Abstract 15655: Thrombin Induces Expression Of Sphingosine Kinase-1 (SPHK-1) In Human Vascular Smooth Muscle Cells Via The mRNA Stabilising Protein HuR. Inhibition By Dabigatran Reduces Vascular SPHK-1 Expression And Atherosclerotic Burden In Vivo
Objective: Sphingosine-1-phosphate (S1P) is a cellular signaling lipid generated by sphingosine kinase-1 (SPHK-1). This study investigated the potential regulation of SPHK-1 in thrombin-stimulated human vascular smooth muscle cells (SMC) and mice treated with the direct thrombin inhibitor dabigatran.
Methods: SPHK-1 expression was determined by Taqman® real-time PCR and Western blotting in human vascular SMC and ApoE-deficient mice. Cell proliferation was determined via DNA synthesis and cell counting. Binding of the mRNA stabiliser HuR was measured by immunoprecipitation (pulldown PCR). ApoE mice were treated with dabigatran etixilate (DE)-supplemented chow (10 mg DE/g chow) or matching placebo for 6 months. Plaque coverage was determined by oil red O staining.
Results: Thrombin induced a time- and concentration-dependent (1-100 nmol/L) increase in SPHK-1 mRNA and protein expression in human saphenous vein SMC, determined by realtime PCR and Western blotting, n=6-7. S1P synthesis and release were enhanced in thrombin-stimulated SMC. Inhibition of SPHK-1 attenuated thrombin-induced SMC proliferation but not SMC migration (n=5). The regulatory action of thrombin on SPHK-1 was mimicked by a synthetic PAR-1 ligand and reduced by siRNA against the mRNA stabilising protein HuR. Thrombin was also shown to promote HuR binding to SPHK-1 mRNA, associated with increased nucleo-cytosolic shuttling of HuR and SPHK-1 mRNA stabilisation in the presence of actinomycin D. In ApoE-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic SPHK-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10).
Conclusions: Thrombin induces SPHK-1 expression and S1P synthesis in vascular SMC via the mRNA stabilising protein HuR. This leads to increased SMC proliferation. Inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing SPHK-1 expression.
- © 2011 by American Heart Association, Inc.