis by regulating ca 2+. On the whole, the findings of the present study indicate that UVB exposure can lead to an imbalance in the intracellular ca 2+ homeostasis in HLEcs and that calb1 protein exerts a negative effect on the expression of pro-apoptotic genes in HLEcs. calb1 may thus inhibit the UVB radiation-induced apoptosis of HLEcs by regulating ca 2+ .
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