Contractile Phenotype Of Vascular Smooth Muscle Cells Research Articles

Abstract 129: BAF60c Prevents Abdominal Aortic Aneurysm Formation Through Maintaining Vascular Smooth Muscle Cell Homeostasis

Background: Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. Vascular smooth muscle cell (VSMC) dysfunction is involved in the dilatation and eventual rupture of AAA, with the precise epigenetic control of this process less studied. BAF60c, a unique subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, serves as a core modulator of physiological and pathological processes, yet little is known about its function in the vasculature and pathogenesis of AAA. Methods and Results: BAF60c is downregulated in AAAs from humans and mice with primary staining to the aortic VSMC, confirmed by single cell RNA sequencing. In vivo studies revealed that VSMC-specific knockout Baf60c markedly aggravated both AngII- and elastase-induced murine AAA formation, with significant increases of elastin degradation, inflammatory cell accumulation, VSMC phenotype switching, and apoptosis. In vitro studies showed that knockdown of BAF60c in human aortic smooth muscle cells resulted in the loss of contractile phenotype while upregulation of inflammatory genes and increased apoptosis. Chromatin immunoprecipitation followed by sequencing and qPCR, as well as CoIP assays showed that BAF60c maintained VSMC contractile phenotype by enhancing serum response factor association with its co-activator p300 and SWI/SNF complex, suppressed VSMC inflammation via precise control of the repressive state of NF-κB-target genes, as well as prevented VSMC apoptosis through transcriptional activation of KLF5-dependent BCL2 expression. Conclusions: Our study indicates that BAF60c is essential for SWI/SNF complex recruitment to chromatin and interaction with diverse transcription factors to precise epigenetic control of VSMC homeostasis and prevention of AAA formation.

Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling.

How the extracellular matrix (ECM) microenvironment modulates the contractile phenotype of vascular smooth muscle cells (VSMCs) and confers vascular homeostasis remains elusive. To explore the key ECM proteins in the maintenance of the contractile phenotype of VSMCs, we applied protein-protein interaction network analysis to explore novel ECM proteins associated with the VSMC phenotype. By combining in vitro and in vivo genetic mice vascular injury models, we identified nidogen-2, a basement membrane glycoprotein, as a key ECM protein for maintenance of vascular smooth muscle cell identity. We collected a VSMC phenotype-related gene dataset by using Gene Ontology annotation combined with a literature search. A computational analysis of protein-protein interactions between ECM protein genes and the genes from the VSMC phenotype-related gene dataset revealed the candidate gene nidogen-2, a basement membrane glycoprotein involved in regulation of the VSMC phenotype. Indeed, nidogen-2-deficient VSMCs exhibited loss of contractile phenotype in vitro, and compared with wild-type mice, nidogen-2-/- mice showed aggravated post-wire injury neointima formation of carotid arteries. Further bioinformatics analysis, coimmunoprecipitation assays, and luciferase assays revealed that nidogen-2 specifically interacted with Jagged1, a conventional Notch ligand. Nidogen-2 maintained the VSMC contractile phenotype via Jagged1-Notch3 signaling but not Notch1 or Notch2 signaling. Nidogen-2 enhanced Jagged1 and Notch3 interaction and subsequent Notch3 activation. Reciprocally, Jagged1 and Notch3 interaction, signaling activation, and Jagged1-triggered VSMC differentiation were significantly repressed in nidogen-2-deficient VSMCs. In accordance, the suppressive effect of Jagged1 overexpression on neointima formation was attenuated in nidogen-2-/- mice compared with wild-type mice. Nidogen-2 maintains the contractile phenotype of VSMCs through Jagged1-Notch3 signaling in vitro and in vivo. Nidogen-2 is required for Jagged1-Notch3 signaling.

MicroRNA‑199a‑5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch.

Varicose veins are among the most common disorders of the vascular system; however, the pathogenesis of varicose veins remains unclear. The present study aimed to investigate the roles of microRNA (miR)-199a-5p in varicose veins and in the phenotypic transition of vascular smooth muscle cells (VSMCs). Bioinformatics analysis confirmed that miR-199a-5p had target sites on the forkhead box C2 (FOXC2) 3′-untranslated region. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were used to detect the expression levels of miR-199a-5p and FOXC2 in varicose vein and normal great saphenous vein tissues. Cell Counting Kit-8 and Transwell migration assays were performed to validate the effects of miR-199a-5p on VSMCs. Contractile markers, such as smooth muscle 22α, calponin, smooth muscle actin and myosin heavy chain 11 were used to detect phenotypic transition. RT-qPCR revealed that miR-199a-5p was downregulated in varicose veins compared with expression in normal great saphenous veins, whereas FOXC2 was upregulated in varicose veins. In addition, biomarkers of the VSMC contractile phenotype were downregulated in varicose veins. Overexpression of miR-199a-5p by mimics suppressed VSMC proliferation and migration, whereas depletion of miR-199a-5p enhanced VSMC proliferation and migration. Notably, the effects caused by miR-199a-5p could be reversed by FOXC2 overexpression. Dual luciferase reporter analysis confirmed that FOXC2 was a target of miR-199a-5p. In conclusion, miR-199a-5p may be a novel regulator of phenotypic switching in VSMCs by targeting FOXC2 during varicose vein formation.

Smooth Muscle Overexpression of PGC1α Attenuates Atherosclerosis in Rabbits.

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MicroRNA-23b prevents aortic aneurysm formation by inhibiting smooth muscle cell phenotypic switching via FoxO4 suppression

AimsPhenotypic switching of vascular smooth muscle cells (VSMCs) is essential for the formation of abdominal aortic aneurysms (AAAs). MicroRNA-23b (miR-23b) has recently been shown to play a vital role in maintaining the VSMC contractile phenotype; however, little is known about the role of miR-23b in the formation of AAAs. Here, we investigated whether miR-23b prevents AAA formation by inhibiting VSMC phenotypic switching. Materials and methodsWe administered angiotensin II (Ang II, 1000 ng/kg/min) or vehicle to 10–12-week-old male apolipoprotein E knockout (ApoE−/−) or C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks. Key findingsThe expression of miR-23b was significantly reduced in the aorta during the early onset of AAA in angiotensin II-treated ApoE−/− mice and in human AAA samples. In vitro experiments showed that the suppression of SMC contractile marker gene expression induced by Ang II was accelerated by miR-23b inhibitors but inhibited by mimics. In vivo studies revealed that miR-23b deficiency in Ang II-treated C57BL/6J mice aggravated the formation of AAAs in these mice compared with control mice; the opposite results were observed in miR-23b-overexpressing mice. Mechanistically, miR-23b knockdown significantly increased the expression of the transcription factor forkhead box O4 (FoxO4) during VSMC phenotypic switching induced by Ang II. In addition, a luciferase reporter assay showed that FoxO4 is a target of miR-23b in VSMCs. SignificanceOur study revealed a pivotal role for miR-23b in protecting against aortic aneurysm formation by maintaining the VSMC contractile phenotype.

Bioinformatics Analysis Reveals MicroRNA-193a-3p Regulates ACTG2 to Control Phenotype Switch in Human Vascular Smooth Muscle Cells.

Aortic dissection (AD) is among the most fatal cardiovascular diseases. However, the pathogenesis of AD remains poorly understood. This study aims to integrate the microRNAs (miRNA) and mRNA profiles and use bioinformatics analyses with techniques in molecular biology to delineate the potential mechanisms involved in the development of AD. We used the human miRNA and mRNA microarray datasets GSE98770, GSE52093, and GEO2R, Venn diagram analysis, gene ontology, and protein–protein interaction networks to identify target miRNAs and mRNAs involved in AD. RNA interference, western blotting, and luciferase reporter assays were performed to validate the candidate miRNAs and mRNAs in AD tissues and human vascular smooth muscle cells (VSMCs). Furthermore, we studied vascular smooth muscle contraction in AD. In silico analyses revealed that miR-193a-3p and ACTG2 were key players in the pathogenesis of AD. miR-193a-3p was upregulated in the AD tissues. We also found that biomarkers for the contractile phenotype in VSMCs were downregulated in AD tissues. Overexpression and depletion of miR-193a-3p enhanced and suppressed VSMC proliferation and migration, respectively. Dual luciferase reporter assays confirmed that ACTG2 was a target of miR-193a-3p. ACTG2 was also downregulated in human AD tissues and VMSCs overexpressing miR-193a-3p. Taken together, miR-193a-3p may be a novel regulator of phenotypic switching in VSMCs and the miR-193a-3p/ACTG2 axis may serve as a promising diagnostic biomarker and therapeutic candidate for AD.

Protective effect of suppressor of cytokine signalling 1-based therapy in experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by chronic inflammation, oxidative stress and proteolytic activity in the aortic wall. Targeting JAK/signal transducer and activator of transcription (JAK/STAT) pathway is a promising strategy for chronic inflammatory diseases. We investigated the vasculo-protective role of suppressor of cytokine signalling-1 (SOCS1), the negative JAK/STAT regulator, in experimental AAA. A synthetic, cell permeable peptide (S1) mimic of SOCS1 kinase inhibitory domain to suppress STAT activation was evaluated in the well-established mouse model of elastase-induced AAA by monitoring changes in aortic diameter, cellular composition and gene expression in abdominal aorta. S1 function was further evaluated in cultured vascular smooth muscle cells (VSMC) and macrophages exposed to elastase or elastin-derived peptides. S1 peptide prevented AAA development, evidenced by reduced incidence of AAA, aortic dilation and elastin degradation, partial restoration of medial VSMC and decreased inflammatory cells and oxidative stress in AAA tissue. Mechanistically, S1 suppressed STAT1/3 activation in aorta, down-regulated cytokines, metalloproteinases and altered the expression of cell differentiation markers by favouring anti-inflammatory M2 macrophage and contractile VSMC phenotypes. In vitro, S1 suppressed the expression of inflammatory and oxidative genes, reduced cell migration and reversed the phenotypic switch of macrophages and VSMC. By contrast, SOCS1 silencing promoted inflammatory response. This preclinical study demonstrates the therapeutic potential of SOCS1-derived peptide to halt AAA progression by suppressing JAK/STAT-mediated inflammation and aortic dilation. S1 peptide may therefore be a valuable option for the treatment of AAA.

Role of Vascular Smooth Muscle Cell Plasticity and Interactions in Vessel Wall Inflammation.

The pathobiology of atherosclerotic disease requires further elucidation to discover new approaches to address its high morbidity and mortality. To date, over 17 million cardiovascular-related deaths have been reported annually, despite a multitude of surgical and nonsurgical interventions and advances in medical therapy. Existing strategies to prevent disease progression mainly focus on management of risk factors, such as hypercholesterolemia. Even with optimum current medical therapy, recurrent cardiovascular events are not uncommon in patients with atherosclerosis, and their incidence can reach 10–15% per year. Although treatments targeting inflammation are under investigation and continue to evolve, clinical breakthroughs are possible only if we deepen our understanding of vessel wall pathobiology. Vascular smooth muscle cells (VSMCs) are one of the most abundant cells in vessel walls and have emerged as key players in disease progression. New technologies, including in situ hybridization proximity ligation assays, in vivo cell fate tracing with the CreERT2-loxP system and single-cell sequencing technology with spatial resolution, broaden our understanding of the complex biology of these intriguing cells. Our knowledge of contractile and synthetic VSMC phenotype switching has expanded to include macrophage-like and even osteoblast-like VSMC phenotypes. An increasing body of data suggests that VSMCs have remarkable plasticity and play a key role in cell-to-cell crosstalk with endothelial cells and immune cells during the complex process of inflammation. These are cells that sense, interact with and influence the behavior of other cellular components of the vessel wall. It is now more obvious that VSMC plasticity and the ability to perform nonprofessional phagocytic functions are key phenomena maintaining the inflammatory state and senescent condition and actively interacting with different immune competent cells.

YAP/TAZ Are Required to Suppress Osteogenic Differentiation of Vascular Smooth Muscle Cells.

SummaryVascular smooth muscle cells (VSMCs) represent the prevailing cell type of arterial vessels and are essential for blood vessel structure and homeostasis. They have substantial potential for phenotypic plasticity when exposed to various stimuli in their local microenvironment. How VSMCs maintain their differentiated contractile phenotype is still poorly understood. Here we demonstrate that the Hippo pathway effectors YAP and TAZ play a critical role in maintaining the differentiated contractile phenotype of VSMCs. In the absence of YAP/TAZ, VSMCs lose their differentiated phenotype and undergo osteogenic differentiation, which results in vascular calcification. Osteogenic transdifferentiation was accompanied by the upregulation of Wnt target genes. The absence of YAP/TAZ in VSMCs led to Disheveled 3 (DVL3) nuclear translocation and upregulation of osteogenesis-associated genes independent of canonical Wnt/β-catenin signaling activation. Our data indicate that cytoplasmic YAP/TAZ interact with DVL3 to avoid its nuclear translocation and osteogenic differentiation, thereby maintaining the differentiated phenotype of VSMCs.

Integrin α9 is involved in the pathopoiesis of acute aortic dissection via mediating phenotype switch of vascular smooth muscle cell

Acute aortic dissection (AAD) is a devastating disease with high mortality; however, the pathogenic mechanisms of AAD remain poorly understood. Our present study aimed to identify genes associated with AAD and explore the molecular function of candidate genes in the pathogenesis of AAD. We used a whole-genome transcriptional microarray to identify putative AAD genes using ascending aortic tissues from four patients with AAD and four healthy organ donors. The differentially expressed genes were further validated in eight patients with AAD and eight healthy organ donors. Functional assessments were conducted to analyze the effects of the identified AAD genes on the phenotype of aortic vascular smooth muscle cells (VSMCs). The whole-genome transcriptional microarray analysis found 129 dysregulated genes in the ascending aortic tissues of AAD (fold change≥2), which were mainly associated with the focal adhesion pathway and actin cytoskeleton regulation pathway. Among these genes, integrin α9 (ITGA9) was identified to be involved in both pathways and downregulated by 50% in AAD patients. The association of ITGA9 with AAD was confirmed by Western blotting analysis (P = 0.003). Functional studies showed that knocking down ITGA9 in VSMCs resulted in a decrease in contractile markers (SM22α and α-SMA) and an increase in synthetic markers (OPN and SMemb), suggesting that the VSMCs switched from a contractile to a synthetic phenotype. After overexpression of ITGA9 by a recombinant adenovirus vector in VSMCs, SM22α and α-SMA were upregulated, while SMemb was downregulated, indicating a phenotypic switch from the synthetic to contractile phenotype of VSMCs. In conclusion, our study identified ITGA9 as a novel AAD gene. This gene is downregulated in patients with AAD and is involved in the regulation of the phenotypic switch of VSMCs from a contractile to a synthetic phenotype.

A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway

ObjectiveArsenic trioxide (ATO or As2O3) has beneficial effects on suppressing neointimal hyperplasia and restenosis, but the mechanism is still unclear. The goal of this study is to further understand the mechanism of ATO's inhibitory effect on vascular smooth muscle cells (VSMCs). Methods and resultsThrough in vitro cell culture and in vivo stent implanting into the carotid arteries of rabbit, a synthetic-to-contractile phenotypic transition was induced and the proliferation of VSMCs was inhibited by ATO. F-actin filaments were clustered and the elasticity modulus was increased within the phenotypic modulation of VSMCs induced by ATO in vitro. Meanwhile, Yes-associated protein (YAP) nuclear translocation was inhibited by ATO both in vivo and in vitro. It was found that ROCK inhibitor or YAP inactivator could partially mask the phenotype modulation of ATO on VSMCs. ConclusionsThe interaction of YAP with the ROCK pathway through ATO seems to mediate the contractile phenotype of VSMCs. This provides an indication of the clinical therapeutic mechanism for the beneficial bioactive effect of ATO-drug eluting stent (AES) on in-stent restenosis (ISR).

Identification of Long Noncoding RNAs Involved in Differentiation and Survival of Vascular Smooth Muscle Cells

Long noncoding RNAs (lncRNAs) have recently been implicated in many pathophysiological cardiovascular processes, including vascular remodeling and atherosclerosis. However, the functional role of lncRNAs in the differentiation, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs) is largely unknown. In this study, differentially expressed lncRNAs in synthetic and contractile human VSMCs were screened using RNA sequencing. Among the seven selected lncRNAs, the expression of MSC-AS1, MBNL1-AS1, and GAS6-AS2 was upregulated, whereas the expression of NR2F1-AS1, FUT8-AS1, FOXC2-AS1, and CTD-2207P18.2 was reduced upon VSMC differentiation. We focused on the NR2F1-AS1 and FOXC2-AS1 lncRNAs and showed that their knockdown significantly reduced the expression of smooth muscle contractile marker genes (ACTA2, CNN1, and TAGLN). Furthermore, FOXC2-AS1 was found to regulate cell proliferation and apoptosis through Akt/mTOR signaling, and affect Notch signaling, which is a key regulator of the contractile phenotype of VSMCs. Taken together, we identified novel lncRNAs involved in VSMC proliferation and differentiation and FOXC2-AS1 as a multifunctional regulator for vascular homeostasis and associated diseases.

Human Umbilical Cord Mesenchymal Stem Cells Attenuate Abdominal Aortic Aneurysm Progression in Sprague-Dawley Rats: Implication of Vascular Smooth Muscle Cell Phenotypic Modulation.

Abdominal aortic aneurysm (AAA) is life-threatening, for which efficient nonsurgical treatment strategy has not been available so far. Several previous studies investigating the therapeutic effect of mesenchymal stem cells (MSCs) in AAA indicated that MSCs could inhibit aneurysmal inflammatory responses and extracellular matrix destruction, and suppress aneurysm occurrence and expansion. Vascular smooth muscle cell (VSMC) phenotypic plasticity is reported to be predisposed in AAA initiation and progression. However, little is known about the effect of MSCs on VSMC phenotypic modulation in AAA. In this study, we investigate the therapeutic efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) in elastase-induced AAA model and evaluate the effect of UC-MSC on VSMC phenotypic regulation. We demonstrate that the intravenous injection of UC-MSC attenuates elastase-induced aneurysmal expansion, reduces elastin degradation and fragmentation, inhibits MMPs and TNF-α expression, and preserves and/or restores VSMC contractile phenotype in AAA. Taken together, these results highlight the therapeutic and VSMC phenotypic modulation effects of UC-MSC in AAA progression, which further indicates the potential of applying UC-MSC as an alternative treatment candidate for AAA.

P0888FIBROBLAST GROWTH FACTOR 23 PRODUCES ARTERIAL STIFFNESS THROUGH CHANGES IN VASCULAR SMOOTH MUSCLE CELL PHENOTYPE

Abstract Background and Aims In patients with chronic kidney disease (CKD), high levels of c-terminal fibroblast growth factor 23 (FGF23) are associated with cardiovascular disease and mortality. Vascular smooth muscle cells (VSMC) may present two clearly differentiated functional phenotypes, contractile and synthetic. The abundance of synthetic phenotype is associated with vascular dysfunction and it is unknown whether in FGF23 may promote transition from a contractile to a synthetic phenotype in VSMC causing vascular stiffness. The present study was conducted to evaluate whether FGF23 affects VSMC phenotype and arterial stiffness. Method and Results High levels of FGF23 promoted VSMC transition from a contractile to a synthetic phenotype. These effects were mediated through FGFR1 and Ras/MAPK signaling activation. Inhibition of both pathways enhanced contractile phenotype of VSMC. The pro-contractile microRNAs, miR-221 and miR-222 were reduced by FGF23 and miR-221 transfection recovered the contractile phenotype of VSMC decreased by FGF23. In experimental rats, exogenous infusion of FGF23 produced an increase in vascular wall thickness with VSMC exhibiting synthetic phenotype and reduction of plasma levels of miR-221. Functional studies performed on aortic arterial rings revealed that passive and active forces were altered in rats treated with FGF23. In a group of CKD stage 2-3 patients with rather high levels of FGF23 it was observed an increased in pulse pressure reflecting vascular stiffness together with low plasma levels of miR-221 and miR-222. Conclusion FGF23 favors the transition of VSMC from contractile to synthetic phenotype causing vascular dysfunction and arterial stiffness; this may be a mechanism by which FGF23 contribute directly to the development of vascular disease in CKD patients.

Prevention of aortic dissection and aneurysm via an ALDH2-mediated switch in vascular smooth muscle cell phenotype.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive. Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs. ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin

Vascular smooth muscle cells (VSMCs) play an essential role in the development and progression of atherosclerosis. The phenotypic switch from contractile to synthetic cells induces excess proliferation and migration of VSMCs into the intima contributing to plaque formation. Adiponectin (Adpn) is an adipokine that regulates glucose metabolism through AMPK and plays beneficial roles in atherosclerosis. We hypothesize that Adpn promotes the contractile phenotype of VSMC reducing proliferation and migration of VSMCs, a process that is protective against plaque formation.Differentiation, proliferation, and signaling through AMPK‐dependent pathways were assessed in VSMCs isolated from aortas of wild type (WT) and Adpn−/− mice. Proliferation markers, including cyclin D1, were increased in Adpn−/− cells, and AMPK activity and differentiation markers were reduced. Cells were analyzed for matrix metalloproteinases (MMPs) which are responsible for extracellular matrix (ECM) remodeling, a contributing factor to the VSMC phenotypic switch. We found that MMP protein expression and activity, measured by zymography, were reduced in Adpn−/− cells. To test whether the phenotypic switch is regulated by AMPK, we treated cells with Metformin, an AMPK activator, or AdipoRon, an activator of Adpn receptors. Metformin and AdipoRon reduced Cyclin D1 and MAPK signaling in Adpn−/− cells, suggesting that AMPK prevents VSMC proliferation; however, none of the treatments yielded an effect on markers of differentiation.These data illustrated that Adpn regulates proliferation through an AMPK‐dependent signaling pathway, while regulating differentiation by an AMPK‐independent mechanism. The role of MMPs in VSMC differentiation in response to Adpn remains to be elucidated.

TRIM28 and TRIM27 are required for expressions of PDGFRβ and contractile phenotypic genes by vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif-containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif-containing 28 (TRIM28) and tripartite motif-containing 27 (TRIM27) in VSMCs. We observed that siRNA-mediated knockdown of TRIM28 and TRIM27 inhibited platelet-derived growth factor (PDGF)-induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor-β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N-terminal RING-B boxes-Coiled-Coil domain. Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs.

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.

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.

Mild Hypercaloric Intake is Associated with an Impaired Vascular Smooth Muscle Phenotype in Absence of Hyperglycemia: Potential Modulation by Anti‐diabetic Drugs

Cardiovascular disease is the leading cause of mortality and morbidity in diabetes. Diabetes‐induced vascular complications are associated with the contractile‐to‐synthetic phenotypic switch of the vascular smooth muscle cells (VSMCs). Emerging experimental and clinical data have shown that functionally impaired VSMCs contribute to the high incidence rates of macrovascular complications in diabetes. Moreover, a significant proportion of diabetic patients show signs of vascular impairment at initial presentation before prolonged exposure to hyperglycemia. The underlying mechanisms leading to VSMC deterioration, particularly in early stages of metabolic dysfunction, remain largely unknown. In this study, we have investigated VSMC phenotypic alterations in a model of early metabolic derangements. Rats fed a mild hypercaloric (MHC) diet were used at time points prior to the development of hyperglycemia. Aortic VSMCs were isolated from control rats, from MHC fed rats sacrificed after 4, 8, or 12 weeks of exposure. At twelve weeks, MHC‐fed rats did not show increased fasting or random blood glucose, impaired glucose tolerance, increased weight, or hypertension. The effect of a two‐week treatment with metformin and pioglitazone prior to sacrifice at the 12‐week time point was examined. A gradual reduction in VSMC activity in MTS assays was observed as a function of MHC‐feeding duration, with the sharpest decrease observed at 12 weeks. Cell cycle assays revealed no changes in cell proliferation indicating that the changes in MTS assays were rather a reduction in metabolic activity. Moreover, expression of VSMC contractile phenotype markers, a‐smooth muscle actin and calponin, decreased progressively with MHC feeding. Interestingly, in vivo treatment with non‐hypoglycemic doses of either metformin and pioglitazone, known to have positive vascular pleiotropic effects, restored VSMCs metabolic activity and contractile marker expression to control levels. Significantly, no changes were detected VSMC metabolic activity and contractile marker expression following in vitro exposure to either a range of concentrations of free fatty acids, to simulate the MHC diet effect, or to metformin or pioglitazone. The contrast between in vitro and in vivo results suggests a direct effect of lipids on VSMC phenotype and function is unlikely; however, the changes observed might be a result of a more complex interaction with more than one tissue type. We hypothesize that the observed effect of MHC diet is mediated by the induction of perivascular adipose inflammation. Studies are underway to verify this claim.Support or Funding InformationSupported by AUB MPP fund #320148This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.