Modulation Of Smooth Muscle Cells Research Articles

Contribution of p62/SQSTM1 to PDGF‐BB‐induced myofibroblast‐like phenotypic transition in vascular smooth muscle cells lacking Smpd1 gene

Accumulating evidence indicate a critical role of acid sphingomyelinase (gene symbol Smpd1), a lysosomal hydrolase metabolizes sphingomyelin into ceramide, in controlling autophagic flux and vascular smooth muscle cell (SMC) homeostasis in atherogenesis. However, it remains elusive whether or not acid sphingomyelinase and autophagy play a modulatory role in SMC transition towards myofibroblast phenotype and extracellular matrix remodeling. In primary cultured SMCs from mouse coronary arteries, we demonstrated that PDGF‐BB stimulation produced an augmented proliferation in Smpd1−/− SMCs compared to Smpd1+/+ SMCs. More interestingly, PDGF‐BB induced more pronounced upregulation of fibroblast specific protein (FSP‐1), deposition of collagen type I, and expression of TGFβ1 in SMCs when Smpd1 is deleted. The increases in fibroblast markers in Smpd1−/− SMCs by PDGF‐BB were also accompanied by markedly elevated inflammatory status as shown by increased release of inflammatory cytokines (IL6 and IL18), expression of ICAM‐1, and monocyte adhesion. These data suggest that PDGF‐BB promotes a phenotypic transition in SMCs toward a myofibroblast‐like phenotype in the absence of acid sphingomyelinase function. Mechanistically, PDGF‐BB induced an exacerbated accumulation of autophagy substrate p62 in Smpd1−/− SMCs compared to Smpd1+/+ SMCs. In addition to impaired autophagic flux, autophagosome formation in Smpd1−/− SMCs was also suppressed by PDGF‐BB due to prolonged activation of Akt‐mTOR signaling, which further contributes to exacerbated p62 accumulation. Lastly, Akt inhibition or p62 gene silencing attenuated PDGF‐BB‐induced phenotypic changes observed in Smpd1−/− SMCs. Together, our data demonstrate, for the time, a p62‐dependent myofibroblast‐like phenotypic transition in Smpd1−/− SMCs, which indicates that acid sphingomyelinase‐regulated autophagy signaling plays a crucial role in maintaining the arterial smooth muscle homeostasis.Support or Funding InformationNational Heart, Lung, and Blood Institute grants (HL122769, HL122937) and the Young Scientists Fund of National Nature Science Foundation of China (No. 81202095).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic and diverse changes in the functional properties of vascular smooth muscle cells in pulmonary hypertension.

Pulmonary hypertension (PH) is the end result of interaction between pulmonary vascular tone and a complex series of cellular and molecular events termed 'vascular remodelling'. The remodelling process, which can involve the entirety of pulmonary arterial vasculature, almost universally involves medial thickening, driven by increased numbers and hypertrophy of its principal cellular constituent, smooth muscle cells (SMCs). It is noted, however that SMCs comprise heterogeneous populations of cells, which can exhibit markedly different proliferative, inflammatory, and extracellular matrix production changes during remodelling. We further consider that these functional changes in SMCs of different phenotype and their role in PH are dynamic and may undergo significant changes over time (which we will refer to as cellular plasticity); no single property can account for the complexity of the contribution of SMC to pulmonary vascular remodelling. Thus, the approaches used to pharmacologically manipulate PH by targeting the SMC phenotype(s) must take into account processes that underlie dominant phenotypes that drive the disease. We present evidence for time- and location-specific changes in SMC proliferation in various animal models of PH; we highlight the transient nature (rather than continuous) of SMC proliferation, emphasizing that the heterogenic SMC populations that reside in different locations along the pulmonary vascular tree exhibit distinct responses to the stresses associated with the development of PH. We also consider that cells that have often been termed 'SMCs' may arise from many origins, including endothelial cells, fibroblasts and resident or circulating progenitors, and thus may contribute via distinct signalling pathways to the remodelling process. Ultimately, PH is characterized by long-lived, apoptosis-resistant SMC. In line with this key pathogenic characteristic, we address the acquisition of a pro-inflammatory phenotype by SMC that is essential to the development of PH. We present evidence that metabolic alterations akin to those observed in cancer cells (cytoplasmic and mitochondrial) directly contribute to the phenotype of the SM and SM-like cells involved in PH. Finally, we raise the possibility that SMCs transition from a proliferative to a senescent, pro-inflammatory and metabolically active phenotype over time.

Uremia does not affect neointima formation in mice

Atherosclerotic cardiovascular disease is a major complication of chronic kidney disease (CKD). CKD leads to uremia, which modulates the phenotype of aortic smooth muscle cells (SMCs). Phenotypic modulation of SMCs plays a key role in accelerating atherosclerosis. We investigated the hypothesis that uremia potentiates neointima formation in response to vascular injury in mice. Carotid wire injury was performed on C57BL/6 wt and apolipoprotein E knockout (Apoe−/−) mice two weeks after induction of uremia by 5/6 nephrectomy. Wire injury led to neointima formation and downregulation of genes encoding classical SMC markers (i.e., myocardin, α-smooth muscle actin, SM22-alpha, and smooth muscle myosin heavy chain) in both wt and Apoe−/− mice. Contrary to our expectations, uremia did not potentiate neointima formation, nor did it affect intimal lesion composition as judged from magnetic resonance imaging and histological analyses. Also, there was no effect of uremia on SMC marker gene expression in the injured carotid arteries, suggesting that there may be different effects of uremia on SMCs in different vascular beds. In conclusion, uremia does not accelerate neointima formation in response to wire injury of the carotid artery in mice.

MicroRNA-182 prevents vascular smooth muscle cell dedifferentiation via FGF9/PDGFRβ signaling.

The abnormal phenotypic transformation of vascular smooth muscle cells (SMCs) causes various proliferative vascular diseases. MicroRNAs (miRNAs or miRs) have been established to play important roles in SMC biology and phenotypic modulation. This study revealed that the expression of miR-182 was markedly altered during rat vascular SMC phenotypic transformation in vitro. We aimed to investigate the role of miR-182 in the vascular SMC phenotypic switch and to determine the potential molecular mechanisms involved. The expression of miR-182 gene was significantly downregulated in cultured SMCs during dedifferentiation from a contractile to a synthetic phenotype. Conversely, the upregulation of miR-182 increased the expression of SMC-specific contractile genes, such as α-smooth muscle actin, smooth muscle 22α and calponin. Additionally, miR-182 overexpression potently inhibited SMC proliferation and migration under both basal conditions and under platelet-derived growth factor-BB stimulation. Furthermore, we identified fibroblast growth factor 9 (FGF9) as the target gene of miR-182 for the phenotypic modulation of SMCs mediated through platelet-derived growth factor receptor β (PDGFRβ) signaling. These data suggest that miR-182 may be a novel SMC phenotypic marker and a modulator that may be used to prevent SMC dedifferentiation via FGF9/PDGFRβ signaling.

Uremia modulates the phenotype of aortic smooth muscle cells

Background and aimsChronic kidney disease leads to uremia and markedly accelerates atherosclerosis. Phenotypic modulation of smooth muscle cells (SMCs) in the arterial media plays a key role in accelerating atherogenesis. The aim of this study was to investigate whether uremia per se modulates the phenotype of aortic SMCs in vivo. MethodsModerate uremia was induced by 5/6 nephrectomy in apolipoprotein E knockout (ApoE-/-) and wildtype C57Bl/6 mice. Plasma analysis, gene expression, histology, and myography were used to determine uremia-mediated changes in the arterial wall. ResultsInduction of moderate uremia in ApoE-/- mice increased atherosclerosis in the aortic arch en face 1.6 fold (p = 0.04) and induced systemic inflammation. Based on histological analyses of aortic root sections, uremia increased the medial area, while there was no difference in the content of elastic fibers or collagen in the aortic media. In the aortic arch, mRNA and miRNA expression patterns were consistent with a uremia-mediated phenotypic modulation of SMCs; e.g. downregulation of myocardin, α-smooth muscle actin, and transgelin; and upregulation of miR146a. Notably, these expression patterns were observed after acute (2 weeks) and chronic (19 and 30 weeks) uremia, both under normo- and hypercholesterolemic settings. Functionally, aortic constriction was decreased in uremic as compared to non-uremic aorta segments, as measured by myography. ConclusionsUremia modulates the phenotype of aortic SMCs as determined by mRNA/miRNA expression, an increased medial area, and decreased aortic contractility. We propose that this phenotypic modulation of SMCs precedes the acceleration of atherosclerosis observed in uremic mice.

Dicer generates a regulatory microRNA network in smooth muscle cells that limits neointima formation during vascular repair.

MicroRNAs (miRNAs) coordinate vascular repair by regulating injury-induced gene expression in vascular smooth muscle cells (SMCs) and promote the transition of SMCs from a contractile to a proliferating phenotype. However, the effect of miRNA expression in SMCs on neointima formation is unclear. Therefore, we studied the role of miRNA biogenesis by Dicer in SMCs in vascular repair. Following wire-induced injury to carotid arteries of Apolipoprotein E knockout (Apoe -/-) mice, miRNA microarray analysis revealed that the most significantly regulated miRNAs, such as miR-222 and miR-21-3p, were upregulated. Conditional deletion of Dicer in SMCs increased neointima formation by reducing SMC proliferation in Apoe -/- mice, and decreased mainly the expression of miRNAs, such as miR-147 and miR-100, which were not upregulated following vascular injury. SMC-specific deletion of Dicer promoted growth factor and inflammatory signaling and regulated a miRNA-target interaction network in injured arteries that was enriched in anti-proliferative miRNAs. The most connected miRNA in this network was miR-27a-3p [e.g., with Rho guanine nucleotide exchange factor 26 (ARHGEF26)], which was expressed in medial and neointimal SMCs in a Dicer-dependent manner. In vitro, miR-27a-3p suppresses ARHGEF26 expression and inhibits SMC proliferation by interacting with a conserved binding site in the 3' untranslated region of ARHGEF26 mRNA. We propose that Dicer expression in SMCs plays an essential role in vascular repair by generating anti-proliferative miRNAs, such as miR-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation.

Abstract 521: Janus Kinase 3 is a Novel Regulator for Smooth Muscle Proliferation and Vascularremodeling

Vascular remodeling due to smooth muscle cell (SMC) proliferation is a common process occurring in a number of vascular diseases such as atherosclerosis, aortic aneurysm, posttransplant vasculopathy, and restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completed understood. Our present study has identified Janus kinase 3 (JAK3), a member of the Janus kinase family, as a novel regulator for SMC proliferation. Platelet-derived growth factor (PDGF)-BB, a SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by shRNA inhibits SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Interestingly, JAK3 does not affect SMC contractile protein expression, suggesting that JAK3 mediate the proliferation, but not the phenotypic modulation of SMC. Mechanistically, JAK3 promotes SMC proliferation by regulating phosphorylation of Signal transducer and activator of transcription 3 and c-Jun N-terminal kinase. In vivo, by using a rat carotid balloon-injury model, we found that knockdown of JAK3 significantly attenuates injury-induced neointima formation. Importantly, JAK3 knockdown blocked the expression of proliferating cell nuclear antigen, suggesting that JAK3 is essential for SMC proliferation in vivo. Moreover, JAK3 knockdown prompts apoptosis in neointima SMC, indicating that JAK3 also stimulates SMC survival during neointima formation. Collectively, our data demonstrate that JAK3 mediates vascular remodeling by promoting both the SMC proliferation and survival.

Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells.

Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β-glycerophosphate with activated vitamin D3 , or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast-like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H-1,2-Dithiole-3-thione was able to inhibit the SMC transition into osteoblast-like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.

Identification and characterization of intimal smooth muscle cell with innate immune capacity in atherosclerosis

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The phenotype transition and modulation of SMC is involved in many atherosclerotic pathological process. To date, more and more evidences indicated that innate immune response play important roles in atherosclerosis. As the first line of host defense and innate immunity, pathogen recognition receptors (PPR) have been shown to be involved in many mechanistic pathways in atherosclerosis. To investigate the role of innate immunity in SMC phenotype modulation in atherosclerosis, we analyzed PPR expression profile between different phenotypes of SMC and immune cells. Our data at the first time identified the high level of PPR expression in intimal SMC and characterized the comprehensive innate immune signatures. With comparison to other phenotypes of SMC and immune cells, we found intimal SMC possess a pro-inflammatory macrophage-like phenotype. To investigate the functional features and regulatory mechanism underlying the distinct phenotype, we used specific ligands for PRRs activation and showed that the innate immune capacity acquired by intimal SMC contribute to chemokine and cytokine secretion, migration promotion and extracellular matrix remodelling. Last but not least, we found the macrophage-like phenotype in intimal SMC and the pro-inflammatory capacity is associated with the elevated level of key transcription factors STAT1a, IRF8, IRF9, KLF4 and HIF2a. In conclusion, we propose a critical role of intimal SMC with innate immune capacity in the development of atherosclerosis. Targeting the phenotypic modulation of intimal SMC might be a new strategy for further clinical treatment.

BRG1 overexpression in smooth muscle cells promotes the development of thoracic aortic dissection.

BackgroundHere we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).MethodsBRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and α-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.ResultsThe expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.ConclusionsBRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.

Smooth muscle cell phenotypic switch: implications for foam cell formation.

It is well accepted that LDLs and its modified form oxidized-LDL (ox-LDL) play a major role in the development of atherosclerosis and foam cell formation. Whereas the majority of these cells have been demonstrated to be derived from macrophages, smooth muscle cells (SMCs) give rise to a significant number of foam cells as well. During atherosclerotic plaque formation, SMCs switch from a contractile to a synthetic phenotype. The contribution of this process to foam cell formation is still not well understood. It has been confirmed that a large proportion of foam cells in human atherosclerotic plaques and in experimental intimal thickening arise from SMCs. SMC-derived foam cells express receptors involved in ox-LDL uptake and HDL reverse transport. In-vitro studies show that treatment of SMCs with ox-LDL induces typical foam-cell formation; this process is associated with a transition of SMCs toward a synthetic phenotype. This review summarizes data regarding the phenotypic switch of arterial SMCs within atherosclerotic lesion and their contribution to intimal foam cell formation.

Control of adipogenic transformation in vascular smooth muscle cells by the transglutaminase 2/b‐catenin signaling axis

In atherosclerosis, smooth muscle cells (SMCs) undergo a unique process known as “phenotype modulation,” transitioning from a quiescent, contractile phenotype to an adipocyte‐like state. Increased b‐catenin signaling has been observed in atherosclerotic plaques, and deficiency of transglutaminase 2 (TG2) – an endogenous activator of b‐catenin – reduces lipid accumulation in the mouse model of atherosclerosis. Here we examined whether inhibition of the TG2/b‐catenin signaling axis in SMCs can prevent their phenotypic transition. Adipogenesis was induced in aortic SMCs using a high‐lipid culture medium, and detected histologically by Oil Red‐O and on the molecular level by expression of adipogenic markers. Pronounced adipogenic differentiation was detected in 6 day‐cultures. Activation of both TG2 enzyme and b‐catenin signaling occurs 4 days earlier, suggesting that this signaling axis may mediate the transition. Inhibition of TG2 by genetic ablation or pharmacologically suppressed both lipid accumulation and b‐catenin activation in aortic SMCs. In addition, direct inhibition of b‐catenin signaling with siRNA for b‐catenin efficiently prevented SMC adipogenesis. Together our results support a key role for the TG2/b‐catenin signaling axis in the transition of SMCs to an adipogenic phenotype. This research was supported by NIH grants R01HL093305 and T32AR007592.

Smooth Muscle Cells: To Be or Not To Be?

The conversion of contractile vascular smooth muscle cells (SMCs) to a synthetic or proliferative phenotype is thought to play a major role in vascular diseases, such as atherosclerosis and restenosis.1–3 Our recent work presents evidence that challenges this widely accepted dogma. Our findings suggest that multipotent vascular stem cells (MVSCs) are a major contributor to vascular remodeling.4 The experimental results demonstrate that the major population of the traditionally defined proliferative/synthetic SMCs is derived from the differentiation of MVSCs rather than the dedifferentiation of mature SMCs. Both in vitro and in vivo results suggest that vascular disease is a stem cell disease, which raises the question on the previous dogma: Is vascular disease a SMC disease? In this issue of Circulation Research , a group of leaders in the area of SMC biology wrote a commentary on this work.5 They present evidence in the literature that seems to support the SMC dedifferentiation hypothesis. However, in many previous studies, it was incorrectly assumed that the vascular cells in the primary culture and in injured blood vessels were mostly derived from SMCs. Thus, the previous experimental findings on vascular cells were often attributed to SMCs, which resulted in data misinterpretation and the overstatement on SMC functions. We agree that further investigations are needed to determine the relative contribution of MVSCs and SMCs to vascular remodeling in various animal models; however, we respectfully disagree on some of the arguments in the commentary. Vascular SMC biology has been studied in vitro and in vivo extensively in the past 50 years since the method for SMC culture was established. The concept of phenotypic modulation of SMCs was originated from the ultrastructural characterization of SMC culture in 1960s to 1970s, which has been summarized in the comprehensive review by Chamley-Campbell et al. …

Local Medial Microenvironment Directs Phenotypic Modulation of Smooth Muscle Cells After Experimental Renal Transplantation

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Abstract 47: Development and Use of a Novel Single-Cell Epigenetic Lineage Tracing Method to Identify Smooth Muscle--Derived Macrophage-Like Cells Within Human Atherosclerotic Lesions

Aim: Smooth muscle cells (SMC) possess remarkable phenotypic plasticity that allows adaptation to changing environmental cues. Lack of definitive SMC lineage tracing studies and inability to identify phenotypically modulated SMCs within lesions due to loss of SMC marker gene expression raise major questions regarding the role of SMC in vivo in atherosclerosis progression. We hypothesize that a subset of cells within lesions that express macrophage markers are derived from SMC not monocytes and play a key role in determining plaque stability. Methods: We developed a novel lineage tracing based on detection of H3K4dime of the SM MHC gene, a SMC-specific epigenetic lineage marker we have previously shown is stable during phenotypic switching in vitro. Detection of H3K4dime of the SM MHC locus was done using a Proximity ligation assay (PLA) developed in our lab with an antibody targeting the biotinylated DNA probe for the SM MHC locus in conjunction with an anti-H3K4dime antibody. Use of secondary antibodies conjugated with oligonucleotides induces formation of circular DNA that serve as template for amplification, allowing visualization of co-localization of H3K4dime and the SM MHC gene (Duolink). Our new lineage tracing is suitable with human paraffin-embedded tissue sections (n=4) allowing investigation of SMC fate within human atherosclerotic lesion. Results: H3K4dime on the SM MHC gene (PLA+ cells) was found to be specific for SMCs and not found in any other cell types including adventitial fibroblasts, or endothelial cells. The method was validated using a SMC-specific lineage tracing mouse model wherein SM MHC Cre mice are crossed to ROSA flox STOP eYFP+/+ ApoE -/- mice. The H3K4dime/SM MHC PLA signal (i.e. PLA+) was exclusively found in eYFP+ cells. Moreover, some of the lesion SMCs were eYFP+/PLA+/SM α-actin-. Similarly, we identified PLA+ cells in human lesions that were positive for the macrophage marker CD68. Conclusion: Our new method permits definitive identification of SMC-derived cells within lesions even if they are not identifiable as SMC due to loss of SMC markers. Moreover, we provide exciting evidence that a significant fraction of macrophage-like cells in human lesions are derived from SMC. We postulate that transition of SMC to a macrophage state may be a key event leading to plaque destabilization and rupture with possible myocardial infarction or stroke.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

Smooth Muscle miRNAs Are Critical for Post-Natal Regulation of Blood Pressure and Vascular Function

Phenotypic modulation of smooth muscle cells (SMCs) plays a key role in vascular disease, including atherosclerosis. Several transcription factors have been suggested to regulate phenotypic modulation of SMCs but the decisive mechanisms remain unknown. Recent reports suggest that specific microRNAs (miRNAs) are involved in SMC differentiation and vascular disease but the global role of miRNAs in postnatal vascular SMC has not been elucidated. Thus, the objective of this study was to identify the role of Dicer-dependent miRNAs for blood pressure regulation and vascular SMC contractile function and differentiation in vivo. Tamoxifen-inducible and SMC specific deletion of Dicer was achieved by Cre-Lox recombination. Deletion of Dicer resulted in a global loss of miRNAs in aortic SMC. Furthermore, Dicer-deficient mice exhibited a dramatic reduction in blood pressure due to significant loss of vascular contractile function and SMC contractile differentiation as well as vascular remodeling. Several of these results are consistent with our previous observations in SM-Dicer deficient embryos. Therefore, miRNAs are essential for maintaining blood pressure and contractile function in resistance vessels. Although the phenotype of miR-143/145 deficient mice resembles the loss of Dicer, the phenotypes of SM-Dicer KO mice were far more severe suggesting that additional miRNAs are involved in maintaining postnatal SMC differentiation.

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.

Blockade of Connexin 43 Hemichannels Reduces Neointima Formation After Vascular Injury by Inhibiting Proliferation and Phenotypic Modulation of Smooth Muscle Cells

Connexins 43 (Cx43) plays a key role in neointimal formation after vascular injury, but the mechanism still needs to be further explored. We hypothesized that the gap junction-dependent function of Cx43 to mediate intercellular communication has a crucial role in the development and progression of vascular diseases. The effect of intercellular communication mediated by Cx43 hemichannels on neointimal formation after vascular injury was investigated. Cx43 was overexpressed or knockdown in rat vascular smooth muscle cell (SMC) by transfection pcDNA-Cx43 plasmid or small interfering RNA (siRNA) against Cx43 (siCx43). SMC proliferation and marker genes expression after Cx43 alteration and blockade of the Cx43 hemichannel were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and RT-PCR. The effect of carbenoxolone on neointimal formation was investigated in carotid artery injured rat model. We demonstrated that overexpression of Cx43 promoted SMC proliferation, meanwhile, mRNA expression level of smooth muscle alpha-actin and calponin, which were important markers of SMC in a contractile state, were down-regulated in smooth muscle. Knockdown of Cx43 inhibited SMC proliferation but increased SMC marker genes expression level. Carbenoxolone (50 muM) improved SMC contractile differentiation and inhibited its proliferation. Our data showed that carbenoxolone reduced neointimal formation after carotid artery injury. In summary, blockade of intercellular communication via Cx43 hemichannels reduces neointimal formation after vascular injury by inhibiting proliferation and phenotypic modulation of SMCs.

Shear Stress Induces Synthetic-to-Contractile Phenotypic Modulation in Smooth Muscle Cells via Peroxisome Proliferator-Activated Receptor α/δ Activations by Prostacyclin Released by Sheared Endothelial Cells

Phenotypic modulation of smooth muscle cells (SMCs), which are located in close proximity to endothelial cells (ECs), is critical in regulating vascular function. The role of flow-induced shear stress in the modulation of SMC phenotype has not been well defined. The objective was to elucidate the role of shear stress on ECs in modulating SMC phenotype and its underlying mechanism. Application of shear stress (12 dyn/cm2) to ECs cocultured with SMCs modulated SMC phenotype from synthetic to contractile state, with upregulation of contractile markers, downregulation of proinflammatory genes, and decreased percentage of cells in the synthetic phase. Treating SMCs with media from sheared ECs induced peroxisome proliferator-activated receptor (PPAR)-alpha, -delta, and -gamma ligand binding activities; transfecting SMCs with specific small interfering (si)RNAs of PPAR-alpha and -delta, but not -gamma, inhibited shear induction of contractile markers. ECs exposed to shear stress released prostacyclin (PGI2). Transfecting ECs with PGI2 synthase-specific siRNA inhibited shear-induced activation of PPAR-alpha/delta, upregulation of contractile markers, downregulation of proinflammatory genes, and decrease in percentage of SMCs in synthetic phase. Mice with PPAR-alpha deficiency (compared with control littermates) showed altered SMC phenotype toward a synthetic state, with increased arterial contractility in response to angiotensin II. These results indicate that laminar shear stress induces synthetic-to-contractile phenotypic modulation in SMCs through the activation of PPAR-alpha/delta by the EC-released PGI2. Our findings provide insights into the mechanisms underlying the EC-SMC interplays and the protective homeostatic function of laminar shear stress in modulating SMC phenotype.