We investigated gene expression profiles of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in differentiated and non-differentiated retinal pigment epithelial (RPE) cells during oxidative stress. Human RPE cells were grown in culture on laminin-coated flasks to obtain differentiated features. Cells cultured on plastic were used as non-differentiated controls. After confluence, hydrogen peroxide (H2O2) was added for 48 h, then, total RNA was extracted and used for RT-PCR and Northern blot analysis. Medium conditioned by RPE was used for ELISA, Western blotting, and in vitro angiogenesis assay. As a result, differentiated RPE cells expressed significantly higher levels of VEGF protein, as compared to their non-differentiated counterparts. The expression pattern remained consistent even after cellular exposure to H2O2. Conversely, while elevated levels of PEDF transcript and protein were seen in differentiated RPE cells, compared to non-differentiated cells, a marked decrease at both PEDF mRNA and protein levels was seen after treatment with H2O2. Moreover, this decrease in PEDF expression was dosage dependent. In in vitro angiogenesis assay, conditioned medium from differentiated human RPE cells after exposure to H2O2 showed a dramatic increase in tubular formation and migratory activity of microvascular endothelial cells. These data suggest that, in physiological conditions, a critical balance between PEDF and VEGF exists, and PEDF may counteract the angiogenic potential of VEGF. Under oxidative stress, PEDF decreases disrupting this balance. This equilibrium shift may be significant in promoting a pathological condition of RPE cells and contributing to choroidal neovascularization in age-related macular degeneration.
MMP-2 may be essential in the regulation of retinal neovascularization. Pharmacologic intervention using MMP inhibitors may be a future therapeutic approach for angiogenic retinal diseases.
We investigated the expression profile of monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (RPE) cells under different culture conditions and evaluated the molecular mechanism responsible for MCP-1 gene expression in RPE cells. After cellular confluence, total RNA was extracted and used for RT-PCR. Medium conditioned by RPE was used for ELISA and Western blotting. The result showed that RPE cells cultured on plastic expressed MCP-1 constitutively in the absence of any stimuli. On the other hand, growing human RPE on laminin-coated flasks instead of plastic reduced the production of MCP-1. In the RPE cells cultured on plastic, IkappaB was degraded and A20 protein increased concomitantly. MCP-1 upregulation in RPE cells on plastic was attenuated by the addition of MG-132, a proteasome inhibitor. Also, the addition of pyrolidine dithiocarbonate (PDTC) and hypoxic conditions (0.5% O2) decreased MCP-1 production in these cells. These findings suggested that the expression profile of MCP-1 is regulated by phenotypic alterations of the RPE cells. And the increased MCP-1 expression in RPE cells cultured on plastic is caused via spontaneous activation of NFkappaB induced by susceptibility to oxidative stress.
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