Vascular Smooth Muscle Cell Phenotype Research Articles

Transcriptional control of a novel long noncoding RNA Mymsl in smooth muscle cells by a single Cis-element and its initial functional characterization in vessels

Differentiated vascular smooth muscle cells (VSMCs) are crucial in maintaining vascular homeostasis. While the coding transcriptome of the differentiated VSMC phenotype has been defined, we know little about its noncoding signature. Herein, we identified a Myocardin-induced muscle specific long noncoding RNA (lncRNA) (Mymsl) downregulated upon VSMC phenotypic modulation. We demonstrated an essential role of a proximal consensus CArG element in response to MYOCD/SRF in vitro. To validate the in vivo role of this CArG element, we generated CArG mutant mice via CRISPR-Cas9 genome editing. While the CArG mutation had no impact on the expression of surrounding genes, it abolished Mymsl expression in SMCs, but not skeletal and cardiac muscle. Chromatin immunoprecipitation assays (ChIPs) showed decreased SRF binding to CArG region in mutants whereas the enrichment of H3K79Me2 remained the same. RNA-seq analysis showed a downregulation of matrix genes in aortas from Mymsl knockout mice, which was further validated in injured carotid arteries. Our study defined the transcriptional control of a novel lncRNA in SMCs via a single transcription factor binding site, which may offer a new strategy for generating SMC-specific knockout mouse models. We also provided in vivo evidence supporting the potential importance of Mymsl in vascular pathophysiology.

The Role of Vascular Smooth Muscle Cells in Arterial Remodeling: Focus on Calcification-Related Processes.

Arterial remodeling refers to the structural and functional changes of the vessel wall that occur in response to disease, injury, or aging. Vascular smooth muscle cells (VSMC) play a pivotal role in regulating the remodeling processes of the vessel wall. Phenotypic switching of VSMC involves oxidative stress-induced extracellular vesicle release, driving calcification processes. The VSMC phenotype is relevant to plaque initiation, development and stability, whereas, in the media, the VSMC phenotype is important in maintaining tissue elasticity, wall stress homeostasis and vessel stiffness. Clinically, assessment of arterial remodeling is a challenge; particularly distinguishing intimal and medial involvement, and their contributions to vessel wall remodeling. The limitations pertain to imaging resolution and sensitivity, so methodological development is focused on improving those. Moreover, the integration of data across the microscopic (i.e., cell-tissue) and macroscopic (i.e., vessel-system) scale for correct interpretation is innately challenging, because of the multiple biophysical and biochemical factors involved. In the present review, we describe the arterial remodeling processes that govern arterial stiffening, atherosclerosis and calcification, with a particular focus on VSMC phenotypic switching. Additionally, we review clinically applicable methodologies to assess arterial remodeling and the latest developments in these, seeking to unravel the ubiquitous corroborator of vascular pathology that calcification appears to be.

Role of the Balance of Akt and MAPK Pathways in the Exercise-Regulated Phenotype Switching in Spontaneously Hypertensive Rats.

The mechanisms regulating vascular smooth muscle cell (VSMC) phenotype switching and the critical signal modulation affecting the VSMCs remain controversial. Physical exercise acts as an effective drug in preventing elevated blood pressure and improving vascular function. This study was designed to explore the influence of aerobic exercise on the suppression of VSMC phenotype switching by balancing of the Akt, also known as PKB (protein kinase B) and mitogen-activated protein kinase (MAPK) signaling pathways. Spontaneously hypertensive rats (SHRs) and normotensive rats were subjected to exercise treatment before measuring the vascular morphological and structural performances. Exercise induced reverse expression of VSMC protein markers (α-SM-actin, calponin, and osteopontin (OPN)) in spontaneously hypertensive rats. It is noteworthy that the low expression of phosphorylated Akt significantly decreased the expression of VSMC contractile phenotype markers (α-SM-actin and calponin) and increased the expression of the VSMC synthetic phenotype marker (OPN). However, the MAPK signal pathway exerts an opposite effect. VSMCs and whole vessels were treated by inhibitors, namely the p-Akt inhibitor, p-ERK inhibitor, and p-p38 MAPK inhibitors. VSMC phenotype markers were reversed. It is important to note that a significant reverse regulatory relationship was observed between the expression levels of MAPK and the contractile markers in both normotensive and spontaneously hypertensive rats. We demonstrate that aerobic exercise regulates the VSMC phenotype switching by balancing the Akt and MAPK signaling pathways in SHRs.

Osteogenic gene markers are epigenetically reprogrammed during contractile-to-calcifying vascular smooth muscle cell phenotype transition

The understanding of vascular calcification-based mechanism is an urgent pending task in vascular biology and this prompted us to better address this issue by investigating whether DNA methylation mechanism might drive osteogenic marker genes modulation in primary human vascular smooth muscle cells (VSMCs) responding to calcium and phosphate levels overload up to 72 h. Firstly, our data shows this calcifying process recapitulates the molecular repertory of osteogenic biomarkers and specifically requiring RUNX2, Osterix and ALP, BSP genes activations along 72 h in vitro, and this behavior was validated here using other lineages. Conversely, both BMPs 4 and 7 were significantly overexpressed, maybe already as a mechanism in response to RUNX2 and Osterix genes activities identified earlier in response to the calcifying condition, and taken into maintain the calcifying phenotype of VSMCs. Additionally, survival signaling was maintained active and accompanied by a dynamic cytoskeleton rearrangement signaling requiring MAPK and AKT phosphorylations. Moreover, during the contractile-to-calcifying transition phenotype of VSMCs, epigenetic machinery was finely modulated, requiring the translocation of DNMT3B and TET2 into nucleus and this prompted us evaluating whether the profile of osteogenic-related gene promoters’ methylation might contribute with this process. By firstly estimating 5meC/5 hmeC ratio changes, we further specifically show the significance of the epigenetic modulation of Osterix and Bone sialoprotein related gene promoters, presenting a positive correlation between the epigenetic signature of their gene promoters and transcriptional patterns. Altogether, our results show for the first time the importance of epigenetic mechanism on modulating osteogenic gene markers reprogramming during calcifying VSMCs phenotype acquisition, which might drive the genesis of vascular ectopic calcification.

Sauchinone inhibits angiotensin II-induced proliferation and migration of vascular smooth muscle cells.

Hypertension is a common type of cardiovascular disease that remains a major cause of death in the world. Vascular remodelling is an important complication of hypertension, and vascular smooth muscle cells (VSMCs) play a major role in vascular remodelling. Sauchinone is one of the active lignins which has been found to possess vascular protective effects. However, the functional role of sauchinone in hypertension has not been investigated. The aim of this study was to evaluate the role of sauchinone in the angiotensin II (Ang II)-induced vascular remodelling model in VSMCs. The results showed that treatment of sauchinone inhibited Ang II-induced VSMCs proliferation and migration in VSMCs. Sauchinone treatment suppressed the reactive oxygen species (ROS) production and NADPH oxidase (NOX) activity in Ang II-induced VSMCs. The inhibitory effects of Ang II on expressions of VSMCs phenotype markers including α-smooth muscle actin (α-SMA), calponin, osteopontin were mitigated by sauchinone treatment. Furthermore, sauchinone inhibited Ang II-induced over-activation of TGF-β1/Smad3 signalling pathway in VSMCs. Taken together, this study identified sauchinone as a potential agent for preventing vascular remodelling in hypertension.

HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype.

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine single nucleotide polymorphisms (SNPs) associated with the extent of abdominal (AAC, n = 9,417) or descending thoracic (TAC, n = 8,422) aortic calcification. Two genetic loci, HDAC9 and RAP1GAP, were associated with AAC at a genome-wide level (P < 5.0 × 10−8). No SNPs were associated with TAC at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells (HASMCs) promoted calcification and reduced contractility, while inhibition of HDAC9 in HASMCs inhibited calcification and enhanced cell contractility. In matrix Gla protein (MGP)-deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a novel role for HDAC9 in the development of vascular calcification.

Autophagy as a novel therapeutic target in vascular calcification.

The autophagy pathway is a key regulator of cellular metabolism and homeostasis, and plays a critical role in maintaining normal vascular cell function. It is well recognised that autophagy can regulate endothelial cell homeostasis, vascular smooth muscle cell (VSMC) phenotype transition, and calcium (Ca2+) homeostasis in VSMCs. Emerging evidence has demonstrated that autophagy directly protects against vascular calcification (VC). Crosstalk between endosomes, dysfunctional mitochondria, autophagic vesicles and Ca2+ and phosphate (Pi) enriched matrix vesicles (MVs) may underpin the pathogenesis of VC. In this review, we summarize the current experimental evidence in understanding how autophagy maintains normal vascular cell function and its protective role against vascular calcification. We also discuss the underlying molecular and cellular mechanisms through which autophagy inhibits vascular calcification. Pharmacological modulation of autophagy may offer an exciting new strategy for the treatment of vascular calcification.

Transcriptional characterisation of human lung cells identifies novel mesenchymal lineage markers.

The lung mesenchyme gives rise to multiple distinct lineages of cells in the mature respiratory system, including smooth muscle cells of the airway and vasculature. However, a thorough understanding of the specification and mesenchymal cell diversity in the human lung is lacking. We completed single-cell RNA sequencing analysis of fetal human lung tissues. Canonical correlation analysis, clustering, cluster marker gene identification and t-distributed stochastic neighbour embedding representation was performed in Seurat. Cell populations were annotated using ToppFun. Immunohistochemistry and in situ hybridisation were used to validate spatiotemporal gene expression patterns for key marker genes. We identified molecularly distinct populations representing "committed" fetal human lung endothelial cells, pericytes and smooth muscle cells. Early endothelial lineages expressed "classic" endothelial cell markers (platelet endothelial cell adhesion molecule/CD31 and claudin 5), while pericytes expressed platelet-derived growth factor receptor-β, Thy-1 membrane glycoprotein and basement membrane molecules (collagen IV, laminin and proteoglycans). We observed a large population of "nonspecific" human lung mesenchymal progenitor cells characterised by expression of collagen I and multiple elastin fibre genes (ELN, MFAP2 and FBN1). We closely characterised the diversity of mesenchymal lineages defined by α2-smooth muscle actin (ACTA2) expression. Two cell populations, with the highest levels of ACTA2 transcriptional activity, expressed unique sets of markers associated with airway or vascular smooth muscle cells. Spatiotemporal analysis of these marker genes confirmed early and persistent spatial specification of airway (HHIP, MYLK and IGF1) and vascular (NTRK3 and MEF2C) smooth muscle cells in the developing human lung. Our data suggest that specification of distinct airway and vascular smooth muscle cell phenotypes is established early in development and can be identified using the markers we provide.

Expression of Long Noncoding RNA LIPCAR Promotes Cell Proliferation, Cell Migration, and Change in Phenotype of Vascular Smooth Muscle Cells.

BackgroundThe long noncoding RNA LIPCAR is a type of transcription product (>200 nucleotides long). Recent studies demonstrated that LIPCAR is a potential biomarker in cardiovascular disease and can predict survival in patients with cardiovascular disease. Therefore, the present study explored the role of LIPCAR in the regulation of proliferation, migration, and change in phenotype of vascular smooth muscle cells.Material/MethodsHuman vascular smooth muscle cells (VSMCs) were treated with 20 g/mL oxidatively modified low-density lipoprotein (ox-LDL) or 20 ng/ml platelet-derived growth factor BB (PDGF-BB) for 24 h, then the expression levels of LIPCAR were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. LIPCAR-overexpressing plasmids were transfected into VSMCs. After transfection, cell proliferation and migration were measured using the Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The levels of α-smooth muscle actin (α-SMA) a molecular marker of the contractile VSMC phenotype, were measured using Western blot and immunofluorescence assays. Protein levels of cyclin-dependent kinase-2 (CDK2), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor A (VEGF-A), and angiopoietin-2 (Ang-2) were assessed by Western blot. The level of tissue factor (TF) was measured by enzyme-linked immunosorbent assay (ELISA).ResultsTreatment with PDGF-BB or ox-LDL significantly increased levels of LIPCAR in VSMCs. Overexpression of LIPCAR markedly promoted cell proliferation and migration. Further, upregulation of LIPCAR increased CDK2, p21, PCNA, MMP2, MMP9, VEGF-A, Ang-2, and TF expression and decreased p21 expression. In addition, LIPCAR significantly decreased α-SAM expression.ConclusionsTogether, our data suggest that overexpression of LIPCAR promotes cell proliferation, migration, and phenotypic switch of vascular smooth muscle cells.

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture.

We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

Artemisinin attenuates the development of atherosclerotic lesions by the regulation of vascular smooth muscle cell phenotype switching

AimsThe purpose of this study was to investigate the therapeutic effect of artemisinin (ART) on atherosclerosis and explore the molecular mechanisms involved by RNA sequencing (RNA-Seq). Main methodsEight-week-old male ApoE−/- mice were treated with ART for eight weeks. Atherosclerotic lesion sizes were determined by Oil Red O staining, and RNA-Seq was used to detect the profile of differentially expressed genes following the administration of ART. The expressions of contractile phenotypic markers were detected by western blot and qRT-PCR, and the ability of the MOVAS cells to migrate and proliferate were assessed using the wound healing and CCK8 assays. Key findingsArtemisinin treatment significantly reduced plaque area in the ApoE−/- mice and increased the expression of contractile phenotypic markers. RNA-Seq of aorta tissue revealed a distinct change in gene expression patterns after the mice were treated with ART. Our bioinformatics analysis demonstrated that the most prominently enriched pathway was a set of genes involved in vascular smooth muscle contractile function. Using an in vitro cell model, we demonstrated that ART could effectively reverse PDGF-activated MOVAS migration and proliferation, and elevate the level of proteins involved in the contractile phenotype. SignificanceWe provide in vivo and in vitro evidence supporting a role for ART in the suppression of atherosclerosis, partly through the inhibition of vascular smooth muscle cell phenotype switching to a de-differentiated phenotype. These data further advances our understanding for a potential role for ART and suggests that ART is an excellent candidate for the treatment of atherosclerosis.

Nrf-2 signaling inhibits intracranial aneurysm formation and progression by modulating vascular smooth muscle cell phenotype and function

BackgroundOxidative stress and vascular smooth muscle cell (VSMC) phenotypic modulation influence intracranial aneurysm (IA) formation and progression. Oxidative stress plays an important role in phenotype switching, and nuclear factor erythroid 2-related factor 2 (Nrf-2) is one of the main antioxidant systems. Unfortunately, little is known about how Nrf-2 signaling influences VSMC phenotype switches during IA pathogenesis.MethodsWe examined the effect of Nrf-2 activation IA on formation and progression in an elastase-induced rat IA model. We also developed a hydrogen peroxide (H2O2)-induced VSMC oxidative damage model. Then, we analyzed VSMC phenotype changes in the setting of Nrf-2 activation or inhibition in vitro. The proliferation, migration ability, and apoptosis rate of VSMCs were tested. Lastly, we measured the expression levels of antioxidant enzymes and inflammatory cytokines downstream of Nrf-2.ResultsNrf-2 activation suppressed IA formation and progression in vivo. We confirmed Nrf-2 nuclear translocation and a VSMC switch from the contractile to synthetic phenotype. Nrf-2 activation inhibited the proliferation, migratory ability, and apoptosis rate enhanced by H2O2. Quantitative real-time polymerase chain reaction (PCR) and western blot analysis revealed that Nrf-2 activation promoted antioxidant enzymes and VSMC-specific marker gene expressions but decreased pro-inflammatory cytokine levels.ConclusionThese results suggest that Nrf-2 exerts protective effects against IA development by preventing VSMCs from changing to a synthetic phenotype.

3312Thrombomodulin expressed on vascular smooth muscle cell influences arterial injury-induced neointima formation in mouse

Abstract Background Thrombomodulin (TM) is a cell membrane-bound anticoagulant protein that only expresses on endothelial cells in normal artery. Vascular smooth muscle cells (SMCs) start to exhibit TM after arterial injury. Our previous study demonstrated that vascular SMC-bound TM expression was associated with SMC synthetic phenotype. TM knockdown not only attenuated aortic SMCs proliferation but also reduced aortic SMC-mediated inflammation. In this study, we investigated the effect of vascular SMC-bound TM on arterial injury-induced neointima formation in mouse. Methods and results Because complete loss of TM in TM knockout transgenic mice causes embryonic lethality, we generated vascular SMC-specific TM-deficient mice (SM22-cretg/TMflox/flox) and their wild-type controls (SM22-cretg/TM+/+) using the Cre-loxP system to explore the role of vascular SMC membrane-bound TM in vivo. The blood pressure and body weight were similar between SM22-cretg/TMflox/flox mice and their wild-type controls. Carotid ligation caused neointima formation in mice. Immunofluorescence staining showed that there was large amount TM expression in the medial and neointimal cells at 4 weeks in SM22-cretg/TM+/+ mice after carotid ligation, but there was no TM staining could be found in SM22-cretg/TMflox/flox mice. There was a progressively increased neointima area from 2 to 4 weeks after carotid ligation both in SM22-cretg/TMflox/flox mice and SM22-cretg/TM+/+ mice, but the neointima area and neointima/media area ratio were significantly smaller in SM22-cretg/TMflox/flox mice than SM22-cretg/TM+/+ mice. Immunofluorescence staining showed that there were less Ki67-positive cells in the media and neointima in SM22-cretg/TMflox/flox mice indicating less proliferating cells in the arterial wall of TM-deficient mice. The α-smooth muscle actin-positive staining area was also larger in the SM22-cretg/TMflox/flox mice, suggesting TM deficiency of SMCs in medial lesion exhibited a more contractile status after carotid ligation. Conclusions Our results indicated that vascular SMC-bound TM not only mediated vascular SMC phenotype change and cell behavior but also significantly influenced arterial injury-induced neointima formation. Acknowledgement/Funding This study was sponsored by grants 104-2314-B-006-083-MY2 and 106-2314-B-006-045-MY3 from the Ministry of Science and Technology, Taipei, Taiwan.

P6495Role of vascular smooth muscles cells in naked mole rats during ageing (Heterocephalus glaber)

Abstract Vascular stiffening and a prothrombotic state consistently increase with age. We have shown previously that Vascular Smooth Muscle Cells (VSMCs) have a major role in both vascular stiffening and thrombin generation in the vascular wall in a context of hypertension (Arterioscler Thromb Vasc Biol 2015). Naked mole rats (NMRs) are the longest-lived rodent species, have a maximum lifespan exceeding 30 years, living approximately ten times longer than a similarly sized mouse, and do not develop arterial stiffening with age (AJP, 2014). The objective was to analyze arterial composition as well as phenotypic VSMC changes from primary cultures in NMRs at 2- and 10-year old. We used Thoracic aortas of young (2-year-old) and adult (10-year-old) NMRs for the study of arterial wall as well as for the preparation of VSMCs' primary cultures. Our results showed no difference in elastin/collagen ratio and no intimal thickening in NMRs at 10-years-old compared to 2-years-old (n=4 in each group). Primary cultures showed positive alpha-actin staining without VSMC morphological changes up to 6 passages using standard conditions (20% fetal bovine serum). Smoothelin and myosin-heavy chain are similar at both ages. VSMCs density was identical in 2- and 10-years-old NMRs, whereas cell hypertrophy was observed in older animals. Focal adhesions assessed by vinculin staining were unchanged between the two groups. There was no increase in plasma thrombin generation and at the surface of VSMCs up to 10 years of age associated with no increase in fibrinogen and factor VIII, both known to increase normally with age in humans. Our findings showed an absence of canonical signs of normal vascular ageing in NMRs that are normally present in rodent and humans. The retaining of a VSMC phenotype and focal adhesion distribution support previous data observed in ageing. These results reinforce the current concept of VSMCs as emergent cells in the process of ageing.

P1936Selective inhibition of the Mek1/2-Erk1/2 signalling pathway induces the differentiation of human cardiac pericyte-like cells into contractile vascular smooth muscle cells

Abstract Background Arteriogenesis is key for tissue repair but whether myocardial stromal cells contribute to this phenomenon remains unknown. Purpose Investigate if cardiac pericytes are a druggable target for therapeutic arteriogenesis. Methods and results The localization of pericyte-like cells (PCs) was assessed in the human and murine heart by immunohistochemistry of typical antigenic markers. CD34+ PCs co-expressing NG2 and PDGFRβ but not endothelial cell (EC) or vascular smooth muscle cell (VSMC) antigens were identified in peri-arterial position in normal hearts. Interestingly, we also found rare PCs co-expressing αSMA in the peri-infarct myocardium, suggesting these cells could represent a transitory phenotype between PCs and VSMCs. Next, we isolated human cardiac PCs by immunosorting for CD31 and CD34 and established the purity of the isolated CD34+CD31- fraction by flow cytometry. Following culture expansion, cardiac PCs maintain the typical antigenic profile except for CD34. Moreover, we confirmed the PCs' ability to promote angiogenesis in-vitro. The withdrawal of EGF and bFGF from the culture media for 10 days induced the differentiation of PCs into mature VSMCs, as documented by a massive upregulation of contractile genes MYH11, CNN1 and ACTA2 (200-, 35- and 15-folds increase versus naïve PCs, p&lt;0.01), which was followed by the induction of SM-MHC, Smoothelin B, αSMA, Calponin and SM22α proteins (p&lt;0.05 versus PCs). In addition, PC-derived cells lost migratory capacity, secreted elastin, and responded to endothelin-1 in a contraction assay, thus phenocopying the behaviour of control coronary artery-derived VSMCs. We excluded contamination of the PC preparation by verifying similar phenomena occur in PCs expanded from single cell clonogeneic assays. Moreover, the process is partially reversible, with PC-derived VSMCs being able to reacquire some intermediate markers following EGF/bFGF re-challenge. ECs secrete EGF and bFGF, with this GF signalling being enhanced by hypoxia, suggesting ECs may control the PC phenotype in a paracrine fashion. Mechanistic studies revealed the Mek1/2-Erk1/2-Elk1 signalling is accountable for the transcriptional repression of VSMC genes in PCs. Accordingly, a selective Mek1/2 inhibitor (PD0325901) was able to switch the definitive VSMC phenotype of PCs maintained in full media. The drug prevented the phosphorylation of Erk1/2 and its downstream target Elk1. This likely relieves the complex SRF/MyocD and abolishes the transcriptional repression at the gene promoter. Conclusions Cardiac PCs have a VSMC potential which is under the inhibitory control of the Mek1/2-Erk1/2-Elk1 signalling. Mek1/2 inhibitors showed promises for the treatment of melanoma and solid tumours. A novel application of this class of compounds to improve arteriogenesis in myocardial ischemia is fascinating. The caveat about their potential cardiotoxicity could be less relevant with short duration treatments of myocardial ischemia. Acknowledgement/Funding British Heart Foundation Centre for Regenerative Medicine Award (II) - “Centre for Vascular Regeneration” (RM/17/3/33381)

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -β/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling.

Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs. This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease.

Nox5 Regulates Vascular Smooth Muscle Cell Phenotype

Nox5 Regulates Vascular Smooth Muscle Cell Phenotype

Impact of Herpud1 in the homocysteine-induced phenotypic switching of vascular smooth muscle cells

Objective: To investigate the impact of homocysteine inducible endoplasmic reticulum(ER) protein with ubiquitin like domain 1 protein (Herpud1) in the homocysteine (Hcy) -induced phenotypic switching of vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from thoracic aortic artery of male Sprague Dawley rats and cultured VSMCs (4-7 passage) were treated with various concentrations of Hcy (0, 100, 500 and 1 000 μmol/L) and applied to immunofluorescence to observe the morphological changes of VSMCs via SM-actin staining. Western blot was used to detect the expression of VSMCs phenotypic markers, including Osteopontin, Calponin and smooth muscle myosin heavy chain (SM-MHC) and the expression of endoplasmic reticulum stress (ERS) related proteins, including C/EBP-homologous protein (CHOP), inositol-requiring kinase 1 (IRE-1) and glucose regulating protein 78 (GRP78) in the absence and presence of non-selective inhibitor of ERS, 4-phenylbutyric acid (4-PBA, 2 mg/ml). The Herpud1 mRNA and protein levels were determined in Hcy-stimulated VSMCs treated with 4-PBA or transfected with specific siRNA targeting Herpud1. Results: Compared with the control group, SM-actin staining results showed that the shape of VSMCs treated with different concentrations of Hcy for 24 hours changed from long fusiform into round form, arrangement of myofilament became irregular and the most significant alteration was found in the 500 μmol/L Hcy group. After intervention of 24 hours, various concentration of Hcy increased protein expression of Osteopontin, and reduced Calponin and SM-MHC protein expressions in VSMCs (all P<0.05). In addition, the results showed that Hcy increased the expression of CHOP, IRE-1 and GRP78 in a dose-dependent manner, which could be reversed by 4-PBA treatment (all P<0.05). However, 4-PBA inhibited Hcy induced upregulation of Osteopontin and downregulation of Calponin and SM-MHC, suggesting that ERS was involved in Hcy-induced phenotypic switching of VSMCs. Herpud1 protein was mostly expressed in the cytoplasm and was also expressed in the nucli, both in the control, Hcy and Hcy+4-PBA groups. Moreover, Hcy increased mRNA and protein levels of Herpud1 (P<0.05), whereas treatment with 4-PBA could significantly reduce Hcy-induced upregulation of Herpud1 (P<0.05). Furthermore, knockdown of Herpud1 abrogated the effects of Hcy on VSMCs phenotype markers. Conclusion: Herpud1 plays an important role in Hcy-induced phenotypic switching of VSMCs.