Abstract 472: Thrombin Induces Release of Fibroblast Growth Factor-2 via the Rho Pathway which is Enhanced in Cholesterol-enriched Human Vascular Smooth Muscle Cells
Thrombin - in addition to its function as coagulation factor - is an agonist for G-protein-coupled protease-activated receptors (PARs) and a mitogen for vascular smooth muscle cells (SMC). The mitogenic response to thrombin in human SMC depends on release of fibroblast growth factor-2 (FGF-2) into the pericellular matrix and consecutive activation of the FGF receptor-1 (FGFR-1). The present study investigates whether the Rho-Rho kinase pathway is involved in thrombin-induced FGF-2 release and the influence of cholesterol-enrichment and -depletion, which have been shown to affect FGF-2 signaling and SMC mitogenesis. Cultured human aortic and saphenous vein SMC were enriched with cholesterol by using a cyclodextrin-cholesterol complex. ELISA, Western blotting and RT-PCR were used for quantification of FGF-2 levels. Immunoprecipitation was used to determine FGFR-1 phosphorylation. DNA synthesis was determined by [3H]-thymidine incorporation, proliferation by cell counting. Stimulation of SMC with thrombin (10 nmol/L) resulted in release of FGF-2 into the pericellular space within 10 minutes. The highly specific Rho kinase inhibitor Y-27632 (1 μmol/L) and the protein kinase C (PKC) delta inhibitor Rottlerin (1 μmol/L) inhibited thrombin-induced release of FGF-2, FGFR-1 phosphorylation and mitogenesis. Furthermore, preincubation with cyclodextrin-cholesterol caused accumulation of cellular cholesterol, strongly increased thrombin-induced FGF-2 release and stimulated FGF-2 de novo synthesis. FGF-2 release in cholesterol-rich SMC was inhibited by Y-27632 and Rottlerin. Thrombin-induced DNA synthesis and proliferation were enhanced in cholesterol-rich SMC. This effect was inhibited by FGF-2-neutralizing antibodies. These data suggest that thrombin releases FGF-2 via the Rho kinase-PKC delta pathway in human SMC. This mechanism is enhanced by increased cellular cholesterol levels, resulting in an enhanced mitogenic response towards thrombin. These findings may be relevant for thrombin induced mitogenesis in hypercholesterolemia in vivo.