Smooth Muscle Cell Plasticity Research Articles

Acute injury to the mouse carotid artery provokes a distinct healing response.

Treatment of vascular stenosis with angioplasty results in acute vascular damage, which may lead to restenosis. Owing to the highly complex cellularity of blood vessels, the healing response following this damage is incompletely understood. To gain further insight into this process, scRNA-seq of mouse carotid tissue after wire injury was performed. Stages of acute inflammation, resolution and remodeling were recapitulated in these data. To identify cell types which give rise to neointima, analyses focused on smooth muscle cell and fibroblast populations, and included data integration with scRNA-seq data from myocardial infarction and atherosclerosis datasets. Following carotid injury, a subpopulation of smooth muscle cells which also arises during atherosclerosis and myocardial infarction was identified. So-called stem cell/endothelial cell/monocyte (SEM) cells are candidates for repopulating injured vessels, and were amongst the most proliferative cell clusters following wire-injury of the carotid artery. Importantly, SEM cells exhibit specific transcriptional profiles which could be therapeutically targeted. SEM cell gene expression patterns could also be detected in bulk RNA-sequencing of neointimal tissue isolated from injured carotid vessels by laser capture microdissection. These data indicate that phenotypic plasticity of smooth muscle cells is highly important to the progression of lumen loss following acute carotid injury. Interference with SEM cell formation could be an innovative approach to combat development of restenosis.

Genomic, Transcriptomic, and Proteomic Depiction of Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells As Emerging Cellular Models for Arterial Diseases.

Vascular smooth muscle cells (SMCs) plasticity is a central mechanism in cardiovascular health and disease. We aimed at providing cellular phenotyping, epigenomic and proteomic depiction of SMCs derived from induced pluripotent stem cells and evaluating their potential as cellular models in the context of complex diseases. Human induced pluripotent stem cell lines were differentiated using RepSox (R-SMCs) or PDGF-BB (platelet-derived growth factor-BB) and TGF-β (transforming growth factor beta; TP-SMCs), during a 24-day long protocol. RNA-Seq and assay for transposase accessible chromatin-Seq were performed at 6 time points of differentiation, and mass spectrometry was used to quantify proteins. Both induced pluripotent stem cell differentiation protocols generated SMCs with positive expression of SMC markers. TP-SMCs exhibited greater proliferation capacity, migration and lower calcium release in response to contractile stimuli, compared with R-SMCs. Genes involved in the contractile function of arteries were highly expressed in R-SMCs compared with TP-SMCs or primary SMCs. R-SMCs and coronary artery transcriptomic profiles were highly similar, characterized by high expression of genes involved in blood pressure regulation and coronary artery disease. We identified FOXF1 and HAND1 as key drivers of RepSox specific program. Extracellular matrix content contained more proteins involved in wound repair in TP-SMCs and higher secretion of basal membrane constituents in R-SMCs. Open chromatin regions of R-SMCs and TP-SMCs were significantly enriched for variants associated with blood pressure and coronary artery disease. Both induced pluripotent stem cell-derived SMCs models present complementary cellular phenotypes of high relevance to SMC plasticity. These cellular models present high potential to study functional regulation at genetic risk loci of main arterial diseases.

Distinct Cellular Origins and Differentiation Process Account for Distinct Oncogenic and Clinical Behaviors of Leiomyosarcomas.

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named 'hLMS'. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

Transcription factor GATA6 promotes migration of human coronary artery smooth muscle cells in vitro.

Vascular smooth muscle cell plasticity plays a pivotal role in the pathophysiology of vascular diseases. Despite compelling evidence demonstrating the importance of transcription factor GATA6 in vascular smooth muscle, the functional role of GATA6 remains poorly understood. The aim of this study was to elucidate the role of GATA6 on cell migration and to gain insight into GATA6-sensitive genes in smooth muscle. We found that overexpression of GATA6 promotes migration of human coronary artery smooth muscle cells in vitro, and that silencing of GATA6 in smooth muscle cells resulted in reduced cellular motility. Furthermore, a complete microarray screen of GATA6-sensitive gene transcription resulted in 739 upregulated and 248 downregulated genes. Pathways enrichment analysis showed involvement of transforming growth factor beta (TGF-β) signaling which was validated by measuring mRNA expression level of several members. Furthermore, master regulators prediction based on microarray data revealed several members of (mitogen activated protein kinase) MAPK pathway as a master regulators, reflecting involvement of MAPK pathway also. Our findings provide further insights into the functional role of GATA6 in vascular smooth muscle and suggest that targeting GATA6 may constitute as a new approach to inhibit vascular smooth muscle migration.

Differential affinity chromatography reveals a link between Porphyromonas gingivalis-induced changes in vascular smooth muscle cell differentiation and the type 9 secretion system.

Porphyromonas gingivalis is implicated in adverse pregnancy outcome. We previously demonstrated that intrauterine infection with various strains of P. gingivalis impairs the physiologic remodeling of the uterine spiral arteries (IRSA) during pregnancy, which underlies the major obstetrical syndromes. Women diagnosed with IRSA also have a greater risk for premature cardiovascular disease in later life. The dysregulated plasticity of vascular smooth muscle cells (VSMCs) is present in both IRSA and premature cardiovascular events. We hypothesized that VSMCs could serve as a bait to identify P. gingivalis proteins associated with dysregulated VSMC plasticity as seen in IRSA. We first confirmed that dams with P. gingivalis A7UF-induced IRSA also show perturbed aortic smooth muscle cell (AoSMC) plasticity along with the P. gingivalis colonization of the tissue. The in vitro infection of AoSMCs with IRSA-inducing strain A7UF also perturbed AoSMC plasticity that did not occur with infection by non-IRSA-inducing strain W83. Far-Western blotting with strain W83 and strain A7UF showed a differential binding pattern to the rat aorta and primary rat AoSMCs. The affinity chromatography/pull-down assay combined with mass spectrometry was used to identify P. gingivalis/AoSMC protein interactions specific to IRSA. Membrane proteins with a high binding affinity to AoSMCs were identified in the A7UF pull-down but not in the W83 pull-down, most of which were the outer membrane components of the Type 9 secretion system (T9SS) and T9SS cargo proteins. Additional T9SS cargo proteins were detected in greater abundance in the A7UF pull-down eluate compared to W83. None of the proteins enriched in the W83 eluate were T9SS components nor T9SS cargo proteins despite their presence in the prey preparations used in the pull-down assay. In summary, differential affinity chromatography established that the components of IRSA-inducing P. gingivalis T9SS as well as its cargo directly interact with AoSMCs, which may play a role in the infection-induced dysregulation of VSMC plasticity. The possibility that the T9SS is involved in the microbial manipulation of host cell events important for cell differentiation and tissue remodeling would constitute a new virulence function for this system.

Molecular Mechanisms of Vascular Health: Insights From Vascular Aging and Calcification.

Cardiovascular disease is the most common cause of death worldwide, especially beyond the age of 65 years, with the vast majority of morbidity and mortality due to myocardial infarction and stroke. Vascular pathology stems from a combination of genetic risk, environmental factors, and the biologic changes associated with aging. The pathogenesis underlying the development of vascular aging, and vascular calcification with aging, in particular, is still not fully understood. Accumulating data suggests that genetic risk, likely compounded by epigenetic modifications, environmental factors, including diabetes and chronic kidney disease, and the plasticity of vascular smooth muscle cells to acquire an osteogenic phenotype are major determinants of age-associated vascular calcification. Understanding the molecular mechanisms underlying genetic and modifiable risk factors in regulating age-associated vascular pathology may inspire strategies to promote healthy vascular aging. This article summarizes current knowledge of concepts and mechanisms of age-associated vascular disease, with an emphasis on vascular calcification.

Phenotypic plasticity of vascular smooth muscle cells in vascular calcification: Role of mitochondria.

Vascular calcification (VC) is an important hallmark of cardiovascular disease, the osteo-/chondrocyte phenotype differentiation of vascular smooth muscle cells (VSMCs) is the main cause of vascular calcification. Accumulating evidence shows that mitochondrial dysfunction may ultimately be more detrimental in the VSMCs calcification. Mitochondrial participate in essential cellular functions, including energy production, metabolism, redox homeostasis regulation, intracellular calcium homeostasis, apoptosis, and signal transduction. Mitochondrial dysfunction under pathological conditions results in mitochondrial reactive oxygen species (ROS) generation and metabolic disorders, which further lead to abnormal phenotypic differentiation of VSMCs. In this review, we summarize existing studies targeting mitochondria as a treatment for VC, and focus on VSMCs, highlighting recent progress in determining the roles of mitochondrial processes in regulating the phenotype transition of VSMCs, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy, mitochondrial energy metabolism, and mitochondria/ER interactions. Along these lines, the impact of mitochondrial homeostasis on VC is discussed.

Lack of PI 3-kinase isoform p110alpha impairs SMC differentiation and proliferation and promotes aortic aneurysm formation

Abstract Background Proliferation and phenotypic modulation of vascular smooth muscle cells (SMCs) significantly contribute to the functionality of the aortic wall. Dysregulation of underlying signal transduction pathways impairs the vessel wall structure and promote the development and progression of abdominal aortic aneurysms (AAA). The PI 3-kinase (PI3K) isoform p110α is activated downstream of receptor tyrosine kinases (RTKs) and represents the most relevant PI3K isoform in SMCs. Aim This project follows the hypothesis that p110α deficiency impairs proliferation and phenotypic modulation of SMCs as well as the structure of the extracellular matrix (ECM) and therefore promotes the development and progression of AAA. It was investigated how p110α deficiency affects the plasticity of SMCs, the production and structure of ECM components, and the formation of AAA. Methods and results Western blot analyses showed that SMCs isolated from smooth muscle specific p110α−/− (sm-p110α−/−) mice were characterized by decreased expression of the differentiation markers sm-α-actin, calponin and sm-MHC. Mechanistically, phosphorylation of key modulators of the SMC phenotype – AKT1, AKT2, FOXO1, -3 and -4 as well as GSK3β – was impaired in p110α−/− SMCs after RTK stimulation. These findings indicate that phenotypic modulation of p110α−/− SMCs is restricted. In addition, protein expression of elastin and fibrillin was reduced in p110α−/− SMCs. In silico analysis (MatLab macro CT-FIRE and Curvalign) of the ECM produced by SMCs in vitro revealed a significantly reduced elastin fiber length and width in p110α−/− SMCs compared to fibers produced by WT SMCs (p<0.05). Consistently, aortas from sm-p110α−/− mice showed a significantly higher number of elastic fiber breaks specifically in the thoracic section than WT controls (289±31 mmm–2 versus 190±9 mmm–2, n=5, p=0.015). Aortic aneurysms in sm-p110α−/− mice and wild-type littermates were analyzed using the established porcine pancreatic elastase (PPE) model. PPE was perfused into the infrarenal aorta to induce AAA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. However, the increase in aortic diameter in sm-p110α−/− mice (70.16±10.82% mm, n=9) was significant more pronounced compared to wild-type animals (42.44±5.99%, n=10) (p<0.05). Three days after PPE perfusion, the number of elastic fiber breaks was significantly increased, and amount of proliferating SMCs were decreased in the infrarenal aorta of sm-p110α−/− mice compared to WT controls. Conclusion p110α deficiency in SMCs impairs aortic wall structure and promotes the development and progression of aortic aneurysms. Mechanistically, p110α activity maintains a differentiated SMC phenotype as well as the expression and assembly of ECM components. These data identify p110α signaling as a modifiable target for preventive and therapeutic strategies for aortic aneurysms. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Bves maintains vascular smooth muscle cell contractile phenotype and protects against transplant vasculopathy via Dusp1-dependent p38MAPK and ERK1/2 signaling

Background and aimsVascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear. MethodsWe used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development. ResultsHere, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation. ConclusionsBves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Human primary plaque cells to study cell functions and the biology behind sex-differences in atherosclerosis

Background and Aims : We previously showed that female key driver genes are involved in vascular smooth muscle cell plasticity in female atherosclerotic plaques. To further study mechanisms on how those sex-driven genes contribute to atherosclerotic disease they need to be first prioritized in a relevant in vitro model system. We hypothesize that human isolated plaque cells retain diseased key driver gene expression and activity and therefore can be used for plasticity experiments to prioritize and study the function of female key driver genes.

SREBP1 regulates Lgals3 activation in response to cholesterol loading

Aberrant smooth muscle cell (SMC) plasticity is etiological to vascular diseases. Cholesterol induces SMC phenotypic transition featuring high LGALS3 (galectin-3) expression. This proatherogenic process is poorly understood for its molecular underpinnings, in particular, the mechanistic role of sterol regulatory-element binding protein-1 (SREBP1), a master regulator of lipid metabolism. Herein we show that cholesterol loading stimulated SREBP1 expression in mouse, rat, and human SMCs. SREBP1 positively regulated LGALS3 expression (and vice versa), whereas Krüppel-like factor-15 (KLF15) acted as a negative regulator. Both bound to the Lgals3 promoter, yet at discrete sites, as revealed by chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. SREBP1 and LGALS3 each abated KLF15 protein, and blocking the bromo/extraterminal domain-containing proteins (BETs) family of acetyl-histone readers abolished cholesterol-stimulated SREBP1/LGALS3 protein production. Furthermore, silencing bromodomain protein 2 (BRD2; but not other BETs) reduced SREBP1; endogenous BRD2 co-immunoprecipitated with SREBP1’s transcription-active domain, its own promoter DNA, and that of Lgals3. Thus, results identify a previously uncharacterized cholesterol-responsive dyad—SREBP1 and LGALS3, constituting a feedforward circuit that can be blocked by BETs inhibition. This study provides new insights into SMC phenotypic transition and potential interventional targets.

Functional Remodeling of the Contractile Smooth Muscle Cell Cortex, a Provocative Concept, Supported by Direct Visualization of Cortical Remodeling.

Simple SummaryAs a key element of the smooth muscle cell contractile apparatus, the actin cytoskeleton participates in the development of force by acting as a molecular track for the myosin cross bridge motor. At the same time, the actin cytoskeleton must transmit the force developed during contraction to the extracellular matrix and, thus, to neighboring cells. This propagation of force to the cell periphery and beyond is initiated in part on specifically localized cellular cortical actin filaments also involved in mechano-chemical transduction. During the contractile process itself and in response to extracellular structural and chemical alterations, the smooth muscle actin cytoskeletal remodels. This indicates that the cytoskeleton is a dynamic cellular organelle that adapts to the changes in cell shape and chemical cues. Current evidence connecting contractile function and mechano-transduction mechanisms to the plasticity of the vascular smooth muscle actin cytoskeleton is reviewed; we then describe new evidence for cytoskeletal remodeling in vascular smooth muscle cells. Here, using immunoelectron microscopy, we visualize the actin binding proteins filamin A, zyxin and talin in these cells and show that they participate in the cortical cell cytoskeletal alteration, thus supporting the premise that smooth muscle cell remodeling occurs during contraction.Considerable controversy has surrounded the functional anatomy of the cytoskeleton of the contractile vascular smooth muscle cell. Recent studies have suggested a dynamic nature of the cortical cytoskeleton of these cells, but direct proof has been lacking. Here, we review past studies in this area suggesting a plasticity of smooth muscle cells. We also present images testing these suggestions by using the technique of immunoelectron microscopy of metal replicas to directly visualize the cortical actin cytoskeleton of the contractile smooth muscle cell along with interactions by representative cytoskeletal binding proteins. We find the cortical cytoskeletal matrix to be a branched, interconnected network of linear actin bundles. Here, the focal adhesion proteins talin and zyxin were localized with nanometer accuracy. Talin is reported in past studies to span the integrin–cytoplasm distance in fibroblasts and zyxin is known to be an adaptor protein between alpha-actinin and VASP. In response to activation of signal transduction with the alpha-agonist phenylephrine, we found that no movement of talin was detectable but that the zyxin-zyxin spacing was statistically significantly decreased in the smooth muscle cells examined. Contractile smooth muscle is often assumed to have a fixed cytoskeletal structure. Thus, the results included here are important in that they directly support the concept at the electron microscopic level that the focal adhesion of the contractile smooth muscle cell has a dynamic nature and that the protein–protein interfaces showing plasticity are protein-specific.

Role of Integrins in Modulating Smooth Muscle Cell Plasticity and Vascular Remodeling: From Expression to Therapeutic Implications.

Smooth muscle cells (SMCs), present in the media layer of blood vessels, are crucial in maintaining vascular homeostasis. Upon vascular injury, SMCs show a high degree of plasticity, undergo a change from a “contractile” to a “synthetic” phenotype, and play an essential role in the pathophysiology of diseases including atherosclerosis and restenosis. Integrins are cell surface receptors, which are involved in cell-to-cell binding and cell-to-extracellular-matrix interactions. By binding to extracellular matrix components, integrins trigger intracellular signaling and regulate several of the SMC function, including proliferation, migration, and phenotypic switching. Although pharmacological approaches, including antibodies and synthetic peptides, have been effectively utilized to target integrins to limit atherosclerosis and restenosis, none has been commercialized yet. A clear understanding of how integrins modulate SMC biology is essential to facilitate the development of integrin-based interventions to combat atherosclerosis and restenosis. Herein, we highlight the importance of integrins in modulating functional properties of SMCs and their implications for vascular pathology.

Endothelial-derived extracellular microRNA-92a promotes arterial stiffness by regulating phenotype changes of vascular smooth muscle cells

Endothelial dysfunction and vascular smooth muscle cell (VSMC) plasticity are critically involved in the pathogenesis of hypertension and arterial stiffness. MicroRNAs can mediate the cellular communication between vascular endothelial cells (ECs) and neighboring cells. Here, we investigated the role of endothelial-derived extracellular microRNA-92a (miR-92a) in promoting arterial stiffness by regulating EC–VSMC communication. Serum miR-92a level was higher in hypertensive patients than controls. Circulating miR-92a level was positively correlated with pulse wave velocity (PWV), systolic blood pressure (SBP), diastolic blood pressure (DBP), and serum endothelin-1 (ET-1) level, but inversely with serum nitric oxide (NO) level. In vitro, angiotensin II (Ang II)-increased miR-92a level in ECs mediated a contractile-to-synthetic phenotype change of co-cultured VSMCs. In Ang II-infused mice, locked nucleic acid-modified antisense miR-92a (LNA-miR-92a) ameliorated PWV, SBP, DBP, and impaired vasodilation induced by Ang II. LNA-miR-92a administration also reversed the increased levels of proliferative genes and decreased levels of contractile genes induced by Ang II in mouse aortas. Circulating serum miR-92a level and PWV were correlated in these mice. These findings indicate that EC miR-92a may be transported to VSMCs via extracellular vesicles to regulate phenotype changes of VSMCs, leading to arterial stiffness.

Dynamic changes in chromatin accessibility are associated with the atherogenic transitioning of vascular smooth muscle cells.

De-differentiation and activation of pro-inflammatory pathways are key transitions vascular smooth muscle cells (SMCs) make during atherogenesis. Here, we explored the upstream regulators of this 'atherogenic transition'. Genome-wide sequencing studies, including Assay for Transposase-Accessible Chromatin using sequencing and RNA-seq, were performed on cells isolated from both murine SMC-lineage-tracing models of atherosclerosis and human atherosclerotic lesions. At the bulk level, alterations in chromatin accessibility were associated with the atherogenic transitioning of lesional SMCs, especially in relation to genes that govern differentiation status and complement-dependent inflammation. Using computational biology, we observed that a transcription factor previously related to coronary artery disease, Activating transcription factor 3 (ATF3), was predicted to be an upstream regulator of genes altered during the transition. At the single-cell level, our results indicated that ATF3 is a key repressor of SMC transitioning towards the subset of cells that promote vascular inflammation by activating the complement cascade. The expression of ATF3 and complement component C3 was negatively correlated in SMCs from human atherosclerotic lesions, suggesting translational relevance. Phenome-wide association studies indicated that genetic variation that results in reduced expression of ATF3 is correlated with an increased risk for atherosclerosis, and the expression of ATF3 was significantly down-regulated in humans with advanced vascular disease. Our study indicates that the plasticity of atherosclerotic SMCs may in part be explained by dynamic changes in their chromatin architecture, which in turn may contribute to their maladaptive response to inflammation-induced stress.

The Expanding Role of Alternative Splicing in Vascular Smooth Muscle Cell Plasticity.

Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to ‘switch’ between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.

Abstract MP11: Histone Methyl Transferase (suv39h1): Function In Smooth Muscle Cell Phenotypic Plasticity

Introduction: The phenotypic plasticity of vascular smooth muscle cells (VSMCs) is central to growth and remodeling processes, but also contributes to cardiovascular disease. This unique ability of VSMCs to reversibly differentiate and de-differentiate is incompletely understood. SUV39H1 is a histone methyltransferase that specifically trimethylates Lysine-9 of histone H3 (H3K9me3), resulting in transcriptional repression through epigenetic gene silencing. Hypothesis: We hypothesized that SUV39H1 may play a role in SMC phenotypic switch. Methods: Using in vitro and in vivo approaches including knockdown, qPCR, western, and chromatin immunoprecipitation (ChIP) assays to determine role of SUV39H1 in SMC plasticity. Results: A qPCR array screen of epigenetic regulators in VSMCs identified SUV39H1 mRNA as upregulated with PDGF induced dedifferentiation and downregulated with rapamycin induced differentiation. This was confirmed at the protein level. SUV39H1 knockdown significantly increased VSMC contractile protein mRNA, protein levels and decreased dedifferentiation associated gene expression, and also decreased PDGF-induced VSMC migration (n=4). Interestingly, we found that expression of KLF4, the master transcriptional regulator of dedifferentiation in SMCs, was dramatically decreased after SUV39H1 knockdown (n=5). We further noted that SUV39H1 knockdown decreased KLF4 mRNA stability (n=3). Mechanistically, SUV39H1 knockdown increased miRNA143, a well-known repressor of KLF4. ChIP assays at contractile gene promoters showed a significant decrease in H3K9me3 mark and an increase in H3K27 acetylation, an activation mark (n=4). Carotid artery ligation induced intimal hyperplastic lesions in wild type C57BL/6 mice, showed a significant increase in SUV39H1 and H3K9me3 expression compared to uninjured vessels. Conclusion: We identify SUV39H1 an epigenetic regulator of VSMC phenotype whose expression and activity increase with dedifferentiation in vitro and in vivo . Mechanistically, SUV39H1 influences VSMC chromatin marks and KLF4 expression to repress contractile phenotype. Understanding the role of SUV39H1 and its targets may have implications for developing new therapeutic strategies for treating vascular diseases.

Abstract P281: Vascular Remodeling And Impaired Vascular Smooth Muscle Cell Plasticity In Age And Sex-dependent Hypertension

Aim: Hypertension (HTN) and aging are associated with the development of vascular dysfunction. We speculated that vascular smooth muscle cell plasticity and vascular remodeling play major roles in age and sex-dependent HTN. Methods: Male and female Sprague-Dawley (SD) rats aged 3 and 16-months-old (N=6/group) were housed under standard conditions. Blood pressure was measured via femoral artery cannulation and sympathetic tone to the vasculature was estimated by ganglion blockade via Hexamethonium (30mg/Kg IV). PBS-perfused abdominal aorta and renal arteries were collected and immunoblotting was performed following protein extraction. Results: Male SD rats, but not females, develop HTN and increased sympathetic tone with age. Aged hypertensive male rats, but not aged normotensive females, exhibit reduced p-Erk1/2 and p-eNos levels in both abdominal aorta and renal arteries. α-Smooth Muscle Actin significantly increased in aged male abdominal aorta. Elevated c-Src was observed in aged female abdominal aorta and p-c-Src was reduced in aged male abdominal aorta. Caveolin-1 changed oppositely in young and aged abdominal aorta and renal arteries of two sexes. Conclusions: Our data suggest that artery-specific changes of key signaling molecules contribute to impaired vascular smooth muscle plasticity and vascular dysfunction in aged hypertensive male but not in aged normotensive female rats.

The atypical cadherin FAT1 limits smooth muscle cell metabolic reprogramming and atherosclerosis

Background and Aims: Phenotypic modulation of smooth muscle cells (SMCs) promotes atherosclerosis, and the transition from quiescence to a proliferative state is a key feature of SMC plasticity. How SMCs regulate energy and biomass utilization during this transition is largely unknown. We previously showed that FAT1 intracellular domain (ICD) fragments localize to mitochondria, and that FAT1 expression in SMCs attenuates neointima formation after injury, in part by restraining cellular respiration.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.