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M. Hollborn, C. Petto, P. Wiedemann, L. Kohen, A. Bringmann; The Effects of Thrombin on RPE Cells Are Mediated by Transactivation of Growth Factor Receptors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3533.
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VEGF is the major angiogenic factor. In various tissues, the serine protease thrombin has been implicated in VEGF-induced angiogenesis. We investigated whether thrombin alters the mRNA expression of various cytokines including VEGF, the expression of VEGF-A protein, and the proliferation and chemotaxis of retinal pigment epithelial (RPE) cells.
Cultured human RPE cells of passages 3 - 5 were used. The mRNA expression was evaluated by real-time PCR and the VEGF protein content of the cultured media was determined by ELISA. Cell proliferation was analyzed by a bromodeoxyuridine immunoassay, and chemotaxis was investigated by a Boyden chamber assay. The phosphorylation levels of ERK1/2, p38, and Akt were investigated by Western blotting.
Both acutely isolated and cultured RPE cells expressed mRNAs for the protease-activated receptors PAR1 and PAR3, as well as for the effector cell protease receptor-1. Exogenous thrombin significantly stimulated the mRNA expression of PDGF, HB-EGF, bFGF and VEGF. Thrombin stimulated dose-dependently the secretion of VEGF protein. This effect was blocked by an anti-TGF-ß1 antibody, by hirudin (a thrombin-specific inhibitor), and by selective inhibitors of ERK1/2, p38, JNK, Akt and mTOR activation. Thrombin increased the chemotaxis in a dose dependent manner but had no influence on the proliferation of RPE cells. The thrombin induced chemotaxis is likely mediated by a transactivation of the PDGF receptor tyrosine kinase and the p38 MAPK signaling pathway.
Thrombin enhances the expression of VEGF and induces chemotaxis in human RPE cells. Thrombin evokes the activation of several signaling pathways which are differentially involved in various cellular responses, i.e., migration and VEGF synthesis. Transactivation of growth factor receptors is involved in these processes. Thrombin may represent a critical factor that promotes neovascularization and formation of epiretinal membranes.
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