Phenotypic switching of vascular smooth muscle cells in atherosclerosis,hypertension, and aortic dissection

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.     

血管损伤的新靶点：平滑肌22 alpha

血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型转化是血管损伤性疾病动脉粥样硬化、高血压和血管成形术后再狭窄等的共同病理生理过程.平滑肌22 alpha (smooth muscle 22 alpha, SM22α) 是一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性. 该蛋白不仅作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节,它还参与VSMC的增殖、炎症和氧化应激等进程. 本文就SM22α 的结构特征及其在VSMC血管损伤中的作用机制进行综述.     

TGF-beta superfamily members do not promote smooth muscle-specific alternative splicing, a late marker of vascular smooth muscle cell differentiation

Smooth muscle (SM) specific alternate splicing of a number of genes is a late marker of the differentiated vascular smooth muscle cell (VSMC) phenotype and is one of the first differentiation characteristics to be lost during de-differentiation and in disease. An understanding of how this aspect of VSMC phenotype is regulated may provide insights into the earliest events of the atherosclerotic process. TGF-beta1 is a potent regulator of VSMC differentiation and can induce expression of SM-specific contractile proteins in both pluripotent stem cells and de-differentiated VSMCs. The purpose of this study was to test the hypothesis that members of the TGFbeta-superfamily can also effect SM-specific alternative splicing. Firstly, we established that SM-specific splicing of alpha-tropomyosin, vinculin and SM-myosin heavy chain (MHC) increases during rat fetal/neonatal development and is decreased in VSMCs following balloon-induced carotid injury in the rat. Treatment of cultured rat VSMCs with TGFbeta-superfamily members resulted in a significant reduction in the ratio of SM to non-muscle (NM) alpha-tropomyosin, but did not effect SM-specific alternative splicing of vinculin or SM-MHC. Treatment of pluripotent C3H10T1/2 cells with TGF-beta1, which increased SM differentiation marker expression, did not increase SM-specific alpha-tropomyosin splicing. Taken together, these results demonstrate differential regulation of SM-specific alternative splicing and indicate that although TGF-beta1 promotes VSMC differentiation marker expression, TGF-beta1 cannot act as the sole trigger of VSMC differentiation.     

Studies on microRNAs that are correlated with the cancer stem cells in chronic myeloid leukemia

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.     

SM22α在细胞骨架组构及血管重塑中的作用

血管平滑肌细胞（VSMC）表型转化是动脉粥样硬化、高血压和血管成形术后再狭窄等血管重塑性疾病的共同病理生理过程。VSMC表型转化过程中平滑肌特异基因的表达变化和细胞骨架的组构是当前研究的热点问题之一。平滑肌22α（SM22α）是近年发现的一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性,该蛋白作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节。本文就SM22α的结构特征及其在VSMC骨架组构和血管重塑中的作用机制进行综述。     

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.     

Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3? untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.     

Involvement of BK channel in differentiation of vascular smooth muscle cells induced by mechanical stretch

The differentiation of vascular smooth muscle cells (VSMCs), which are exposed to mechanical stretch in vivo, plays an important role in vascular remodeling during hypertension. Here, we demonstrated the mechanobiological roles of large conductance calcium and voltage-activated potassium (BK) channels in this process. In comparison with 5% stretch (physiological), 15% stretch (pathological) induced the de-differentiation of VSMCs, resulting in significantly decreased expressions of VSMC markers, i.e., α-actin, calponin and SM22. The activity of BK channels, assessed by patch clamp recording, was significantly increased by 15% stretch and was accompanied by an increased alternative splicing of BK channel α-subunit at the stress axis-regulated exons (STREX). Furthermore, transfection of whole BK or STREX-deleted BK plasmids revealed that STREX was important for BK channels to sense mechanical stretch. Using thapsigargin (TG) which induces endoplasmic reticulum (ER) stress, and xbp1-targeted siRNA transfection which blocks ER stress, the results revealed that ER stress was contribute to stretch-induced alternative splicing of STREX. Our results suggested that during hypertension, pathological stretch may induce the ER stress in VSMCs, which affects the alternative splicing and activity of BK channels, and subsequently modulates VSMC differentiation.     

Creatinine metabolite,HMH (5-hydroxy-1-methylhydantoin; NZ-419), modulates bradykinin-induced changes in vascular smooth muscle cells

Abstract

A creatinine metabolite, 5-hydroxy-1-methylhydantoin (HMH: NZ-419), a hydroxyl radical scavenger, has previously been shown to confer renoprotection by inhibiting the progression of chronic kidney disease in rats. In the current study, we demonstrate that HMH modulates the effects of glucose and bradykinin (BK) in vascular smooth muscle cell (VSMC). HMH a novel anti-oxidant drug completely suppressed the expression of B2-kinin receptors (B2KR) in response to high glucose (25?mM) stimulation in VSMC and was also shown to attenuate the effects of BK on VSMC remodeling. HMH inhibited the BK-induced increase in MAPK phosphorylation and attenuated the increase in connective tissue growth factor (CTGF) protein levels in VSMC. These findings suggest that HMH may confer vascular protection against high glucose concentrations and BK-stimulation to ameliorate vascular injury and remodeling through its anti-oxidant properties.     

Coculture with endothelial cells enhances vascular smooth muscle cell adhesion and spreading via activation of beta1-integrin and phosphatidylinositol 3-kinase/Akt

The interactions between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) play significant roles in the homeostasis of the blood vessel during vascular remodeling. Cell adhesion and spreading are an essential process for VSMC migration, survival and proliferation in the events of vascular physiology and pathophysiology. However, effects of ECs on adhesion and spreading of VSMCs have not been characterized yet. Here, the interaction of ECs and VSMCs on adhesion and spreading of VSMCs were investigated by using a coculture system. The results showed that VSMCs cocultured with ECs exhibited a significant increase in the number of adherent and spreading cells, and much more mRNA (twofold, P<0.01) and protein (threefold, P<0.05) expression of beta(1)-integrin comparing to the control, i.e., VSMCs cultured alone. Furthermore, the enhanced functional activity of beta(1)-integrin expression was confirmed by FACS. A beta(1)-integrin blocking antibody (P5D2) could inhibit the EC-induced VSMC adhesion and spreading. It was demonstrated that in correspondence with enhanced cell adhesion, ECs also prompted focal adhesion complex assembly and stress fiber formation of VSMCs. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway was more pronouncedly activated in response to VSMC attachment. Our results for the first time show that coculture with ECs enhances VSMC adhesion and spreading by up-regulating beta(1)-integrin expression and activating the PI3K/Akt pathway, suggesting that the interaction between ECs and VSMCs serves an important role in vascular homeostasis and remodeling.     

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.     

Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodeling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. To study the direct consequences of VSMC apoptosis, we generated transgenic mice expressing the human diphtheria toxin receptor (hDTR, encoded by HBEGF) from a minimal Tagln (also known as SM22alpha) promoter. Despite apoptosis inducing loss of 50-70% of VSMCs, normal arteries showed no inflammation, reactive proliferation, thrombosis, remodeling or aneurysm formation. In contrast, VSMC apoptosis in atherosclerotic plaques of SM22alpha-hDTR Apoe-/- mice induced marked thinning of fibrous cap, loss of collagen and matrix, accumulation of cell debris and intense intimal inflammation. We conclude that VSMC apoptosis is 'silent' in normal arteries, which have a large capacity to withstand cell loss. In contrast, VSMC apoptosis alone is sufficient to induce features of plaque vulnerability in atherosclerosis. SM22alpha-hDTR Apoe-/- mice may represent an important new model to test agents proposed to stabilize atherosclerotic plaques.     

血管再狭窄发生过程中SMα肌动蛋白和SMemb基因表达的变化及其影响机制研究

为研究血管再狭窄发生过程中 VSMC表型转化的规律及机制 ,采用大鼠主动脉内皮剥脱后血管再狭窄动物模型和体外培养的 VSMC,通过 Northern印迹分析及 3H- Td R参入实验 ,动态观察血管再狭窄发生过程中 VSMC表型标志基因α肌动蛋白和 SMemb的表达变化及 b FGF、TNF-α和 IL - 1β对两种基因表达的影响及其与 VSMC增殖之间的关系 .结果表明 ,血管内皮剥脱后 3d,分化型标志基因α肌动蛋白表达活性开始降低 ,去分化型标志基因 SMemb表达明显上调 ,至第 7d,前者的下调与后者的上调均达到最大 ,此后 ,两者的表达活性趋于向正常恢复 .b FGF可明显下调 α肌动蛋白的表达和诱导 SMemb表达 ,对分化型和去分化型 VSMC均有促增殖作用 ,但对后者的作用大于前者 ,TNF- α和 IL- 1 β对 VSMC的促转化及促增殖作用较弱 .提示 b FGF等生长因子介导血管内皮损伤所诱发的 VSMC表型转化并促进其增殖 ,内皮损伤 7d后 ,在发生表型转化并进行增殖的 VSMC中 ,一部分细胞再分化 ,一部分细胞仍处于去分化状态并继续进行增殖并持续较长时间 .     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Suppressed nuclear envelope proteins activate autophagy of vascular smooth muscle cells during cyclic stretch application

Dysfunctions of vascular smooth muscle cells (VSMCs) play crucial roles in vascular remodeling in hypertension, which correlates with pathologically elevated cyclic stretch due to increased arterial pressure. Recent researches reported that autophagy, a life-sustaining process, was increased in hypertension. However, the mechanobiological mechanism of VSMC autophagy and its potential roles in vascular remodeling are still unclear. Using renal hypertensive rats in vivo and FX5000 stretch application Unit in vitro, the autophagy of VSMCs was detected. The results showed that LC3II remarkably enhanced in hypertensive rats and 15% cyclic stretch (mimic the pathologically increased mechanical stretch in hypertension), and the activity of mammalian target of rapamycin (mTOR) was suppressed in 15% cyclic stretch. Administration of autophagy inhibitors, bafilomycin A1 and chloroquine, repressed VSMC proliferation efficiently, but did not affect the degradation of two important nuclear envelope (NE) proteins, lamin A/C and emerin. Using RNA interference to decline the expression of lamin A/C and emerin, respectively, we discovered that autophagy was upregulated under both static and 5% cyclic stretch conditions, accompanying with the decreased mTOR activity. During 15% cyclic stretch application, mTOR inhibition was responsible for autophagy elevation. Chloroquine administration in vivo inhibited the expression of PCNA (marker of proliferation) of abdominal aorta in hypertensive rats. Altogether, these results demonstrated that pathological cyclic stretch suppresses the expression of lamin A/C and emerin which subsequently represses mTOR pathway so as to induce autophagy activation. Blocking autophagic flux may be a practicable way to relieve the pathological vascular remodeling in hypertensive.     

Injury-induced expression of cytokeratins 8 and 18 by vascular smooth muscle cells requires concurrent activation of cytoskeletal and growth factor receptors

Cytokeratins are not present in the vascular smooth muscle cells (VSMCs) of normal arteries, but they are detectable in the VSMCs of atherosclerotic lesions. A correlation between cytokeratin expression and VSMC phenotype is proposed, but an examination of VSMCs after mechanical injury has yet to be performed. Immunohistochemistry was used to monitor proteins in arterial sections. Western blotting enabled quantification of protein levels. Angioplasty of porcine femoral artery in vivo and porcine coronary artery in vitro served as models of vascular injury. Cytokeratins 8 and 18 were expressed by VSMCs in porcine femoral artery lesions 14 days after balloon angioplasty. Cytokeratins were also present in the neointima of porcine coronary artery segments placed into organ culture for 4 days. Cytokeratin expression was decreased in the presence of inhibitors that affect MAP kinase, PI3 kinase, Src kinase, and G protein, but not in the presence of an AT1 receptor antagonist. Cytokeratin expression also occurred when VSMCs were plated onto collagen in the presence of serum. We conclude that mechanical injury induces expression of cytokeratin 8 and 18 both in vitro and in vivo by synthetic VSMCs that migrate into the neointima. Furthermore, cytokeratin expression requires cellular attachment to extracellular matrix proteins in conjunction with mitogenic stimulation.     

Circ_0002984 induces proliferation,migration and inflammation response of VSMCs induced by ox-LDL through miR‑326-3p/VAMP3 axis in atherosclerosis

Atherosclerosis can result in multiple cardiovascular diseases. Circular RNAs (CircRNAs) have been reported as significant non-coding RNAs in atherosclerosis progression. Dysfunction of vascular smooth muscle cells (VSMCs) is involved in atherosclerosis. However, up to now, the effect of circ_0002984 in atherosclerosis is still unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low-density lipoprotein (ox-LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNF-α and IL-6 levels. The data implied that lack of circ_0002984 obviously repressed ox-LDL–stimulated VSMC inflammation. Meanwhile, miR-326-3p, which was predicted as a target of circ_0002984, was obviously down-regulated in VSMCs treated by ox-LDL. Additionally, after overexpression circ_0002984 in VSMCs, a decrease in miR-326-3p was observed. Subsequently, miR-326-3p was demonstrated to target vesicle-associated membrane protein 3 (VAMP3). Therefore, we hypothesized that circ_0002984 could modulate expression of VAMP3 through sponging miR-326-3p. Furthermore, we confirmed that up-regulation of miR-326-3p rescued the circ_0002984 overexpressing-mediated effects on VMSC viability, migration and inflammation. Additionally, miR-326-3p inhibitor-mediated functions on VSMCs were reversed by knockdown of VAMP3. In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR-326-3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.