SM Α-actin Research Articles

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

Cyclosporine upregulates KLF4 in vascular smooth muscle cells and drives phenotypic modulation in vivo

Cyclosporine‐A (CSA, a calcineurin inhibitor) is a fungus‐derived immunosuppressant that blocks proliferation of lymphocytes. CSA has also been shown to block both vascular smooth muscle cell (SMC) proliferation in culture and vessel neointimal formation following injury in vivo. The molecular and pathological effects of CSA on SMCs are not fully understood. We show here that CSA increased SMC expression of Krüppel like factor 4 (KLF4) mRNA and protein. KLF4 is a transcription factor that regulates SMC phenotype. KLF4 inhibits proliferation, promotes migration, and suppresses SMC differentiation marker genes. We show that CSA suppressed SMC differentiation markers SM α‐actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD), but increased cyclin‐dependent kinase inhibitor‐1A (CDKN1A) & matrix metalloproteinase‐3 (MMP3). CSA treatment of rat carotid arteries in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, & SM myosin heavy chain (MYH11) mRNA. Conversely, tumor suppressor genes KLF4, p53, & CDKN1A were increased, as well as MMP3, MMP9, & collagen‐VIII (COL8). CSA‐treated arteries showed breakdown of the internal elastic lamina and pathological reorientation of SMCs. KLF4 immunostaining in SMCs and endothelial cells was increased. In conclusion, CSA increases KLF4 expression and promotes SMC phenotypic modulation in vivo. Sources of support: APS UGSRF, APS post‐doc, NIH HL81682, AHA SDG.

Overexpression of The Cellular Repressor of E1A-stimulated Genes Inhibits The Apoptosis of Human Vascular Smooth Muscle Cells <I>via</I> Blocking p38/JNK MAP Kinase Activation*

The cellular repressor of E1A-stimulated genes (CREG) is a recently described glycoprotein which plays a critical role in keeping cells or tissue in homeostasis states.The current study examined whether and how CREG may regulate VSMC apoptosis.Both loss-of-function (CREG-DW by retrovirus expressing CREG shRNAs) and gain-of-function (CREG-UP by retroviral infection with vector pLNCX-CREG) studies were performed to support this notion.Western blot showed that CREG knockdown stimulated with STS or VP-16 was identified to increase cleaved caspase-3,a marker for apoptosis.It was also observed that the number of cells undergoing apoptosis remarkably increased in CREG-DW cells by annexin V/PI dual-color flow cytometry and TUNEL assays.Moreover,p38 and JNK mitogen-activated protein kinases were significantly upregulated in CREG-DW and significantly reduced in CREG-UP VSMCs.More importantly,CREG-DW-induced VSMC apoptosis was blocked by a p38-specific inhibitor (SB203580),or by overexpression of a dominant negative p38α (p38α AGF).The data also showed that inactivation of p38 decreased the amount of phosphorylated JNK,indicated that the p38 fusion proteins are functionally active in regulating JNK activity and induced JNK phosphorylation contributes positively to VSMC apoptosis.In addition,CREG-UP increased expression of the VSMC differentiation markers SM α-actin and SM-MHC,and reduced cell-associated fibronectin in cultured VSMCs.These results demonstrate for the first time that CREG plays a key role in modulating VSMC apoptosis by p38/JNK signaling transduction pathway in vitro.Accordingly,CREG might have potential applicational perspective in attenuating the progression of atherosclerotic plaques and restenosis after percutaneous coronary intervention.

Thyroid hormone induces artery smooth muscle cell proliferation: discovery of a new TRα1‐Nox1 pathway

Thyroid hormone (T3) can stimulate protein synthesis and cell growth. NOX1 is a mitogenic oxidase. The aim of this study was to test a novel hypothesis that T3 induces artery smooth muscle cell proliferation by up-regulating NOX1. Immunofluoresence confocal microscopy was used to visualize the sub-cellular localization of NOX1 and TRα1 in rat aorta smooth muscle (RASM) cells. Optical sectioning showed that TRα1 and NOX1 co-localized around the nucleus. T3 promoted RASM cell proliferation as determined by the fact that T3 significantly increased the number of cytokinesis cells, proliferating cellular nuclear antigen (PCNA) and smooth muscle α-actin (SM α-actin). T3 increased NOX1 expression at both the transcription (mRNA) and translation (protein) levels as evaluated by RT-PCR and Western blot, respectively. T3 also significantly increased the intracellular ROS production based on the oxidation of 2’,7’-dichlorodihydrofluoresein (H2DCF) to a fluorescent 2’,7’-dichlorofluoresein (DCF). RNAi silence of TRα1 or NOX1 abolished T3-induced intracellular ROS generation and PCNA and SM α-actin expression, indicating that TRα1 and NOX1 mediated T3-induced RASM cell proliferation. Notably, RNAi silence of TRα1 blocked the T3-induced increase in NOX1 expression, whereas silence of NOX1 did not affect TRα1 expression, disclosing a new pathway, i.e. T3-TRα1-NOX1-cell proliferation. TRα1 and NOX1 co-localized around the nucleus. T3 induced RASM cell proliferation by up-regulating NOX1 in a TRα1-dependent manner.

AP-1 Inhibits Expression of MMP-2/9 and Its Effects on Rat Smooth Muscle Cells

We designed AP-1 small interfering RNA (siRNA) to study the relationship between AP-1 expression and matrix metalloproteinase (MMP)-2 activation. To suppress the expression of AP-1 gene, we used RNA interference to silence the AP-1 gene post-transcriptionally in rat smooth muscle cells. Effects of AP-1 siRNA on mRNA expression of AP-1 were examined using reverse transcriptase polymerase chain reaction (RT-PCR). Phosphorylation levels of c-Jun, c-Fos, and the activity of AP-1 were determined by Western blotting and AP-1 DNA-binding activity assay. To observe the expression of SM alpha-actin and downstream genes of AP-1, we simultaneously examined the activity of cell matrix metal proteinases and the migration ability of smooth muscle cells using a modified Boyden-chamber assay. Effects of AP-1 siRNA on rat vascular smooth muscle cells (VSMC) in vitro were evaluated using cell cycle analysis, MTT-tests, BrdU-ELISA and immunofluorescence. AP-1 siRNA decreased not only AP-1 mRNA and AP-1 binding activity, but also c-Jun and c-Fos phosphorylation levels and the expression levels of urokinase-type plasminogen (u-PA), MMPs, TGF-beta1 and bFGF in VSMCs. The AP-1 siRNA also significantly inhibited PDGF/IL-1-induced MMP-2 and MMP-9 gelatinolytic activity in VSMCs. The invasion and migration of VSMC were also induced greatly. AP-1 inhibited expression of MMP-2/9 and AP-1 siRNA was able to effectively inhibit the proliferation, migration and de-differentiation of rat smooth muscle cells.

Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells

Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM α-actin and SM22α are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside–outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC—the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.

Adenovirus-mediated intra-arterial delivery of cellular repressor of E1A-stimulated genes inhibits neointima formation in rabbits after balloon injury

This study examined the effect on neointimal hyperplasia of adenovirus-mediated delivery of cellular repressor of E1A-stimulated genes (CREG) to the artery after balloon injury. Sixty rabbits were randomized into three groups and underwent balloon injury in the left common carotid arteries. The injured arterial segment was isolated by two inflated balloon catheters. Saline or recombinant adenovirus expressing CREG or green fluorescent protein was injected into the lumen of the isolated arterial segments and incubated for 30 minutes. The rabbits were euthanized for immunohistochemistry, Western blotting, and morphometric analysis at 3, 7, 14, and 28 days after balloon injury and in vivo gene transfer (5 rabbits for each time point). Common carotid artery angiography was performed before euthanasia. Immunohistochemistry and Western blot analysis demonstrated that CREG expression was significantly down-regulated in the acute phase of vascular injury and was gradually restored in the resolution phase. The changes of CREG expression were in parallel with those of the smooth muscle cell (SMC) differentiation markers SM alpha-actin and SM myosin heavy chain in the injured arteries. Adenovirus-mediated CREG transfer markedly increased CREG expression in the injured artery. Consequently, morphometric analysis revealed an approximate 50% reduction in the neointima and the intima/media ratio in CREG-transferred arteries compared with the saline and green fluorescent protein controls. Assay with 5-bromo-2-deoxyuridine showed that CREG transfer significantly inhibited SMC proliferation. In contrast, endothelialization of the injured artery was not affected by CREG transduction as assessed by CD31 immunostaining. These data suggest that forced expression of CREG in the artery wall after acute vascular injury inhibits SMC proliferation, induces cellular differentiation, and attenuates neointimal hyperplasia. CREG delivery may have therapeutic potential for the prevention of restenosis after vascular angioplasty.

Human Adipose Stem Cells: A Potential Cell Source for Cardiovascular Tissue Engineering

Background/Aims: A crucial step in providing clinically relevant applications of cardiovascular tissue engineering involves the identification of a suitable cell source. The objective of this study was to identify the exogenous and endogenous parameters that are critical for the differentiation of human adipose stem cells (hASCs) into cardiovascular cells. Methods: hASCs were isolated from human lipoaspirate samples, analyzed, and subjected to two differentiation protocols. Results: As shown by fluorescence-activated cell sorter (FACS) analysis, a population of hASCs expressed stem cell markers including CXCR4, CD34, c-kit, and ABCG2. Further, FACS and immunofluorescence analysis of hASCs, cultured for 2 weeks in DMEM-20%-FBS, showed the expression of smooth muscle cell (SMC)-specific markers including SM α-actin, basic calponin, h-caldesmon and SM myosin. hASCs, cultured for 2 weeks in endothelial cell growth medium-2 (EGM-2), formed a network of branched tube-like structures positive for CD31, CD144, and von Willebrand factor. The frequency of endothelial cell (EC) marker-expressing cells was passage number-dependent. Moreover, hASCs attached and formed a confluent layer on top of electrospun collagen-elastin scaffolds. Scanning electron microscopy and DAPI staining confirmed the integration of hASCs with the fibers and formation of a cell-matrix network. Conclusion: Our results indicate that hASCs are a potential cell source for cardiovascular tissue engineering; however, the differentiation capacity of hASCs into SMCs and ECs is passage number- and culture condition-dependent.

The Histone Demethylase, Jmjd1a, Interacts With the Myocardin Factors to Regulate SMC Differentiation Marker Gene Expression

We and others have previously shown that the myocardin transcription factors play critical roles in the regulation of smooth muscle cell (SMC) differentiation marker gene expression. In a yeast 2-hybrid screen for proteins that interact with myocardin-related transcription factor-A (MRTF-A), we identified the histone 3 lysine 9 (H3K9)-specific demethylase, Jmjd1a. GST pull-down assays demonstrated that Jmjd1a bound all 3 myocardin family members, and further mapping studies showed that the jumonjiC domain of Jmjd1a was sufficient to mediate this interaction. Overexpression of Jmjd1a in multipotential 10T1/2 cells decreased global levels of di-methyl H3K9, stimulated the SM alpha-actin and SM22 promoters, and synergistically enhanced MRTF-A- and myocardin-dependent transactivation. Using chromatin immunoprecipitation assays, we also demonstrated that TGF-beta-mediated upregulation of SMC differentiation marker gene expression in 10T1/2 cells was associated with decreased H3K9 dimethylation at the CArG-containing regions of the SMC differentiation marker gene promoters. Importantly, knockdown of Jmjd1a in 10T1/2 cells and primary rat aortic SMCs by retroviral delivery of siRNA attenuated TGF-beta-induced upregulation of endogenous SM myosin heavy chain expression. These effects were concomitant with increased H3K9 dimethylation at the SMC differentiation marker gene promoters and with inhibition of MRTF-A-dependent transactivation of the SMC-specific transcription. These results suggest, for the first time, that SMC differentiation marker gene expression is regulated by H3K9 methylation and that the effects of the myocardin factors on SMC-specific transcription may involve the recruitment of Jmjd1a to the SMC-specific promoters.

A Novel Role of Brg1 in the Regulation of SRF/MRTFA-dependent Smooth Muscle-specific Gene Expression

Serum response factor (SRF) is a key regulator of smooth muscle differentiation, proliferation, and migration. Myocardin-related transcription factor A (MRTFA) is a co-activator of SRF that can induce expression of SRF-dependent, smooth muscle-specific genes and actin/Rho-dependent genes, but not MAPK-regulated growth response genes. How MRTFA and SRF discriminate between these sets of target genes is still unclear. We hypothesized that SWI/SNF ATP-dependent chromatin remodeling complexes, containing Brahma-related gene 1 (Brg1) or Brahma (Brm), may play a role in this process. Results from Western blotting and qRT-PCR analysis demonstrated that dominant negative Brg1 blocked the ability of MRTFA to induce expression of smooth muscle-specific genes, but not actin/Rho-dependent early response genes, in fibroblasts. In addition, dominant negative Brg1 attenuated expression of smooth muscle-specific genes in primary cultures of smooth muscle cells. MRTFA overexpression did not induce expression of smooth muscle-specific genes in SW13 cells, which lack endogenous Brg1 or Brm. Reintroduction of Brg1 or Brm into SW13 cells restored their responsiveness to MRTFA. Immunoprecipitation assays revealed that Brg1, SRF, and MRTFA form a complex in vivo, and Brg1 directly binds MRTFA, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated the ability of MRTFA to increase SRF binding to the promoters of smooth muscle-specific genes, but not early response genes. Together these data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in regulating expression of SRF/MRTFA-dependent smooth muscle-specific genes but not SRF/MRTFA-dependent early response genes.

Diaphanous 1 and 2 Regulate Smooth Muscle Cell Differentiation by Activating the Myocardin-Related Transcription Factors

We have previously shown that smooth muscle cell (SMC) differentiation marker gene expression is regulated by the small GTPase, RhoA. The objective of the present study was to determine the contributions of the RhoA effectors, diaphanous 1 and 2 (mDia1 and mDia2), to this regulatory mechanism. mDia1 and mDia2 are expressed highly in aortic SMCs and in a number of SMC-containing organs including bladder, lung, and esophagus. Activation of mDia1/2 signaling by RhoA strongly stimulated SMC-specific promoter activity in multiple cell-types including primary aortic SMCs, and stimulated endogenous SM alpha-actin expression in 10T1/2 cells. Expression of a dominant negative Dia1 variant that inhibits both mDia1 and mDia2 significantly decreased SMC-specific transcription in SMCs. The effects of mDia1 and mDia2 required the presence of SRF and the activity of the myocardin transcription factors and were dependent on changes in actin polymerization. Importantly, stimulation of mDia1/2 signaling synergistically enhanced the activities of the myocardin-related transcription factors, MRTF-A and MRTF-B, and this effect was attributable to increased nuclear localization of these factors. These results indicate that RhoA-dependent signaling through mDia1/2 and the MRTFs is important for SMC-specific gene expression in SMCs.

Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors

Novel tubular scaffolds of marine source collagen and PLGA fibers were fabricated by freeze drying and electrospinning processes for vascular grafts. The hybrid scaffolds, composed of a porous collagen matrix and a fibrous PLGA layer, had an average pore size of 150±50 μm. The electrospun fibrous PLGA layer on the surface of a porous tubular collagen scaffold improved the mechanical strength of the collagen scaffolds in both the dry and wet states. Smooth muscle cells (SMCs)- and endothelial cells (ECs)-cultured collagen/PLGA scaffolds exhibited mechanical properties similar to collagen/PLGA scaffolds unseeded with cells, even after culturing for 23 days. The effect of a mechanical stimulation on the proliferation and phenotype of SMCs and ECs, cultured on collagen/PLGA scaffolds, was evaluated. The pulsatile perfusion system enhanced the SMCs and ECs proliferation. In addition, a significant cell alignment in a direction radial to the distending direction was observed in tissues exposed to radial distention, which is similar to the phenomenon of native vessel tissues in vivo. On the other hand, cells in tissues engineered in the static condition were randomly aligned. Immunochemical analyses showed that the expressions of SM α-actin, SM myosin heavy chain, EC von Willebrand factor, and EC nitric oxide were upregulated in tissues engineered under a mechano-active condition, compared to vessel tissues engineered in the static condition. These results indicated that the co-culturing of SMCs and ECs, using collagen/PLGA hybrid scaffolds under a pulsatile perfusion system, leads to the enhancement of vascular EC development, as well as the retention of the differentiated cell phenotype.

Nox4 Is Required for Maintenance of the Differentiated Vascular Smooth Muscle Cell Phenotype

The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process. Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle alpha-actin (SM alpha-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM alpha-actin-based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-alpha actin, SM-MHC, and calponin, as well as SM-alpha actin stress fibers. Nox1 depletion did not decrease these parameters. Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.

Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22alpha expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (DeltaB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM alpha-actin, SM22alpha, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-beta on SMC-specific transcription. Our data confirmed the importance of the NH(2)-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM alpha-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

Excitation–Transcription Coupling in Arterial Smooth Muscle

The primary function of the vascular smooth muscle cell (SMC) is contraction for which SMCs express a selective repertoire of genes (eg, SM alpha-actin, SM myosin heavy chain [SMMHC], myocardin) that ultimately define the SMC from other muscle cell types. Moreover, the SMC exhibits extensive phenotypic diversity and plasticity, which play an important role during normal development, repair of vascular injury, and in vascular disease states. Diverse signals modulate ion channel activity in the sarcolemma of SMCs, resulting in altered intracellular calcium (Ca) signaling, activation of multiple intracellular signaling cascades, and SMC contraction or relaxation, a process known as "excitation-contraction coupling" (EC-coupling). Over the past 5 years, exciting new studies have shown that the same signals that regulate EC-coupling in SMCs are also capable of regulating SMC-selective gene expression programs, a new paradigm coined "excitation-transcription coupling" (ET-coupling). This article reviews recent progress in our understanding of the mechanisms by which ET-coupling selectively coordinates the expression of distinct gene subsets in SMCs by disparate transcription factors, including CREB, NFAT, and myocardin, via selective kinases. For example, L-type voltage-gated Ca2+ channels modulate SMC differentiation marker gene expression, eg, SM alpha-actin and SMMHC, via Rho kinase and myocardin and also regulate c-fos gene expression independently via CaMK. In addition, we discuss the potential role of IK channels and TRPC in ET-coupling as potential mediators of SMC phenotypic modulation, ie, negatively regulate SMC differentiation marker genes, in vascular disease.

Increased Neointima Formation in Cysteine-Rich Protein 2–Deficient Mice in Response to Vascular Injury

In response to arterial injury, medial vascular smooth muscle cells (VSMCs) proliferate and migrate into the intima, contributing to the development of occlusive vascular disease. The LIM protein cysteine-rich protein (CRP) 2 associates with the actin cytoskeleton and may maintain the cytoarchitecture. CRP2 also interacts with transcription factors in the nucleus to mediate SMC gene expression. To test the hypothesis that CRP2 may be an important regulator of vascular development or function we generated Csrp2 (gene symbol of the mouse CRP2 gene)-deficient (Csrp2(-/-)) mice by targeted mutation. Csrp2(-/-) mice did not have any gross vascular defects or altered expression levels of SM alpha-actin, SM22alpha, or calponin. Following femoral artery injury, CRP2 expression persisted in the vessel wall at 4 days and then decreased by 14 days. Intimal thickening was enhanced 3.4-fold in Csrp2(-/-) compared with wild-type (WT) mice 14 days following injury. Cellular proliferation was similar between WT and Csrp2(-/-) VSMC both in vivo and in vitro. Interestingly, Csrp2(-/-) VSMC migrated more rapidly in response to PDGF-BB and had increased Rac1 activation. Our data demonstrate that CRP2 is not required for vascular development. However, an absence of CRP2 enhanced VSMC migration and increased neointima formation following arterial injury.

NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-β1–Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts

Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (SM alpha-actin) de novo and produce extracellular matrix. We hypothesized that TGF-beta1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-beta1 potently upregulates the contractile marker SM alpha-actin mRNA (7.5+/-0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-beta1, Nox4 mRNA is dramatically upregulated by 16.2+/-0.8-fold (n=3, P<0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80+/-5%, inhibits NADPH-driven superoxide production in response to TGF-beta1 by 65+/-7%, and reduces TGF-beta1-induced expression of SM alpha-actin by 95+/-2% (n=6, P<0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-beta1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-beta1 stimulation of Smad 2/3 phosphorylation by 75+/-5% and 68+/-3%, respectively (n=7, P<0.0001). We conclude that Nox 4 mediates TGF-beta1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure.

NAD(P)H Oxidases and TGF-β–Induced Cardiac Fibroblast Differentiation

See related article, pages 900–907 Under normal conditions, cardiac fibroblasts play a pivotal role in the maintenance of the structural integrity of the myocardium by regulating extracellular matrix (ECM) production. In response to myocardial infarction and pressure overload, paracrine and autocrine signals in the interstitial milieu stimulate fibroblast proliferation and differentiation into myofibroblasts. The phenotype of these cells is characterized by an upregulation of smooth muscle α-actin (SM α-actin), collagen gel contraction in vitro, increased proliferation, decreased secretion of matrix degrading enzymes (matrix metalloproteinases), and enhanced production of extracellular matrix proteins, plasminogen activator inhibitor-1, and tissue inhibitors of matrix metalloproteineases. Thus, fibroblast transformation into myofibroblasts is associated with enhanced matrix production and decreased matrix turnover, resulting in interstitial fibrosis. This adverse ECM structural remodeling facilitates abnormalities of both systolic and diastolic cardiac function, because fibrosis leads to increase in chamber stiffness.1 The renin–angiotensin system and transforming growth factor-β1 (TGF-β1) play a critical role in the development of cardiac fibrosis. Angiotensin II (Ang II)–dependent upregulation of TGF-β1 expression in cardiac fibroblasts is absolutely required for Ang II–induced fibrosis.2 TGF-β1 induces the proliferation of cardiac fibroblasts, their phenotypic conversion to myofibroblasts, deposition of ECM proteins such as collagen, fibronectin, and proteoglycans, and hypertrophic growth of cardiomyocytes, thereby mediating Ang II–induced structural remodeling of the ventricular wall in an autocrine/paracrine manner.2 The profibrotic effects of TGF-β1 are primarily attributable to the differentiation of fibroblasts to myofibroblasts, because acute exposure (<24 hours) of primary rat cardiac fibroblasts fails to increase collagen production.3 On the other hand, chronic exposure to TGF-β1 caused maximal collagen production that was accompanied by an increase in SM α-actin and the phenotypic conversion of fibroblasts to myofibroblasts. The signaling cascades that control cardiac fibroblast differentiation into myofibroblasts have …

PIAS1 Activates the Expression of Smooth Muscle Cell Differentiation Marker Genes by Interacting with Serum Response Factor and Class I Basic Helix-Loop-Helix Proteins

Although a critical component of vascular disease is modulation of the differentiated state of vascular smooth muscle cells (SMC), the mechanisms governing SMC differentiation are relatively poorly understood. We have previously shown that E-boxes and the ubiquitously expressed class I basic helix-loop-helix (bHLH) proteins, including E2-2 and E12, are important in regulation of the SMC differentiation marker gene, the SM alpha-actin gene. The aim of the present study was to identify proteins that bind to class I bHLH proteins in SMC and modulate transcriptional regulation of SMC differentiation marker genes. Herein we report that members of the protein inhibitor of activated STAT (PIAS) family interact with class I bHLH factors as well as serum response factor (SRF). PIAS1 interacted with E2-2 and E12 based on yeast two-hybrid screens, mammalian two-hybrid assays, and/or coimmunoprecipitation assays. Overexpression of PIAS1 significantly activated the SM alpha-actin promoter and mRNA expression, as well as SM myosin heavy chain and SM22alpha, whereas a small interfering RNA for PIAS1 decreased activity of these promoters, as well as endogenous mRNA expression, and SRF binding to SM alpha-actin promoter within intact chromatin in cultured SMC. Of significance, PIAS1 bound to SRF and activated SM alpha-actin promoter expression in wild-type but not SRF(-/-) embryonic stem cells. These results provide novel evidence that PIAS1 modulates transcriptional activation of SMC marker genes through cooperative interactions with both SRF and class I bHLH proteins.