Purpose: The purpose of the present study was to investigate the 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-alpha, monocytes, and TGF-beta2. After stimulation, conditioned medium and lysed cells were subjected to ELISA, Western blot analysis, immunocytochemistry, and RT-PCR analyses. Inhibitors specific for various signal transduction pathways were used to determine the signaling 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 protease-activated receptor (PAR)-1 dependent. Approximately 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-kappaB (NF-kappaB), and reactive oxygen species (ROS). Analyses of VEGF protein production and mRNA synthesis revealed that VEGF induction by thrombin plus TNF-alpha or coculture with monocytes was additive, whereas that by co-incubation with TGF-beta2 was synergistic. The costimulated VEGF production by TGF-beta2 plus thrombin was an average of three times higher than the sum of that induced by each agent alone. Furthermore, BAPTA [bis-(o-aminophenoxy)ethane-N,N',N'-tetraacetic acid], a calcium chelator, blocked the VEGF secretion induced by thrombin and thrombin plus TGF-beta2 by 65% and 20%, respectively, but had no effect on that induced by TGF-beta2 alone.
Conclusions: Thrombin alone and in combination with TNF-alpha, monocytes, and TGF-beta2 potently stimulated VEGF expression in hRPE cells via multiple signaling pathways. The thrombin-induced calcium mobilization may play an important permissive role in maximizing TGF-beta2-induced VEGF expression in RPE cells.