Thrombin Induces VEGF Expression in Human Retinal Pigment

PURPOSE—The aim of the present study was to investigate effects of thrombin and thrombin in combination with other proangiogenic factors on VEGF expression in hRPE cells. METHODS—hRPE cells were stimulated with thrombin TNF-α, monocytes and TGF-β2. Following stimulation, conditioned medium and lysed cells were subjected to ELISA, Western blotting, immunocytochemistry and RT-PCR analyses. Inhibitors specific for various signal transduction pathways were employed to determine the signal pathways involved. RESULTS—Treatment of RPE cells with thrombin resulted in dose- and time-dependent increases in VEGF mRNA levels and protein production. hRPE VEGF expression is predominantly PAR-1 dependent. About 80 % of thrombin-induced VEGF secretion was abrogated by inhibitors of MAPK/ ERK kinase (MEK), p38, c-Jun NH2-terminal kinase (JNK), protein tyrosine kinase (PTK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), nuclear factor-κB (NF-κB), and reactive oxygen species (ROS). Analyses of VEGF protein production and mRNA synthesis revealed that VEGF induction by thrombin plus TNF-α or co-culture with monocytes was additive, while that by co-incubation with TGF-β2 was synergistic. The co-stimulated VEGF production by TGF-β2 plus thrombin averaged threefold higher than the sum of that induced by each agent alone. Furthermore, BAPTA, a calcium chelator, blocked the VEGF secretion induced by thrombin and thrombin plus TGF-β2 by 65 % and 20%, respectively, but had no effect on that by TGF-β2 alone. CONCLUSIONS—Thrombin alone and in combination with TNF-α, monocytes and TGF-β2 potently stimulated VEGF expression in hRPE cells via multiple signal pathways. The thrombin-induced calcium mobilization may play an important permissive role in maximizing TGF-β2– induced VEGF expression in RPE cells