Sanjay Sinha

Myocardin Is a Key Regulator of CArG-Dependent Transcription of Multiple Smooth Muscle Marker Genes

Abstract— The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM &agr;-actin, SM-myosin heavy chain, and SM22&agr; by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.

Vascular Smooth Muscle Cells in Atherosclerosis

The historical view of vascular smooth muscle cells (VSMCs) in atherosclerosis is that aberrant proliferation of VSMCs promotes plaque formation, but that VSMCs in advanced plaques are entirely beneficial, for example preventing rupture of the fibrous cap. However, this view has been based on ideas that there is a homogenous population of VSMCs within the plaque, that can be identified separate from other plaque cells (particularly macrophages) using standard VSMC and macrophage immunohistochemical markers. More recent genetic lineage tracing studies have shown that VSMC phenotypic switching results in less-differentiated forms that lack VSMC markers including macrophage-like cells, and this switching directly promotes atherosclerosis. In addition, VSMC proliferation may be beneficial throughout atherogenesis, and not just in advanced lesions, whereas VSMC apoptosis, cell senescence, and VSMC-derived macrophage-like cells may promote inflammation. We review the effect of embryological origin on VSMC behavior in atherosclerosis, the role, regulation and consequences of phenotypic switching, the evidence for different origins of VSMCs, and the role of individual processes that VSMCs undergo in atherosclerosis in regard to plaque formation and the structure of advanced lesions. We think there is now compelling evidence that a full understanding of VSMC behavior in atherosclerosis is critical to identify therapeutic targets to both prevent and treat atherosclerosis.

Generation of human vascular smooth muscle subtypes provides insight into embryological origin–dependent disease susceptibility

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin–specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11+ and ACTA2+), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.

L-type Voltage-Gated Ca2+ Channels Modulate Expression of Smooth Muscle Differentiation Marker Genes via a Rho Kinase/Myocardin/SRF–Dependent Mechanism

Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM &agr;-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM &agr;-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM &agr;-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM &agr;-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM &agr;-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM &agr;-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM &agr;-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

A G/C Element Mediates Repression of the SM22α Promoter Within Phenotypically Modulated Smooth Muscle Cells in Experimental Atherosclerosis

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22&agr; is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22&agr; promoter required for this process. Transgenic mice carrying the SM22&agr; promoter–&bgr;-galactosidase (&bgr;-gal) reporter transgene were crossed to apolipoprotein E (ApoE)−/− mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of &bgr;-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22&agr; promoter prevented the decrease in SM22&agr; promoter–&bgr;-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22&agr; in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB–induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22&agr; promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22&agr;. Together, these results support a model whereby a G/C repressor element within the SM22&agr; promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

A Transforming Growth Factor-β Control Element Required for SM α-Actin Expression in Vivo Also Partially Mediates GKLF-dependent Transcriptional Repression

We previously demonstrated that a conserved transforming growth factor-β control element (TCE) within the 5′-region of the smooth muscle cell (SMC) differentiation marker gene SM α-actin could mediate both transcriptional activation and repression in cultured SMCs through interaction with members of the zinc finger Kruppel-like transcription factor (KLF) family. The aims of the present studies were to: 1) determine the role of the SM α-actin TCE in vivo through mutagenesis studies in transgenic mice and 2) further characterize the possible role and mechanisms by which the TCE-binding factor GKLF/KLF4 induces repression of SMC marker genes in various SMC model systems in vitro. Our results showed that the TCE was required for SM α-actin promoter activity in transgenic mice in vivo. Results of transient transfection studies showed that GKLF-induced repression of a SM α-actin promoter/luciferase reporter gene partially depended on the TCE. Furthermore, a GKLF overexpressing adenovirus inhibited whereas GKLF morpholino antisense oligos increased expression of endogenous SMC marker genes. Results of chromatin immunoprecipitation assays showed GKLF binding to TCE containing regions of various SMC marker gene promoters within intact chromatin. Finally, results of co-transfection studies showed that overexpression of IKLF/KLF5 reversed GKLF-dependent repression thus supporting a model of reciprocal activation-repression of SMC gene expression by different members of the KLF gene family.

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

Assessment of Contractility of Purified Smooth Muscle Cells Derived from Embryonic Stem Cells

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin‐resistance gene under the control of either a smooth muscle α‐actin (SMαA) or smooth muscle‐myosin heavy chain (SM‐MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription‐polymerase chain reaction, and flow cytometry and were designated APSCs (SMαA‐puromycin‐selected cells) or MPSCs (SM‐MHC‐puromycin‐selected cells), respectively. Both SMC lines displayed agonist‐induced Ca2+ transients, expressed functional Ca2+ channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin‐1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC‐derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Human embryonic stem cell-derived vascular smooth muscle cells in therapeutic neovascularisation

Ischemic diseases remain one of the major causes of morbidity and mortality throughout the world. In recent clinical trials on cell-based therapies, the use of adult stem and progenitor cells only elicited marginal benefits. Therapeutic neovascularisation is the Holy Grail for ischemic tissue recovery. There is compelling evidence from animal transplantation studies that the inclusion of mural cells in addition to endothelial cells (ECs) can enhance the formation of functional blood vessels. Vascular smooth muscle cells (SMCs) and pericytes are essential for the stabilisation of nascent immature endothelial tubes. Despite the intense interest in the utility of human embryonic stem cells (ESCs) for vascular regenerative medicine, ESC-derived vascular SMCs have received much less attention than ECs. This review begins with developmental insights into a range of smooth muscle progenitors from studies on embryos and ESC differentiation systems. We then summarise the methods of derivation of smooth muscle progenitors and cells from human ESCs. The primary emphasis is on the inherent heterogeneity of smooth muscle progenitors and cells and the limitations of current in vitro characterisation. Essential transplantation issues such as the type and source of therapeutic cells, mode of cell delivery, measures to enhance cell viability, putative mechanisms of benefit and long-term tracking of cell fate are also discussed. Finally, we highlight the challenges of clinical compatibility and scaling up for medical use in order to eventually realise the goal of human ESC-based vascular regenerative medicine.

Myocardin Regulates Vascular Response to Injury Through miR-24/-29a and Platelet-Derived Growth Factor Receptor β

Objective—Myocardin, a potent transcriptional coactivator of serum response factor, is involved in vascular development and promotes a contractile smooth muscle phenotype. Myocardin levels are reduced during vascular injury, in association with phenotypic switching of smooth muscle cells (SMCs). However, the direct role of myocardin in vascular disease is unclear. Approach and Results—We show that re-expression of myocardin prevents the vascular injury response in murine carotid arteries, with reduced neointima formation due to decreased SMC migration and proliferation. Myocardin reduced SMC migration by downregulating platelet-derived growth factor receptor-&bgr; (PDGFRB) expression. Pdgfrb was regulated by myocardin-induced miR-24 and miR-29a expression, and antagonizing these microRNAs restored SMC migration. Furthermore, using miR-24 and miR-29a mimics, we demonstrated that miR-29a directly regulates Pdgfrb expression at the 3′ untranslated region while miR-24 has an indirect effect on Pdgfrb levels. Myocardin heterozygous-null mice showed an augmented neointima formation with increased SMC migration and proliferation, demonstrating that endogenous levels of myocardin are a critical regulator of vessel injury responses. Conclusions—Our results extend the function of myocardin from a developmental role to a pivotal regulator of SMC phenotype in response to injury, and this transcriptional coactivator may be an attractive target for novel therapeutic strategies in vascular disease.