Vascular Smooth Muscle Cells Phenotypic Modulation Research Articles

Abstract 176: O-GlcNAc Transferase Promotes Vascular Smooth Muscle Cell De-differentiation

Vascular smooth muscle cells (VSMCs) in mature vessels have a differentiated, contractile phenotype but retain the ability to reversibly de-differentiate in response to environmental cues. Plasticity is necessary for development and wound healing, but also contributes to pathologies such as atherosclerosis and intimal hyperplasia. O- linked N- acetylglucosamine ( O -GlcNAc) transferase (OGT) mediates nutrient-sensitive post-translational modification of serine and threonine residues and has been implicated in multiple diseases, but its role in VSMC phenotypic modulation is not known. In human coronary SMCs, we found that OGT knockdown significantly increased contractile marker expression, including MYH11 and ACTA2, at the mRNA and protein levels. OGT knockdown also decreased proliferation rates after serum treatment in VSMCs. In addition, rapamycin, a differentiating agent, decreased OGT protein expression and O- GlcNAc levels. Conversely, de-differentiation with PDGF treatment increased OGT expression and global O- GlcNAc. Neither treatment affected OGT mRNA levels. OGT has been shown to increase the protein stability of EZH2, the enzyme that generates the inhibitory histone epigenetic mark H3K27me3. We found that OGT knockdown did not affect EZH2 mRNA levels but significantly decreased EZH2 protein expression as well as global H3K27me3 in VSMCs. Similar to OGT, EZH2 expression was decreased upon rapamycin treatment and increased with PDGF. A decrease in ubiquitination of EZH2 was observed after PDGF treatment, suggesting an increase in protein stability. Importantly, EZH2 knockdown increased protein expression of SMC contractile markers while EZH2 overexpression repressed these markers. Wire injury of mouse femoral arteries increased H3K27me3 levels after 21 days. OGT expression was elevated in the innermost layer of the neointima at the same time point. These data demonstrate that OGT inhibits VSMC differentiation and that OGT and its target EZH2 are both dynamically regulated during phenotypic modulation. Future work will illuminate if OGT promotes VSMC de-differentiation through EZH2-mediated histone modification at contractile gene promoters.

0551 : Slug: a common target of invasive carcinoma and atherosclerosis?

Phenotypic transition (or transdifferentiation) of vascular smooth muscle cells (VSMC) plays a pivotal role in atherosclerosis. This phenomenom allows these cells to migrate from the media to the intima of arteries where they contribute to atherosclerotic lesions development and secrete numerous pro-inflammatory mediators. Migration and inflammation are also features of cancer cells. Molecules that regulate these properties, especially in carcinoma cells, have been identified. It is the case of the transcription factor Slug (or Snail2) which, by allowing epithelial to mesenchymal transition of pre-cancerous epithelial cells, participates in their transformation into invasive carcinoma cells. Our results show for the first time that Slug is well detectable in differentiated and transdifferentiated VSMC. Silencing of the gene encoding Slug inhibits by about 50% to 60% interleukine-1β (IL-1β) induction of pro-migratory and pro-inflammatory genes such as MMP (matrix metalloproteinases) 9 and 13, type IIa secretory phospholipase A2, inducible nitric oxide synthase and CCL3 ( CC-motif ligand 3 ) and CXL2 ( CXC motif ligand 2 ) chemokines in VSMC. These results indicate that Slug could be involved in IL-1β-induced VSMC phenotypic transition especially as we show that IL-1β up-regulates Slug protein level in VSMC. This effect is independent from a genic effect. Slug is described to be a very unstable protein in non cancerous epithelial cells and its stabilization by growth factors during malignant transformation usually involves glycogen synthase kinase-3β inhibition. However we demonstrate that IL-1β doesn’t inhibit this enzyme in VSMC which excludes its involvment in IL-1β-induced Slug protein level in VSMC. All together, our results highlight the potential role of Slug in atherogenesis, a role which would come to be added to the well established one that it plays in carcinoma cells invasion. The author hereby declares no conflict of interest

Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2

Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification.

An overview of potential molecular mechanisms involved in VSMC phenotypic modulation.

The fully differentiated medial vascular smooth muscle cells (VSMCs) of mature vessels keep quiescent and contractile. However, VSMC can exhibit the plasticity in phenotype switching from a differentiated and contractile phenotype to a dedifferentiated state in response to alterations in local environmental cues, which is called phenotypic modulation or switching. Distinguishing from its differentiated state expressing more smooth muscle (SM)-specific/selective proteins, the phenotypic modulation in VSMC is characterized by an increased rate of proliferation, migration, synthesis of extracellular matrix proteins and decreased expression of SM contractile proteins. Although it has been well demonstrated that phenotypic modulation of VSMC contributes to the occurrence and progression of many proliferative vascular diseases, little is known about the details of the molecular mechanisms of VSMC phenotypic modulation. Growing evidence suggests that variety of molecules including microRNAs, cytokines and biochemical factors, membrane receptors, ion channels, cytoskeleton and extracellular matrix play important roles in controlling VSMC phenotype. The focus of the present review is to provide an overview of potential molecular mechanisms involved in VSMC phenotypic modulation in recent years. To clarify VSMC differentiation and phenotypic modulation mechanisms will contribute to producing cell-based therapeutic interventions for aberrant VSMC differentiation-related diseases.

Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration.

Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by platelet-derived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease.

Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of artery restenosis following percutaneous coronary intervention. Digoxin has long been used in the treatment of heart failure and has been shown to inhibit the proliferation of cancer cells through multiple pathways. However, the potential role of digoxin in the regulation of VSMC proliferation and migration and its effectiveness in the treatment of cardiovascular diseases, such as restenosis, remains unexplored. In the present study, we demonstrate that digoxin-induced growth inhibition is associated with the downregulation of CDK activation and the restoration of p27Kip1 levels in platelet-derived growth factor (PDGF)-stimulated VSMCs. In addition, we found that digoxin restored the PDGF-BB-induced inhibition of integrin linked kinase (ILK) expression and prevented the PDGF-BB-induced activation of glycogen synthase kinase (GSK)-3β. Furthermore, digoxin inhibited adhesion molecule and extracellular matrix relative protein expression. Finally, we found that digoxin significantly inhibited neointima formation, accompanied by a decrease in cell proliferation following vascular injury in rats. These effects of digoxin were shown to be mediated, at least in part, through an increase in ILK/Akt signaling and a decrease in GSK-3β signaling in PDGF-BB-stimulated VSMCs. In conclusion, our data demonstrate that digoxin exerts an inhibitory effect on the PDGF-BB-induced proliferation, migration and phenotypic modulation of VSMCs, and prevents neointima formation in rats. These observations indicate the potential therapeutic application of digoxin in the treatment of cardiovascular diseases, such as restenosis.

MiR-135b-5p and MiR-499a-3p Promote Cell Proliferation and Migration in Atherosclerosis by Directly Targeting MEF2C.

Proliferation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are critical processes involved in atherosclerosis. Recent studies have revealed that microRNAs (miRNAs) can be detected in circulating blood with a stable form and the expression profiles differ in many cellular processes associated with coronary artery disease (CAD). However, little is known about their role, especially serum-derived miRNAs, in ECs and VSMCs phenotype modulation during atherosclerosis. We compared the miRNA expressions in serum samples from 13 atherosclerotic CAD patients and 5 healthy control subjects and identified 36 differentially expressed miRNAs. The expression of selected miRNAs (miR-135b-5p and miR-499a-3p) was further validated in 137 serum samples. Interestingly, miR-135b-5p and miR-499a-3p directly regulated a common target gene: myocyte enhancer factor 2C (MEF2C) which plays an important role in modulating cell phenotype of cardiovascular systems. Furthermore, our results indicated that the 2 elevated miRNAs could jointly promote ECs and VSMCs proliferation and migration by repressing MEF2C expression. Together, our findings demonstrated a serum-based miRNA expression profile for atherosclerotic CAD patients, potentially revealing a previously undocumented mechanism for cell proliferation and migration mediated by miR-135b-5p and miR-499a-3p, and might provide novel insights into the role of circulating miRNAs in atherosclerosis pathogenesis.

Essential role of krüppel-like factor 5 during tumor necrosis factor α-induced phenotypic conversion of vascular smooth muscle cells

Tumor necrosis factor α (TNFα) plays an essential role in the regulation of vascular smooth muscle cell (VSMC) phenotype. In the present study, we provide evidence that krüppel-like factor 5 (KLF5) plays an essential role in TNFα-induced phenotypic conversion of VSMCs. Ectopic expression of KLF5 completely blocked phenotypic conversion of VSMCs from synthetic to contractile type. In addition, stimulation of VSMCs with TNFα facilitated expression of KLF5, whereas expression of smooth muscle marker genes such as SM22α and smooth muscle actin (SMA) was significantly down-regulated. TNFα significantly enhanced the promoter activity of KLF5 as well as mRNA level, which is significantly suppressed by the inhibition of the MAPK pathway. Silencing of KLF5 suppressed TNFα-induced phenotypic conversion of VSMCs, whereas overexpression of KLF5 stimulated phenotypic conversion of VSMCs and facilitated the loss of angiotensin II (AngII)-dependent contraction. Finally, overexpression of KLF5 significantly attenuated the promoter activity of SM22α and SMA. Therefore, we suggest that TNFα-dependent induction of KLF5 may play an essential role in phenotypic modulation of VSMCs.

Abstract 497: 5-Methoxytryptophan Protects Against Vascular Remodeling

Vascular smooth muscle cells (VSMCs) in the blood vessel wall exhibit a differentiated phenotype; their main function is contraction and to regulate vascular tone. In response to injury, VSMCs undergo a phenotypic transition whereby they proliferate and migrate from the medial layer into the intima, contributing to lesion formation and atherosclerosis. 5-methoxytryptophan (5-MTP), a recently identified novel tryptophan metabolite, has been shown to inhibit cancer cell growth, migration, and cancer metastasis. We hypothesized that 5-MTP might play an analogous role in vascular disease as in tumorigenesis. To test our hypothesis, we subjected 12 weeks old C57BL/6 mice to a carotid artery cessation of blood flow model to induce neointima formation. Following surgery, mice were treated with vehicle (PBS) or 100 mg/kg of 5-MTP by intraperitoneal injection 3 times a week. Four weeks later, carotid arteries were harvested for histological analysis. H&E and elastin staining revealed robust neointima in ligated carotids of vehicle-treated mice. In contrast, 5-MTP significantly attenuated neointima formation. Given that proinflammatory cytokine interleukin 1 beta (IL-1β) is increased in the injured vessel wall, we examined whether IL-1β affected VSMC phenotypic modulation. Indeed, IL-1β downregulated VSMC marker SM α-actin expression and increased VSMC proliferation and migration. Importantly, in VSMCs, 5-MTP attenuated IL-1β-mediated SM α-actin downregulation, proliferation, and migration, suggesting a potential role of 5-MTP in controlling VSMC phenotypic modulation. Furthermore, 5-MTP inhibited IL-1β-mediated p38 MAPK activation. Inhibiting p38 MAPK activation by SB203580 dose-dependently decreased IL-1β-induced VSMC proliferation. Taken together, our results suggest an important role of 5-MTP in vascular disease.

Abstract 719: PFKFB3 Mediated Glycolysis in Vascular Smooth Muscle Cells Is Essential for Arterial Neointima Formation

Background: Mature vascular smooth muscle cells (VSMCs) exhibit limited proliferation and migration ability. However, in response to stresses, VSMCs undergo phenotypic modulation characterized by increased proliferation and motility. This phenotypic modulation is associated with several vascular diseases, including atherosclerosis, arterial neointima formation. Elevated glycolysis has been described in proliferative VSMCs. The isoform 3 of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3) is a regulatory factor for VSMC glycolysis. PFKFB3 catalyze the synthesis of fructose 2, 6-bishosphate (F2, 6 BP). The latter is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme in glycolytic flux. It is unclear whether PFKFB3 mediated glycolysis play a role in VSMC phenotypic modulation andarterial neointima formation. Methods and results: PDGF treatment increased PFKFB3 expression in human aortic smooth muscle cells (HASMCs). This was accompanied with enhanced proliferation and migration of HASMCs. Knockdown of PFKFB3 with adenovirus carrying PFKFB3 short hairpin RNA suppressed PDGF-induced HASMC proliferation and migration. Examination of intracellular signaling indicated PI3K/Akt participated into PFKFB3-mediated glycolysis, and further cooperated with PFKFB3 to modulate phenotype of HASMCs. In a model of the left common carotid artery ligation, both PFKFB3 heterozygotes and mice with deficiency of PFKFB3 in VSMCs showed the decreased arterial neointima compared with controls mice. Conclusions: PFKFB3 mediates glycolysis and cooperates with PI3K/AKT, thereafter promoting proliferation and migration of VSMCs and leading to the formation of an increased arterial neointima. Thus, PFKFB3 is a promising therapeutic target for the treatment of atherosclerosis, arterial neointima and other peripheral arterial diseases.

ADAR1‐mediated RNA editing, a novel mechanism controlling phenotypic modulation of vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down‐regulation of SMC contractile genes. Platelet‐derived growth factor‐BB (PDGF‐BB), a well‐known stimulator of SMC phenotypic modulation, down‐regulates SMC gene expression via posttranscriptional regulation of the related genes. The post‐transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down‐regulation of SMC contractile genes is caused by abnormal RNA splicing of their precursor mRNAs (pre‐mRNAs). This abnormal pre‐mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF‐BB induces ADAR1 while down‐regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF‐BB‐blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre‐mRNA is accumulated while their mature mRNA is decreased in balloon‐injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Our study suggests that ADAR1‐mediated RNA splicing may also play an important role in VSMC phenotypic modulation and vascular remodeling in vivo.

Phenotypic modulation of vascular smooth muscle cells in diabetes mellitus and intervention of traditional Chinese medicines

Proliferation and migration of vascular smooth muscle cells (VSMC) are common pathological features of diabetic vascular complications,such as atherosclerosis and hypertension. Phenotypic modulation of VSMC is the basis for VSMC proliferation and migration. Therefore, studies on VSMC phenotypic modulation and its mechanisms in diabetes mellitus were of important significance to the prevention and therapy of diabetic vascular complications. This paper introduces VSMC phenotypic modulation and the underlying mechanisms in diabetes mellitus, and summarizes advance of studies on traditional Chinese medicine intervention upon VSMC phenotypic modulation, so as to provide reference for preventing and treating diabetic vascular complications with traditional Chinese medicines.

Abstract 16650: Role of Copper Transport Proteins in VSMC Phenotypic Modulation and Vascular Remodeling

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in vascular repair and vascular remodeling after injury. This involves transition from quiescent contractile (C) to de-differentiated phenotype (S)(C to S) induced by PDGF, followed by re-differentiation to contractile phenotype (S to C). Copper (Cu), an essential micronutrient, is involved in vascular remodeling, and its bioavailability is controlled by Cu transport proteins (importer CTR1 and exporter ATP7A) and Cu chaperone (antioxidant-1, Atox1), which activates Cu enzymes such as amine oxidase, SSAO. However, role of Cu transport proteins in VSMC phenotypic modulation is entirely unknown. Here we show that PDGF stimulation in primary VSMC induced de-differentiation (C to S) by downregulating contractile SMC protein, which was accompanied by a robust increase in Cu uptake and CTR1, ATP7A and SSAO levels, and inhibited by Cu chelators. Knockdown of CTR1, ATP7A, SSAO, but not siAtox1, suppressed PDGF-induced VSMC de-differentiation (69%, 72% and 92%, respectively). Of note, ATP7A depletion inhibited PDGF-induced SSAO while SSAO siRNA suppressed ATP7A induction, suggesting that there is a positive feedback loop whereby ATP7A-SSAO pathway promotes de-differentiation. Mechanistically, PDGF-induced expression of KLF4, a repressor of SMC protein expression, was blocked by depletion of CTR1, ATP7A and SSAO. We reported that Atox1 also functions as a Cu-dependent transcription factor independent of ATP7A. Consistently, Atox1 siRNA suppressed serum starvation-induced VSMC re-differentiation, and overexpression of Atox1 increased SMC contractile gene expression. In vivo vascular injury model showed that SSAO and Atox1 were increased in neointimal SMCs at early de-differentiation phase and at later re-differentiation phase, respectively, in response to injury. Summary and Conclusion: CTR1-ATP7A-SSAO pathway is involved in PDGF-induced VSMC de-differentiation via regulating KLF4 while Cu dependent transcription factor function of Atox1 promotes VSMC re-differentiation via regulating SMC contractile gene transcription. Cu transport proteins and SSAO are potential therapeutic targets for modulating VSMC phenotype switching and vascular remodeling.

Modulation of cysteine-rich protein 2 expression in vascular injury and atherosclerosis.

Vascular smooth muscle cells (VSMCs) of the arterial wall normally display a differentiated and contractile phenotype. In response to arterial injury, VSMCs switch to a synthetic phenotype, contributing to vascular remodeling. Cysteine-rich protein 2 (CRP2) is a cytoskeletal protein expressed in VSMCs and blunts VSMC migration in part by sequestering the scaffolding protein p130Cas at focal adhesions. CRP2 deficiency in mice increases neointima formation following arterial injury. The goal of this study was to use Csrp2 promoter-lacZ transgenic mice to analyze CRP2 expression during VSMC phenotypic modulation. In a neointima formation model after carotid artery cessation of blood flow, lacZ reporter activity and smooth muscle (SM) α-actin expression in the media were rapidly downregulated 4days after carotid ligation. Fourteen days after ligation, there was a high level expression of both Csrp2 promoter activity and SM α-actin protein expression in neointimal cells. In atherosclerosis prone mice fed an atherogenic diet, Csrp2 promoter activity was detected within complex atherosclerotic lesions. Interestingly, Csrp2 promoter activity was also present in the fibrous caps of complicated atherosclerotic lesions, indicating that CRP2 might contribute to plaque stability. These findings support the concept that CRP2 contributes to the phenotypic modulation of VSMCs during vascular disease. Modulating transcription to increase CRP2 expression during vascular injury might attenuate vascular remodeling. In addition, increased CRP2 expression at the fibrous caps of advanced lesions might also serve to protect atherosclerotic plaques from rupture.

Abstract 574: Regulation of Vascular Smooth Muscle Cells Phenotype by the Adhesion of Platelets to Endothelial Cells

Objective: Platelets can regulate endothelial cell genes expression through their adhesion to the subendothelium in response to vascular injury. Recently it has been reported that some endothelial cells (ECs)-secreted genes can modulate vascular smooth muscle cells (VSMCs) phenotypic transformation by ECs/VSMCs co-culture. However, little is known about the effects of platelets adhesion to ECs interaction on VSMCs phenotype. In this study, we investigated the role of some genes secreted by platelets adhesion to EC in regulating SMC phenotypic transformation. Methods and Results: By Q-RT-PCR, expression of PAI-1, MMP-2 and MMP-9 were up-regulated in ECs after platelets adhesion to ECs comparing to ECs monoculture alone. In migration (scratch) and proliferation (CCK-8) assays, platelets adhesion to ECs increased EC migration-promoting activity and proliferative activity. After adhesion of platelets to ECs/VSMCs co-culture, expression of VSMCs contractile apparatus SM-MHC, Smoothelin-B, SMA and SM22a were significantly decreased comparing to ECs/VSMCs co-culture by Q-RT-PCR, but decreasing slightly in adhesion with platelets from type 2 diabetes. Conversely, it increased the expression of synthetic marker Smemb, CCND-1, CCND-2. Further analysis found that platelets adhesion to ECs/VSMCs co-culture significantly increased VSMCs migration-promoting activity and proliferative activity. In Gelatin Zymography assays, there were high levels of MMP-2 and MMP-9 after adhesion of platelets to ECs/VSMCs co-culture comparing to ECs/VSMCs co-culture alone. Conclusion: These results offer important insights into the mechanisms controlling phenotypic modulation of VSMCs by platelets adhesion to ECs, and help to explain the effects of platelets-ECs-VSMCs interaction on VSMCs contractile/synthetic, migration, and proliferation, suggesting that the adhesion of platelets to ECs is a key process in regulating phenotypic switch of VSMCs.

MiR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

ObjectivesVascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid. Approach and resultsPrimary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbf β1, osteocalcin and osteopontin were significantly increased and SM-22β expression was decreased. ALP activity was induced by β-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification. ConclusionOur study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1.

Resveratrol Induces Vascular Smooth Muscle Cell Differentiation through Stimulation of SirT1 and AMPK

Phenotypic plasticity in vascular smooth muscle cells (VSMC) is necessary for vessel maintenance, repair and adaptation to vascular changes associated with aging. De-differentiated VSMC contribute to pathologies including atherosclerosis and intimal hyperplasia. As resveratrol has been reported to have cardio- protective effects, we investigated its role in VSMC phenotypic modulation. We demonstrated the novel finding that resveratrol promoted VSMC differentiation as measured by contractile protein expression, contractile morphology and contraction in collagen gels. Resveratrol induced VSMC differentiation through stimulation of SirT1 and AMPK. We made the novel finding that low or high dose resveratrol had an initially different mechanism on induction of differentiation. We found that low dose resveratrol stimulated differentiation through SirT1-mediated activation of AKT, whereas high dose resveratrol stimulated differentiation through AMPK-mediated inhibition of the mTORC1 pathway, allowing activation of AKT. The health effects of resveratrol in cardiovascular diseases, cancer and longevity are an area of active research. We have demonstrated a supplemental avenue where-by resveratrol may promote health by maintaining and enhancing plasticity of the vasculature.

DHEA inhibits vascular remodeling following arterial injury: a possible role in suppression of inflammation and oxidative stress derived from vascular smooth muscle cells

Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-κB activity was detected by NF-κB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-κB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-κB signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition.

Differential expression of vascular smooth muscle-modulating microRNAs in human peripheral blood mononuclear cells: novel targets in essential hypertension

Vascular smooth muscle cell (VSMC) phenotypic plasticity has a critical role in the pathophysiology of arterial remodeling in essential hypertension. MicroRNAs are emerging as potential biomarkers and therapeutic targets in cardiovascular disease. We assessed the expression levels of the microRNAs miR-143, miR-145, miR-21, miR-133 and miR-1, which are implicated in VSMC phenotypic modulation, in 60 patients with essential hypertension and 29 healthy individuals. All patients underwent 24-h ambulatory blood pressure (BP) monitoring. MicroRNA levels in peripheral blood mononuclear cells were quantified by real-time reverse transcription polymerase chain reaction. Hypertensive patients showed lower miR-143 (2.20±0.25 versus 4.19±0.57, P<0.001), miR-145 (13.51±1.73 versus 22.38±3.31, P=0.010) and miR-133 (8.15±1.32 versus 37.03±8.18, P<0.001) and higher miR-21 (3.08±0.32 versus 2.06±0.31, P=0.048) and miR-1 (33.94±5.19 versus 12.35±2.13 P=0.006) expression levels compared with controls. In hypertensive patients, we observed correlations of miR-143 (r = -0.380, P=0.003), miR-145 (r=-0.405, P=0.001), miR-21 (r=-0.486, P<0.001) and miR-133 (r=0.479, P<0.001) expression levels with 24-h diastolic BP. Furthermore, we observed correlations of miR-21 (r=-0.291, P=0.024), miR-1 (r=-0.312, P=0.015) and miR-133 (r=0.310, P=0.016) levels with the dipping status. Associations of miR-143 (r=-0.292, P=0.025), miR-145 (r=-0.399, P=0.002), miR-21 (r=-0.343, P=0.008) and miR-133 (r=0.370, P=0.004) levels with 24-h mean pulse pressure were also found. Our data provide important evidence that VSMC-modulating microRNAs are closely related to essential hypertension in humans and they may represent potential therapeutic targets in essential hypertension.

Simvastatin Decreases Stent‐Induced In‐Stent Restenosis Rate Via Downregulating the Expression of PCNA and Upregulating that of p27kip1

The inhibition of the proliferation and migration of vascular smooth muscle cells (VSMC) is one cholesterol-independent effect of statins that could lower the rate of in-stent restenosis (ISR), even if the exact mechanism remains unclear. The aim of this study was to explore the exact inhibitory mechanisms of simvastatin on ISR in vivo. Forty-five rabbits were randomized into 3 feeding groups of equal size (n=15): normal rabbit chow (normal group), a high-cholesterol diet (control group), and a simvastatin-enriched high-cholesterol diet (simvastatin group). Balloon de-endothelialization was first performed in the control and simvastatin groups after 3 days, followed by stent deployment at week 14. All rabbits were killed at week 18, and the histological changes of the ISR segments were observed. The expressions of cyclin-dependent kinase inhibitor p27 (p27kip1), proliferating cell nuclear antigen (PCNA), and α-smooth muscle (α-SM) actin were measured. In the simvastatin group compared to the control group, the neointimal thickness, neointimal area, and degree of stenosis decreased, while the residual lumen area increased significantly (P<0.05). Moreover, the expression of α-SM actin in the control group decreased by 55.4% compared to the normal group, while it increased by 29.7% with respect to the simvastatin group (P<0.05). Finally, the expression of p27kip1 increased, while that of PCNA decreased significantly in the simvastatin group compared to the control group (P<0.05). Simvastatin may inhibit VSMC phenotype modulation and proliferation by downregulating the expression of PCNA and upregulating that of p27kip1.