The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D₃ and β-glycerophosphate, respectively. In vivo, the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited β-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.     

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

BMP‐9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP‐9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP‐9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre‐dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP‐9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP‐9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP‐9‐induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5‐Dimethoxy‐N‐(quinolin‐3‐yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP‐9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP‐9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4‐siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP‐9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.     

Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT-1 expression

Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. ‘Uraemic’ and control EV (EV^UR; EV^CTRL) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EV^CTRL and EV^UR differed in size and miRNA contents. Contrary to EV^CTRL, EC- and plasma-derived EV^UR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EV^UR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EV^UR. Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EV^UR. In conclusion, EV^UR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD.     

Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

Vascular smooth muscle cells (VSMCs) play a significant role in atherosclerosis. As a multifunctional protein, nucleolin (NCL) is involved in many important physiological and pathological processes. In this study, we aimed to investigate the role of nucleolin in VSMCs proliferation and cell cycle. The expression of nucleolin increased in VSMCs of mice with aortas advanced plaques. With the left common carotid-artery ligation-injury model, immunofluorescence staining revealed that nucleolin and Ki67 expression increased in VSMCs in mice left carotid artery compared with right carotid artery after surgery. POVPC or ox-LDL up-regulated nucleolin mRNA and protein expression in a dose- and time-dependent manner in HAVSMCs. POVPC (5μg/ml) or ox-LDL (50μg/ml) promoted the proliferation of HAVSMCs. Nucleolin ablation relieved the pro-proliferation role of VSMCs. The cell cycle assay and cell ability results showing that POVPC or ox-LDL increased the proliferation, but nucleolin ablation inhibited the proliferation of HAVSMCs. And nucleolin ablation can prevent DNA replication at S phase and induce cell cycle arrest in S phase. The bioinformatics database predicts protein-protein interactions with nucleolin and aurora B. Nucleolin overexpression and ablation affected the expression of aurora B. These findings indicate for the first time that nucleolin actively involved the proliferation of VSMCs via aurora B.     

The role of microRNAs in the involvement of vascular smooth muscle cells in the development of atherosclerosis

MicroRNAs (miRNAs) are a class of nonprotein‐encoding RNAs of ~22 nucleotides in length that bind to or complement each other with a target gene messenger RNA (mRNA) to promote mRNA degradation or inhibit translation of the target mRNA. The protein required [such as Toll‐like receptor (TLR) proteins] is controlled at an optimal level. By affecting protein translation, miRNAs have become powerful regulators of biological processes, including development, differentiation, cell proliferation, and apoptosis. MiRNAs are involved in the regulation of proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), thereby affecting the formation of atherosclerosis (AS). In recent years, the role and mechanism of miRNAs involved in AS development in VSMCs have been studied extensively. In the current study, the results and progress in miRNA research are reviewed.     

MicroRNAs are critical in regulating smooth muscle cell mineralization and apoptosis during vascular calcification

Vascular calcification refers to the pathological deposition of calcium and phosphate minerals into the vasculature. It is prevalent in atherosclerosis, ageing, type 2 diabetes mellitus and chronic kidney disease, thus, increasing morbidity and mortality from these conditions. Vascular calcification shares similar mechanisms with bone mineralization, with smooth muscle cells playing a critical role in both processes. In the last decade, a variety of microRNAs have been identified as key regulators for the differentiation, phenotypic switch, proliferation, apoptosis, cytokine production and matrix deposition in vascular smooth muscle cells during vascular calcification. Therefore, this review mainly discusses the roles of microRNAs in the pathophysiological mechanisms of vascular calcification in smooth muscle cells and describes several interventions against vascular calcification by regulating microRNAs. As the exact mechanisms of calcification remain not fully elucidated, having a better understanding of microRNA involvement in vascular calcification may give impetus to development of novel therapeutics for the control and treatment of vascular calcification.     

Osteogenic transdifferentiation of vascular smooth muscle cells isolated from spontaneously hypertensive rats and potential menaquinone-4 inhibiting effect

Vascular calcification (VC) is an active and cell-mediated process that shares many common features with osteogenesis. Knowledge demonstrates that in the presence of risk factors, such as hypertension, vascular smooth muscle cells (vSMCs) lose their contractile phenotype and transdifferentiate into osteoblastic-like cells, contributing to VC development. Recently, menaquinones (MKs), also known as Vitamin K2 family, has been revealed to play an important role in cardiovascular health by decreasing VC. However, the MKs' effects and mechanisms potentially involved in vSMCs osteoblastic transdifferentiation are still unknown. The aim of this study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of MKs family, in the modulation of the vSMCs phenotype. To achieve this, vascular cells from spontaneously hypertensive rats (SHR) were used as an in vitro model of cell vascular dysfunction. vSMCs from Wistar Kyoto normotensive rats were used as control condition. The results showed that MK-4 preserves the contractile phenotype both in control and SHR-vSMCs through a γ-glutamyl carboxylase-dependent pathway, highlighting its capability to inhibit one of the mechanisms underlying VC process. Therefore, MK-4 may have an important role in the prevention of vascular dysfunction and atherosclerosis, encouraging further in-depth studies to confirm its use as a natural food supplement.     

Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia

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A morphometric study of vascular smooth muscle cells in culture

Summary Cultured arterial smooth muscle cells derived from different times in culture, different passages, and different species were evaluated by a combination of transmission electron microscopy and morphometry. The morphometric studies focused on point counting and monitored the following cellular components: lysosomes, myofilaments, mitochondria, ribosomes, and rough endoplasmic reticulum (RER). Percent volume composition values for the organelles involved in protein synthesis, namely ribosomes and RER, show significant fluctuations with time. Consistent with these observations, the cells showed increasing myofilaments during the early weeks in culture, which subsequently decreased significantly. The data also indicate that rabbit cells in culture may become synthetically quiescent with time and the distribution of cellular components is altered with each succeeding passage. Cultured calf (bovine) cells exhibit similar activity periods compared to rabbit but show a significantly higher lysosomal and lower myofilament content than rabbit. Calf cells could not be maintained for longer than 21 days in the absence of ascorbate, whereas ascorbate affects the ultrastructure of rabbit cells less dramatically. Age, passage, and donor, among others, are important considerations for studying in vitro smooth muscle cells. With proper morphologic and morphometric monitoring, these smooth muscle cell culture systems can be important tools in the study of aging or pathologic processes, or both. This work was presented as partial fulfillment for the degree of Ph.D. This work was supported by National Institutes of Health Grants HL-13262, HL-19717, and AG-00001.     

Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells

Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components—CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, ⁴⁵Ca²⁺ uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (p<0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r=0.83, 0.92 and 0.93, respectively, all p's<0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components—CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification.     

血管平滑肌细胞的分化与血管内壁的构建

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的发育与血管壁的构建是目前相关领域中的重要学科前沿．国内外同行的工作多集中在血管发育初始阶段内皮细胞及其前体细胞在血管新生中的作用、调节因素及生物学机制．VSMCs参与血管壁早期构建,特别是VSMCs的募集与分化机制已经成为血管新生研究中的一个新领域． 本期发表的《 抑制Rac1蛋白活化阻碍胚胎发育早期血管新生 》(见696～701页)报道了韩雅玲教授及其合作者在这一领域取得的最新研究结果．Rac1是真核细胞内重要的一类信号传递分子,在细胞信号传递过程中发挥分子开关作用．他们采用胚胎干细胞(ESCs)为模型,建立稳定表达持续型Rac1和显性失活型Rac1编码序列的小鼠ESCs并制备胚胎小体,诱导分化后观察其对内皮细胞分化和迁移的影响,发现抑制Rac1可以干扰血管内皮细胞连接成血管网状结构,细胞骨架F-actin排列紊乱,细胞的迁移受到明显抑制,表明Rac1在胚胎早期血管发育过程中与内皮细胞的迁移有关[1]． 近年来,韩雅玲教授及其研究集体在VSMCs发育与血管构建、胚胎干细胞来源的拟胚体血管平滑肌发育与血管新生机制以及胚胎主动脉VSMCs起源等方面开展了研究,取得了一系列有价值的成果[2～11],可能为闭塞性和增生性血管病的发生及防治提供理论依据和候选基因．详见“相关链接”．     

MiR155 modulates vascular calcification by regulating Akt-FOXO3a signalling and apoptosis in vascular smooth muscle cells

microRNA-155 (miR155) is pro-atherogenic; however, its role in vascular calcification is unknown. In this study, we aim to examine whether miR155 regulates vascular calcification and to understand the underlying mechanism. Quantitative real-time PCR showed that miR155 is highly expressed in human calcific carotid tissue and positively correlated with the expression of osteogenic genes. Wound-healing assay and TUNEL staining showed deletion of miR155 inhibited vascular smooth muscle cell (VSMC) migration and apoptosis. miR155 deficiency attenuated calcification of cultured mouse VSMCs and aortic rings induced by calcification medium, whereas miR155 overexpression promoted VSMC calcification. Compared with wild-type mice, miR155^−/− mice showed significant resistance to vitamin D3 induced vascular calcification. Protein analysis showed that miR155 deficiency alleviated the reduction of Rictor, increased phosphorylation of Akt at S473 and accelerated phosphorylation and degradation of FOXO3a in cultured VSMCs and in the aortas of vitamin D3-treated mice. A PI3K inhibitor that suppresses Akt phosphorylation increased, whereas a pan-caspase inhibitor that suppresses apoptosis reduced VSMC calcification; and both inhibitors diminished the protective effects of miR155 deficiency on VSMC calcification. In conclusion, miR155 deficiency attenuates vascular calcification by increasing Akt phosphorylation and FOXO3a degradation, and thus reducing VSMC apoptosis induced by calcification medium.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Cell sex determines anoikis resistance in vascular smooth muscle cells

Sexual dimorphism, detectable in vascular smooth muscle cells freshly isolated from aorta of male and female rats, is associated with a different susceptibility to radiation-induced apoptosis. In this work we investigated the mechanism underlying this difference and discovered that, in comparison with cells from male rats, cells from female rats show adhesion-associated resistance to apoptosis, the so called anoikis resistance. This is apparently due to a more adhering phenotype, characterized by a well organized actin microfilament cytoskeleton and to an increased phosphorylated focal adhesion kinase, and, more importantly, to a higher propensity to undergo survival by autophagy.     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.