Smooth Muscle Cell Contractile Genes Research Articles

Abstract 1054: Toll-like Receptor 4 Is A New Regulator Of Smooth Muscle Cell Phenotypic Transitions

Objectives: There is growing evidence that smooth muscle cell ( SMC ) phenotypic transitions have a critical role in cardiovascular pathologies, including atherosclerosis. However, molecular mechanisms regulating different aspects of these transitions are still not fully elucidated. We previously found that the pluripotency factor OCT4 plays a protective role in SMC in atherogenesis and neointioma formation after vascular injury. Using a bioinformatics approach, we identified Toll-like receptor 4 ( TLR4 ), an integral component of the innate immune system, pivotal in eliciting inflammatory responses, as an indirect target of OCT4 in SMC. However, it is not yet elucidated whether OCT4/TLR4 axis has a role in SMC transitions. Methods and Results: First, we used blocking siRNA against Oct4 and different downstream targets of the TLR4 signaling ( Trif , Irf3 ), as well as a TLR4 inhibitor TAK242 (Resatorvid) in cultured mouse aortic SMC treated with the oxidized phospholipid, POVPC. These experiments demonstrated that blocking TLR4 signaling in wild-type SMC resulted in suppression of SMC contractile genes and up-regulation of LGALS3 (SMC transition marker). In contrast, TAK242 treatment decreased LGALS3 levels in OCT4 deficient SMC, indicating that TLR4, at least in part, is responsible for the SMC phenotype in OCT4 deficient SMC. Next, using the bioinformatics approach and Chromatin Immunoprecipitation assays, we identified the EST Proto-oncogene 1 ( ETS1 ) transcriptional factor as a target of OCT4 that directly regulates TLR4. Interestingly, on the other hand, we found that overexpression of TLR4 led to OCT4 activation in SMC, and, oppositely, the inhibition of TLR4 signaling by TAK242 resulted in significant decreases in the POVPC-induced OCT4 activation, indicating that TLR4 signaling is critical for OCT4 activation in SMC. Using an SMC-lineage tracing mouse model combined with a TLR4-IRES-YFP reporter, we demonstrated that TLR4 is highly expressed in the healthy arteries of young mice but not in the phenotypically modulated SMC within the atherosclerotic plaques. Conclusions: These results suggest a negative feedback loop between TLR4 and OCT4 regulating SMC phenotypic transitions.

Smooth muscle cells-specific loss of OCT4 accelerates neointima formation after acute vascular injury.

There is growing evidence that smooth muscle cell (SMC) phenotypic transitions play critical roles during normal developmental and tissue recovery processes and in pathological conditions such as atherosclerosis. However, the molecular mechanisms responsible for these transitions are not well understood. Recently, we found that the embryonic stem cell/induced pluripotent stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, and regulates angiogenesis after corneal alkali burn and hindlimb ischemia by mediating microvascular SMC and pericyte migration. However, the kinetics of OCT4 activation in arterial SMC and its role in acute pathological conditions are still unknown. Here, using an Oct4-IRES-GFP reporter mouse model, we found that OCT4 is reactivated in the carotid artery 18 hours post-acute ligation-induced injury, a common in vivo model of the SMC phenotypic transitions. Next, using a tamoxifen-inducible Myh11-CreERT2 Oct4 knockout mouse model, we found that the loss of OCT4, specifically in SMC, led to accelerated neointima formation and increased tunica media following carotid artery ligation, at least in part by increasing SMC proliferation within the media. Bulk RNA sequencing analysis on the cultured SMC revealed significant down-regulation of the SMC contractile markers and dysregulation of the genes belonging to the regulation of cell proliferation and, positive and negative regulation for cell migration ontological groups following genetic inactivation of Oct4. We also found that loss of Oct4 resulted in suppression of contractile SMC markers after the injury and in cultured aortic SMC. Further mechanistic studies revealed that OCT4 regulates SMC contractile genes, ACTA2 and TAGLN, at least in part by direct binding to the promoters of these genes. These results demonstrate that the pluripotency factor OCT4 is quickly activated in SMC after the acute vascular injury and inhibits SMC hyperproliferation, which may be protective in preventing excessive neointima formation.

Long noncoding RNA TUG1 regulates smooth muscle cell differentiation via KLF4-myocardin axis.

Abdominal aortic aneurysms (AAAs) are asymptomatic vascular diseases that have life-threatening outcomes. Smooth muscle cell (SMC) dysfunction plays an important role in AAA development. The contribution of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction, is relatively unexplored. We investigated the role of lncRNA TUG1 in SMC dysfunction. To identify potential lncRNAs relevant to SMC functionality, lncRNA profiling was performed in angiotensin-II-treated SMCs. AAA was induced by angiotensin-II treatment in mice. Transcriptional regulation of TUG1 was studied using promoter luciferase and chromatin-immuno-precipitation experiments. Gain-or-loss-of-function experiments were performed in vitro to investigate TUG1-mediated regulation of SMC function. Immunoprecipitation experiments were conducted to elucidate the mechanism underlying TUG1-mediated SMC dysfunction. TUG1 was upregulated in SMCs following angiotensin-II treatment. Similarly, TUG1 levels were elevated in abdominal aorta in a mouse model of angiotensin-II-induced AAA. Further investigations showed that angiotensin-II-induced TUG1 expression could be suppressed by inhibiting Notch-signaling pathway, both in vitro and in mouse AAA model and that TUG1 is a direct transcriptional target of the Notch pathway. In aneurysmal tissues, TUG1 expression was inversely correlated with the expression of SMC contractile genes. Overexpression of TUG1 repressed SMC differentiation in vitro, whereas siRNA/shRNA-mediated TUG1 knockdown showed an opposite effect. Mechanistically, TUG1 interacts with transcriptional repressor KLF4 and facilitates its recruitment to myocardin promoter ultimately leading to the repression of SMC differentiation. In summary, our study uncovers a novel role for the lncRNA TUG1 wherein it modulates SMC differentiation via the KLF4-myocardin axis, which may have potential implications in AAA development.NEW & NOTEWORTHY TUG1 is an angiotensin-II-induced long noncoding RNA that mediates smooth muscle cell (SMC) dysfunction through interaction with transcriptional repressor KLF4.

Drug-eluting stent specifically designed to target vascular smooth muscle cell phenotypic modulation attenuated restenosis through the YAP pathway

Vascular smooth muscle cell (SMC) phenotypic modulation contributes to the development of restenosis. A sorafenib-eluting stent was specifically designed to target SMC phenotypic modulation to inhibit in-stent restenosis in the present study. SMC contractile protein from the freshly isolated rat aorta was expressed at a high level, but its expression was dramatically reduced after SMCs were cultured in 10% FBS for 1 wk. After sorafenib treatment, SMC contractile protein expression was markedly upregulated. We further observed that Yes-associated protein (YAP) expression was attenuated after sorafenib treatment in a dose-dependent manner. Overexpression of YAP by lentivirus reversed the expression of sorafenib-induced SMC contractile protein and increased the expression of cyclin D. Mechanistically, sorafenib regulated the serum response factor-myocardin (SRF-Myocd) complex through competitive binding of YAP to Myocd and increased SRF binding to CArG-containing regions of SMC-specific contractile genes within intact chromatin, thereby controlling the activity of smooth muscle-specific gene transcription. In a rabbit carotid model, the sorafenib-eluting stent (SFES) dramatically inhibited in-stent restenosis and upregulated SMC contractile protein expression. Overexpression of YAP blocked the antirestenosis effect of SFES and repressed contractile smooth muscle-specific genes in vivo, indicating that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. We demonstrated that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. Targeting SMC phenotypic modulation by drug-eluting stent represents an attractive therapeutic approach for the treatment of occlusive vascular diseases.NEW & NOTEWORTHY In the present study, we demonstrated that sorafenib regulates smooth muscle cell (SMC) phenotypic modulation from a proliferative to a contractile state. Sorafenib induced a myocardin-serum response factor interaction and increased SMC contractile gene transcription through the Yes-associated protein pathway. Moreover, local delivery of sorafenib regulating SMC phenotypic modulation represents a promising strategy in the design of drug-eluting stents.

GRAF3 serves as a blood volume-sensitive rheostat to control smooth muscle contractility and blood pressure

ABSTRACTVascular resistance is a major determinant of BP and is controlled, in large part, by RhoA-dependent smooth muscle cell (SMC) contraction within small peripheral arterioles and previous studies from our lab indicate that GRAF3 is a critical regulator of RhoA in vascular SMC. The elevated contractile responses we observed in GRAF3 deficient vessels coupled with the hypertensive phenotype provided a mechanistic link for the hypertensive locus recently identified within the GRAF3 gene. On the basis of our previous findings that the RhoA signaling axis also controls SMC contractile gene expression and that GRAF3 expression was itself controlled by this pathway, we postulated that GRAF3 serves as an important counter-regulator of SMC phenotype. Indeed, our new findings presented herein indicate that GRAF3 expression acts as a pressure-sensitive rheostat to control vessel tone by both reducing calcium sensitivity and restraining expression of the SMC-specific contractile proteins that support this function. Collectively, these studies highlight the potential therapeutic value of GRAF3 in the control of human hypertension.

ADAR1-Mediated RNA Editing, A Novel Mechanism Controlling Phenotypic Modulation of Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (SMC) phenotypic modulation is characterized by the downregulation of SMC contractile genes. Platelet-derived growth factor-BB, a well-known stimulator of SMC phenotypic modulation, downregulates SMC genes via posttranscriptional regulation. The underlying mechanisms, however, remain largely unknown. To establish RNA editing as a novel mechanism controlling SMC phenotypic modulation. Precursor mRNAs (pre-mRNA) of SMC myosin heavy chain and smooth muscle α-actin were accumulated while their mature mRNAs were downregulated during SMC phenotypic modulation, suggesting an abnormal splicing of the pre-mRNAs. The abnormal splicing resulted from SMC marker pre-mRNA editing that was facilitated by adenosine deaminase acting on RNA 1 (ADAR1), an enzyme converting adenosines to inosines (A→I editing) in RNA sequences. ADAR1 expression inversely correlated with SMC myosin heavy chain and smooth muscle α-actin levels; knockdown of ADAR1 restored SMC myosin heavy chain and smooth muscle α-actin expression in phenotypically modulated SMC, and editase domain mutation diminished the ADAR1-mediated abnormal splicing of SMC marker pre-mRNAs. Moreover, the abnormal splicing/editing of SMC myosin heavy chain and smooth muscle α-actin pre-mRNAs occurred during injury-induced vascular remodeling. Importantly, heterozygous knockout of ADAR1 dramatically inhibited injury-induced neointima formation and restored SMC marker expression, demonstrating a critical role of ADAR1 in SMC phenotypic modulation and vascular remodeling in vivo. Our results unraveled a novel molecular mechanism, that is, pre-mRNA editing, governing SMC phenotypic modulation.

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN–SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Abstract 18897: Extracellular Signal-related Kinase Signaling and Smooth Muscle Cell Contractile Gene Expression are Uniformly Enhanced in the Aortic Root and Ascending Aorta in Marfan Syndrome

Introduction: Enhanced TGF-β signaling drives aortic root aneurysm development in Marfan syndrome (MFS) via the non-canonical (ERK) pathway in murine models, but ERK not been studied in human tissue. Conversely, blockade of canonical (Smad) TGF-β signaling is detrimental in MFS mice. Recently, TGF-β-induced increases in smooth muscle cell (SMC) contractile genes and collagen I (COL1) expression have been proposed to lead to aortic stiffness and dilatation in MFS. Hypothesis: We hypothesized that ERK signaling is enhanced in human MFS tissue and dictates the predilection of the aortic root to develop aneurysm by altering SMC gene expression. Methods and Results: Western blot of aortic root tissue from MFS patients with root aneurysms (4.67±0.09 cm) revealed 3.1-fold increased ERK activation relative to control aortic root (p=0.03, n=8). When compared to non-dilated MFS ascending (ASC) aorta (2.94±0.14 cm), there was no difference in ERK activation between the two regions. While RT-PCR of MFS root showed increased expression of the contractile gene regulator myocardin (2.4±0.07 fold, p=0.01), and of MYH11 (3.05±0.13 fold, p=0.03) ACTA2 (2.93±0.10 fold, p<0.01), and SM22-α (1.88±0.13 fold, p=0.02) versus controls, these genes were not differentially expressed between MFS root and ASC. COL1 expression was increased in MFS root relative to controls (5.57±0.06 fold, p<0.01) and MFS ASC (2.25±0.06 p<0.01). In vitro treatment of cultured MFS aortic root SMCs (n=5 patients) with TGF-β and Smad-specific inhibitor (SB431542) failed to increase expression of MYOCD, MYH11, ACTA2, SM22-α or COL1, while co-treatment with TGF-β and ERK inhibitor (PD98059) induced significant increases in expression of these genes (5.4-, 2.6-, 2.7-, 1.4-, and 1.4- fold, respectively p<0.05). Conclusions: ERK signaling is uniformly enhanced in MFS aortic root and ASC tissue, indicating that further study into aortic region-specific responses to cellular signals is needed to assess the development of focal aortic root aneurysm in MFS. Increased contractile gene expression is Smad-dependent and non-focal and thus does not clarify focal root aneurysm. COL1, while focally enhanced in the MFS root, is Smad-driven and suppressed by ERK, and may represent a compensatory response.

TGF-β/Smad3 Stimulates Stem Cell/Developmental Gene Expression and Vascular Smooth Muscle Cell De-Differentiation

Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3∶1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p<0.05) in TGF-β/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-β/Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed up-regulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-β/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-β/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.

Measuring, reversing, and modeling the mechanical changes due to the absence of Fibulin-4 in mouse arteries

Mice with a smooth muscle cell (SMC)-specific deletion of Fibulin-4 (SMKO) show decreased expression of SMC contractile genes, decreased circumferential compliance, and develop aneurysms in the ascending aorta. Neonatal administration of drugs that inhibit the angiotensin II pathway encourages the expression of contractile genes and prevents aneurysm development, but does not increase compliance in SMKO aorta. We hypothesized that multidimensional mechanical changes in the aorta and/or other elastic arteries may contribute to aneurysm pathophysiology. We found that the SMKO ascending aorta and carotid artery showed mechanical changes in the axial direction. These changes were not reversed by angiotensin II inhibitors, hence reversing the axial changes is not required for aneurysm prevention. Mechanical changes in the circumferential direction were specific to the ascending aorta; therefore, mechanical changes in the carotid do not contribute to aortic aneurysm development. We also hypothesized that a published model of postnatal aortic growth and remodeling could be used to investigate mechanisms behind the changes in SMKO aorta and aneurysm development over time. Dimensions and mechanical behavior of adult SMKO aorta were reproduced by the model after modifying the initial component material constants and the aortic dilation with each postnatal time step. The model links biological observations to specific mechanical responses in aneurysm development and treatment.

Abstract W P365: Critical Role of Smooth Muscle Cell Phenotypic Modulation in Cerebral Aneurysm Formation and Rupture

Objective: Little is known about smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. TNF-α has been associated with aneurysms, but a direct role has not been established. Methods: Cultured cerebral SMC were treated with TNF-α for PCR, western blot, chromatin immune-precipitation (CHIP), and adenovirus promoter transfection. In vivo experiments were carried out in the following models: application of TNF-α to the surface of carotid arteries, cerebral model of hypertension and hemodynamic stress, and cerebral model of aneurysm formation and rupture. The TNF-α inhibitor 3,6’dithiothalidomide (DTH) was synthesized. Results: Cultured cerebral SMC over-expressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC), while dominant negative suppressed expression. TNF-α treatment inhibited this contractile phenotype and induced pro-inflammatory genes (MCP-1, MMPs, VCAM-1, IL-1β). TNF-α increased expression of KLF4 and KLF4 siRNA abrogated TNF-α induced phentotypic modulation. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to TNF-α and early in a model of cerebral hypertension and hemodynamic stress prior to cerebral aneurysm formation. Treatment with DTH reversed these pathological vessel wall alterations. TNF-α knock-out mice and DTH pre-treatment decreased the incidence of aneurysm formation and rupture. As compared with sham mice, TNF-α expression was not significantly different in TNF-α knock-out mice or those pre-treated with DTH, but was elevated in unruptured and ruptured aneurysms. Initiation of DTH 7 days after aneurysm induction did not alter aneurysm incidence, but resulted in stabilization and decreased rupture. CHIP assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4 dependent alterations in promoter regions of myocardin, SMC’s, and inflammatory genes. Conclusion: TNF-α induces phenotypic modulation of cerebral SMC through myocardin and KLF4 regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory phenotype. These data suggests a critical role of TNF-α in the formation and rupture of aneurysms.

Cigarette smoke modulates vascular smooth muscle phenotype: implications for carotid and cerebrovascular disease.

BackgroundThe role of smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation and pathogenesis of stroke has not been determined. Cigarette smoke is a major risk factor for atherosclerosis, but potential mechanisms are unclear, and its role in SMC phenotypic modulation has not been established.Methods and ResultsIn cultured cerebral vascular SMCs, exposure to cigarette smoke extract (CSE) resulted in decreased promoter activity and mRNA expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC) and the transcription factor myocardin in a dose-dependent manner. CSE also induced pro-inflammatory/matrix remodeling genes (MCP-1, MMPs, TNF-α, IL-1β, NF-κB). CSE increased expression of KLF4, a known regulator of SMC differentiation, and siKLF4 inhibited CSE induced suppression of SMC contractile genes and myocardin and activation of inflammatory genes. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to CSE. Chromatin immune-precipitation assays in vivo and in vitro demonstrated that CSE promotes epigenetic changes with binding of KLF4 to the promoter regions of myocardin and SMC marker genes and alterations in promoter acetylation and methylation.ConclusionCSE exposure results in phenotypic modulation of cerebral SMC through myocardin and KLF4 dependent mechanisms. These results provides a mechanism by which cigarette smoke induces a pro-inflammatory/matrix remodeling phenotype in SMC and an important pathway for cigarette smoke to contribute to atherosclerosis and stroke.

TNF-α Induces Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Pathology

Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-α) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-α activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-α and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-α inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-α induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.

Mitogen-Activated Protein Kinase 14 Is a Novel Negative Regulatory Switch for the Vascular Smooth Muscle Cell Contractile Gene Program

Several studies have shown through chemical inhibitors that p38 mitogen-activated protein kinase (MAPK) promotes vascular smooth muscle cell (VSMC) differentiation. Here, we evaluate the effects of knocking down a dominant p38MAPK isoform on VSMC differentiation. Knockdown of p38MAPKα (MAPK14) in human coronary artery SMCs unexpectedly increases VSMC differentiation genes, such as miR145, ACTA2, CNN1, LMOD1, and TAGLN, with little change in the expression of serum response factor (SRF) and 2 SRF cofactors, myocardin (MYOCD) and myocardin-related transcription factor A (MKL1). A variety of chemical and biological inhibitors demonstrate a critical role for a RhoA-MKL1-SRF-dependent pathway in mediating these effects. MAPK14 knockdown promotes MKL1 nuclear localization and VSMC marker expression, an effect partially reversed with Y27632; in contrast, MAP2K6 (MKK6) blocks MKL1 nuclear import and VSMC marker expression. Immunostaining and Western blotting of injured mouse carotid arteries reveal elevated MAPK14 (both total and phosphorylated) and reduced VSMC marker expression. Reduced MAPK14 expression evokes unanticipated increases in VSMC contractile genes, suggesting an unrecognized negative regulatory role for MAPK14 signaling in VSMC differentiation.

Abstract 18326: p38MAPK is a Novel Negative Regulator for Smooth Muscle Differentiation

p38MAPK belongs to members of the MAPK family which can be activated by various environmental stressors and inflammatory cytokines. While more and more studies have defined the critical roles of p38MAPK in different biological processes involving cell growth, differentiation and apoptosis, its actual contribution to smooth muscle cell (SMC) phenotypic modulation is largely unexplored. Using the mouse complete carotid artery ligation model, we found both total p38 and phosphorylated p38 to be predominantly located in the neointima of the vessel wall where SMCs undergo hyper-proliferation and SMC differentiation is compromised, suggesting a negative regulatory role of p38MAPK in SMC differentiation. To test this hypothesis, we applied siRNA studies in primary human coronary artery SMC (HCASM) cultures and examined SMC contractile gene expression. Depletion of p38MAPK evoked a dramatic increase in SMC contractile gene expression. The stimulatory effect of SMC contractile gene expression upon knockdown of p38MAPK was reproduced in a variety of SMC culture models including human aortic SMCs, rat aortic SMCs, PAC1 cells and primary human myofibroblasts. Consistent with the siRNA studies, cotransfecting p38 with its upstream kinase MKK3 or MMK6 abolished promoter activity of several SMC-restricted genes, while little effect was found with the dominant negative mutants of p38. Furthermore, adenovirus carrying the constitutively active MKK6 completely abrogated TGF-beta1 induced SMC differentiation as assessed by Western blotting and immunfluorescence mirocsopy, indicating that phosphorylated p38 is required for the repressive effect of p38MAPK in SMC differentiation. Interestingly, in HCASMs, we found myocardin-related transcription factor A (MRTFA) was mainly located in the nucleus when the endogenous p38MAPK was knocked down; conversely, over-expression of constitutively active MKK6 was found to shift the majority of MRTFA to the cytoplasm of HCASMs, suggesting that phosphorylated p38MAPK is able to block nuclear import of MRTFA in SMCs. Taken together, our studies revealed an unexpected role for p38MAPK in the negative regulation of SMC differentiation.

Transforming Growth Factor-β1 (TGF-β1) Utilizes Distinct Pathways for the Transcriptional Activation of MicroRNA 143/145 in Human Coronary Artery Smooth Muscle Cells

MicroRNA 143/145 (miR143/145) is restricted to adult smooth muscle cell (SMC) lineages and mediates, in part, the expression of several SMC contractile genes. Although the function of miR143/145 has begun to be elucidated, its transcriptional regulation in response to various signaling inputs is poorly understood. In an effort to define a miR signature for SMC differentiation, we screened human coronary artery SMCs for miRs modulated by TGF-β1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-β1-induced miRs, including miR143/145. Validation studies showed that TGF-β1 stimulated miR143/145 expression in a dose- and time-dependent manner. We utilized several chemical inhibitors and found that SB203580, a specific inhibitor of p38MAPK, significantly decreased TGF-β1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-β1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-β1 stimulated a 580-bp human miR143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD-binding element for full TGF-β1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-β1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an anti-miR145 partially blocked TGF-β1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-β1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-β1-induced human vascular SMC differentiation.

Fibulin-4 Deficiency Results in Ascending Aortic Aneurysms

Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4(GKO) and Fbln4(SMKO), respectively). Fbln4(GKO) and Fbln4(SMKO) aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4(GKO) and Fbln4(SMKO) mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4(GKO) SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.

“FRNKly, Smooth Muscle, I Don’t Give a CArG!”

Studies spanning the last two decades have begun to uncover the molecular mechanisms underlying smooth muscle cell (SMC) differentiation, specifically the critical role of the ubiquitously expressed serum response factor (SRF) transcription factor. SRF selectively binds to conserved cis-regulatory “CArG box” elements, CC(A/T)6GG consensus sequence, in the promoters or first introns of numerous smooth muscle-selective genes. The SMC contractile marker genes smooth muscle alpha actin (SMαA), smooth muscle myosin heavy chain (SM-MHC), and smooth muscle 22 alpha (SM22α) all require multiple CArG boxes for proper transcriptional activation. On SRF binding, the coactivator myocardin is recruited to the promoter through direct interaction with SRF and is required for expression of CArG containing SMC-selective genes.1,2 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Sayers et al detail the in vivo regulation of a SMC-selective CArG-independent gene, focal adhesion kinase related nonkinase (FRNK), that plays a critical role in CArG-dependent contractile gene expression during SMC differentiation and after vascular injury.3 See accompanying article on page 2115 Unlike other muscle cell types, adult SMCs exist in a highly plastic nonterminally differentiated state. SMCs can assume a proliferative and migratory synthetic phenotype, allowing for both complete investment of the endothelial tube during vascular development and repair of the vessel wall after arterial injury. Various environmental cues direct SMCs to phenotypically modulate into a highly …

Notch Signaling Represses Myocardin-induced Smooth Muscle Cell Differentiation

Notch signaling is essential for vascular patterning and response of the vasculature to injury and growth factor stimulation. Despite these findings, the molecular basis of Notch signaling in the vasculature is poorly understood. Here we report that activation of Notch signaling mediated through members of the HRT family of basic helix-loop-helix transcription factors represses smooth muscle cell (SMC) differentiation and expression of genes encoding smooth muscle cell contractile markers. Activation of Notch receptors by Jagged1 or forced expression of the constitutively active Notch1 intracellular domain in C3H10T1/2 fibroblasts inhibited myocardin-dependent transcription of SMC-restricted genes and activity of multiple SMC-restricted transcriptional regulatory elements. Consistent with these findings, forced expression of HRT2 inhibited myocardin-induced expression of SMC-restricted genes and activity of SMC-restricted transcriptional regulatory elements. Moreover, forced expression of HRT2 repressed transcription of multiple SMC-restricted transcriptional regulatory elements in A10 SMCs. The repressive function of HRT2 was not mediated via the capacity of HRT2 to bind SMC CArG elements or by disruption of myocardin-SRF protein complexes. Structure-function analyses of HRT2 indicated that repression required the basic DNA binding domain and additional C-terminal sequence. Taken together, these results demonstrate that Notch signaling represses myocardin-dependent SMC transcription. These data are consistent with a model wherein Notch signaling represses SMC differentiation and maintenance of the contractile SMC phenotype.