Thrombin Stimulates Smooth Muscle Cell Differentiation From Peripheral Blood Mononuclear Cells via Protease-Activated Receptor-1, RhoA, and Myocardin

Abstract

Rationale: Smooth muscle precursor cells have previously been reported to reside in bone marrow and in the circulation, but little is currently known regarding the proximate stimuli for smooth muscle cell differentiation of these putative progenitors. Objective: Because local thrombin generation occurs as an initial response to vascular injury, we hypothesized that thrombin may influence the differentiation of circulating smooth muscle progenitor cells. Methods and Results: Peripheral blood mononuclear cells were cultured on type I collagen using a protocol optimized to stimulate smooth muscle cell outgrowth. Thrombin-stimulated upregulation of the transcription factor myocardin and smooth muscle myosin heavy chain, and both were inhibited by hirudin or the RhoA inhibitor Y27632. After 10 days of culture, smooth muscle outgrowth colonies formed, which stained positive for ␣-smooth muscle actin, smooth muscle myosin heavy chain, and calponin, in addition to having a contractile response to 100 nmol/L angiotensin II. Coincubation of peripheral blood mononuclear cells with thrombin, 10 mol/L protease-activated receptor-1, but not protease-activated receptor-4 activating peptide significantly increased the number of smooth muscle outgrowth colonies formed. Thrombin-induced enhancement of smooth muscle outgrowth colony formation was inhibited by hirudin, Y27632, and an antibody against protease-activated receptor-1. Conclusions: These data illustrate a novel thrombininduced pathway for smooth muscle differentiation from putative smooth muscle progenitors in peripheral blood. W e have previously described circulating smooth muscle outgrowth cells (SOCs). 1,2 Moreover, we have shown in human subjects who have undergone bone marrow transplantation, smooth muscle cells (SMCs) of donor origin are markedly enriched in coronary atherosclerotic plaque compared to the nondiseased vessel wall. 3 A number of potential agents may mediate SMC differentiation from blood borne progenitors including transforming growth factor-␤; plateletderived growth factor-BB 4 ; and sphingosine-1-phosphate, which induces RhoA-dependent myocardin expression and SMC differentiation in mesenchymal stem cells. 5 Thrombin generation occurs at sites of injury and could conceivably contribute to smooth muscle differentiation of circulating progenitor cells based on several lines of evidence. Under various in vitro conditions in mature SMCs, thrombin stimulates upregulation of the SMC marker genes smooth muscle myosin heavy chain (SM-MHC) 6 and calponin. 7 Thrombin also enhances the angiogenic potential of endothelial progenitor cells 8 and may influence vascular progenitor phenotype and function. 9 In the context of circulating progenitors, no study to date has fully investigated the downstream effects of thrombin on circulating mononuclear cells. Here, we show that thrombin induces robust sequential upregulation of the master smooth muscle cell transcription factor myocardin, as well as smooth muscle specific protein expression in peripheral blood mono...

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