Cholesterol enhances thrombin-induced release of fibroblast growth factor-2 in human vascular smooth muscle cells

Objective - The mitogenic response to the G protein - coupled receptor agonist thrombin in human vascular smooth muscle cells (SMCs) depends on release of fibroblast growth factor-2 (FGF-2). Yet, intracellular mechanisms triggering FGF-2 release are unknown. The present study investigates possible effects of cholesterol enrichment and depletion, which have been shown to influence FGF-2-dependent signaling and SMC mitogenesis, on thrombin-induced FGF-2 release. Methods and Results - Cultured human aortic and saphenous vein SMCs were enriched with cholesterol by using a cyclodextrin-cholesterol complex. Cholesterol accumulation was determined by a fluorometric assay. ELISA, Western blotting, and RT-PCR were used for quantification of FGF-2 levels. DNA synthesis was determined by [H-3]-thymidine incorporation, proliferation by cell counting. Stimulation of SMCs with thrombin (30 nmol/L) resulted in release of FGF-2 into the pericellular space within 10 minutes. Preincubation with cyclodextrin-cholesterol caused accumulation of cellular cholesterol, increased thrombin-induced FGF-2 release, and stimulated FGF-2 de novo synthesis. Thrombin-induced DNA synthesis and proliferation were enhanced in cholesterol-rich SMCs. This effect was inhibited by FGF-2-neutralizing antibodies. Conclusions - Enhanced cellular cholesterol stimulates thrombin-induced release of FGF-2 and increases the mitogenic response toward thrombin in human SMCs. This mechanism might also be relevant for thrombin-induced mitogenesis in hypercholesterolemia in vivo.