Thrombin Stimulates Smooth Muscle Cell Differentiation From Peripheral Blood Mononuclear Cells via Protease-Activated Receptor-1, RhoA, and Myocardin
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 α-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 μmol/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 thrombin-induced pathway for smooth muscle differentiation from putative smooth muscle progenitors in peripheral blood.
We 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-β; platelet-derived growth factor-BB4; 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 cells8 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 mononuclear cells (PBMNCs). Moreover, these transcriptional events precede marked augmentation of colony outgrowth of cells with morphological and ultrastructural characteristics of smooth muscle cells and full contractile competence. We further characterize the signaling events leading to SMC marker transcription and formation of contractile SOCs, linking thrombin activation of PAR-1 and myocardin upregulation via the RhoA pathway.
PBMNCs were isolated from blood using Biocoll density gradients and cultured using a protocol optimized for smooth muscle cell outgrowth. Details of the experimental procedures used in this study are provided in the Online Data Supplement at http://circres.ahajournals.org. All data presented represent at least 4 to 5 individual experiments performed in triplicate.
Results and Discussion
Thrombin Induces Myocardin and Smooth Muscle Myosin Heavy Chain Protein Expression in PBMNCs via RhoA
Because myocardin is recognized as a master transcription factor for smooth muscle–specific gene expression, the effect of thrombin on myocardin and SM-MHC expression in cultured PBMNCs was initially investigated. Myocardin and SM-MHC upregulation by thrombin peaked at three and 5 days (Figure 1A) after initial seeding culture, and protein levels were, respectively, 3- and 2-fold greater than control treatment (P<0.05; Figure 1B and 1C). Downregulation of SM-MHC expression observed from day 7 may be indicative of the phenotype modulation before SOC colony expansion (burst), which is characteristic of adult SMCs, whereby proliferating cells downregulate their structural proteins (reviewed elsewhere10). Thrombin-induced upregulation of myocardin and SM-MHC (Figure 1D) was blocked by hirudin and Y27632, a RhoA inhibitor (P<0.001; Figure 1E and 1F). Normalization of myocardin and SM-MHC antigen to proliferating-cell nuclear antigen (PCNA) expression and proliferation indices (5-bromodeoxyuridine incorporation; Online Figure II) in thrombin and untreated groups indicated that thrombin-stimulated myocardin upregulation was attributable to a differentiation rather than a proliferative stimulus. Myocardin associates with serum response factor, inducing CArG SMC gene transcription. Most contractile SMC marker genes such as α-smooth muscle actin (α-SMA), SM-MHC, and calponin contain at least 1 CArG element located within the promoter–enhancer region of the gene.11 These data suggest, for the first time, a link between thrombin/RhoA signaling, myocardin expression, and smooth muscle cell differentiation.
Thrombin-Induced SOC Colony Formation Is Inhibited by Hirudin and RhoA Blockade
To explore further whether thrombin could augment smooth muscle differentiation of PBMNCs, extended culture of these cells was performed under smooth muscle permissive conditions in the presence or absence of thrombin. After 10 days of culture, SOC colonies with characteristic “hill and valley” morphology that stained positive for α-SMA were evident (Figure 2A). Colony number was increased ≈2.5-fold by 1 U/mL thrombin compared to control treatment (P<0.01; Figure 2B). Both hirudin and Y27632 significantly inhibited thrombin-induced augmentation of SOC outgrowth from PBMNCs (Figure 2B). Hirudin or Y27632 administered in the absence of thrombin had no significant effect on SOC colony formation (Figure 2B). Flow cytometric analysis of SOCs revealed a SMC protein expression profile similar to mature aortic (Ao)SMCs, with cells staining positive for the SMC markers, α-SMA, SM-MHC, and calponin but staining negative for endothelial nitric oxide synthase (Figure 2C). SOCs had contractile activity, in response to 100 nmol/L angiotensin II (20.8±2.1% contraction), comparable to that of AoSMCs (14.9±3.3, versus SOCs; P=NS; Figure 2D). The calcium channel blocker nifedipine inhibited contraction of both SOCs (−0.5±3.4%, P<0.001) and AoSMCs (−3.1±2.7%, P<0.001) equally. Ultrastructural analysis of SOCs revealed a myofilamentous staining pattern for α-SMA and calponin (Figure 2E) similar to contractile AoSMCs. Taken together, these results indicate that thrombin promotes the formation of SOCs from peripheral blood, which possess the capacity to form functional SMCs.
PAR-1, but Not PAR-4 Activation, Enhances SOC Colony Formation From the PBMNCs
Because PAR receptors on PBMNCs are the most likely to mediate thrombin signaling, RT-PCR analysis was performed and confirmed strong expression of PAR-1 but not PAR-4 receptor on these cells (Figure 3A). Indeed, 10 μmol/L PAR-1 activating peptide12 but not PAR-4AP significantly stimulated SOC colony formation (≈2-fold increase in SOC colony formation units versus control treatment, P<0.01; Figure 3B). This increase was similar to thrombin-induced enhancement, suggesting that thrombin may act via PAR-1 to stimulate SOC colony formation in PBMNCs. PAR1-AP–induced augmentation was completely inhibited by Y27632 (P<0.05) but not by hirudin (Figure 3C), which is unsurprising because activation of PAR-1 using the tethered ligand peptide is insensitive to hirudin.13 However, an antibody raised against the N terminus of PAR-1, which includes the thrombin cleavage and tethered ligand sequences inhibited both thrombin-induced SOC colony formation stimulation (P<0.01; Figure 3D) and SM-MHC expression (P<0.05; Figure 3E).
These data provide evidence for thrombin as a differentiation factor for putative smooth muscle progenitor cells within peripheral blood. Moreover, a PAR-1–dependent SMC differentiation pathway linking RhoA signaling, myocardin upregulation, and smooth muscle–specific protein expression (Figure 3F) may have implications for understanding how coagulation proteases contribute to cell fate in the context of thrombosis and mononuclear cell infiltration of the injured vessel wall.
We thank Sanja Trinki for graphic design assistance.
Sources of Funding
This work was supported by grants from the Health Research Board (Ireland) and Science Foundation Ireland.
↵*Both authors contributed equally to this work.
Original received April 28, 2009; revision received June 19, 2009; accepted June 23, 2009.
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