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.     

Jagged-1/Notch3 signaling transduction pathway is involved in apelin-13-induced vascular smooth muscle cells proliferation

The apelin/apelin receptor （APJ, apelin-angiotensin receptor-like 1） system is a newly deorphanized G protein- coupled receptor system. Both apelin and APJ that are important regulatory factors are expressed in the cardio- vascular system. Our previous studies demonstrated that apelin-13 significantly stimulated vascular smooth muscle cell （VSMC） proliferation. In this paper, our data sug- gested that the Jagged-l/Notch3 signaling transduction pathway is involved in apelin-13-induced VSMC prolifer- ation by promoting the expression of Cyclin D1. Results indicated that apelin-13 stimulates the proliferation of VSMC and the expression of Jagged-1 and Notch3 in con- centration- and time-dependent manners. The increased expression of Jagged-1 and Notch3 induced by apelin-13 could be abolished by extracellular signal-regulated protein kinase （ERK） blockade. PD98059 （ERK inhibitor） can inhibit the activation of Jagged-I/Notch3 induced by apelin- 13. Down-regulation of Notch3 using small interfering RNA inhibits the expression of Cyclin DI and prevents apelin- 13-induced VSMC proliferation. In conclusion, Jagged-I/ Notch3 signaling transduction pathway is involved in VSMC proliferation induced by apelin-13.     

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.     

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

The present study was designed to investigate the role of endogenous sulfur dioxide (SO₂) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO₂ inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO₂ synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO₂ suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO₂ did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO₂ dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO₂ simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO₂ could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO₂-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO₂ exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells

Accumulation of transglutaminase 2 (TG2) is often associated with mineral deposits in vasculature. Here, we demonstrate that purified TG2 stimulated a 3-fold increase in matrix mineralization and up-regulation of osteoblastic markers in cultured primary vascular smooth muscle cells (VSMCs). Extracellular TG2 interacts with the low density lipoprotein related-protein 5 receptor and activates beta-catenin signaling in VSMCs. These results suggest that TG2 may promote vascular calcification by activating the beta-catenin signaling pathway.     

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.     

Preliminary study on the mechanism of long noncoding RNA SENCR regulating the proliferation and migration of vascular smooth muscle cells

The proliferation and migration of vascular smooth muscle cells (VSMCs) are one of the key regulatory links of atherosclerosis (AS). Long noncoding RNAs (lncRNAs) are emerging as key regulators in AS development. In this study, we first assessed the expression level of smooth muscle and endothelial cell-enriched migration/differentiation-associated lncRNA (SENCR) in the plasma of patients with coronary heart disease (CHD) and its predictive and diagnostic value. Second, we investigated the role of SENCR in the regulation network of human aortic-VSMCs (HA-VSMCs) proliferation and migration and determined its downstream regulatory mechanism. The results showed that SENCR was downregulated in the peripheral blood of CHD, and negatively related to the Gensini score. SENCR was enriched in HA-VSMCs and mainly distributed in cytoplasm. Overexpression of SENCR significantly inhibited HA-VSMCs proliferation, migration, and block cell cycle, while the knockdown of SENCR had the opposite effects. Moreover, bioinformatics analysis and luciferase reporter assay demonstrated that miR-4731-5p could directly bind to SENCR. Besides, we proved that FOXO3a inhibited HA-VSMCs proliferation and migration by binding to the 3′-untranslated region of miR-4731-5p. In summary, our research suggested that SENCR affects HA-VSMCs proliferation and migration via regulating the miR-4731-5p/FOXO3a pathway.     

Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases

Vascular smooth muscle cells (VSMCs) proliferation is involved in vascular atherosclerosis and restenosis. Recent studies have demonstrated that lipopolysaccharide (LPS) promotes VSMCs proliferation, but the signalling pathways which are involved are not completely understood. The purpose of this review was to summarize the existing knowledge of the role and molecular mechanisms involved in controlling VSMCs proliferation stimulated by LPS and mediated by toll‐like receptor 4 (TLR4) signalling pathways. Moreover, the potential inhibitors of TLR4 signalling for VSMCs proliferation in proliferative vascular diseases are discussed.     

白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4 2丝裂素活化蛋白激酶的表达     

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells

Abstract

Context: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca²⁺ signalling in vascular smooth muscle cells (VSMC). Objective: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca²⁺ concentration [Ca²⁺]i. RGS2 involvement in VSMC proliferation had not been examined. Methods: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca²⁺]i before and after UTP and thapsigargin. [³H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca²⁺ transporters was performed in primary aortic VSMC. Results and discussion: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca²⁺ homeostasis, RGS2KO VSMC showed lower resting [Ca²⁺]i but higher thapsigargin-induced [Ca²⁺]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca²⁺ ATPase-4 (PMCA4) and Na⁺ Ca²⁺ Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca²⁺ ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca²⁺ efflux activity, ⁴⁵Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La³⁺, but not by replacing extracellular Na⁺ with choline, implicating differences in the activity of PMCA and not NCX. Conclusion: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca²⁺-handling mechanisms in response to PDGF stimulation.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

The effects of peptides with estrogen‐like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells

Hormone replacement therapy (HRT) for post‐menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post‐menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen‐mimetic peptide 1 (EMP‐1) (VSWFFE) in comparison to estrogen (E₂) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP‐1‐like E₂ inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP‐1 did not affect E₂ stimulation of DNA, but inhibited E₂ inhibition of cell proliferation at high concentration. All effects by the combination of EMP‐1 and E₂ were abolished at hg. A44‐stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co‐incubated with E₂ at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E₂ stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45‐stimulated CK‐specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP‐1. EMP‐1 and A44 similar to E₂‐stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP‐1 provided it acts similarly in vivo can replace E₂ for treatment of post‐menopausal women in hyperglycemia due to diabetes. J. Cell. Biochem. 110: 1142–1146, 2010. Published 2010 Wiley‐Liss, Inc.     

17β—雌二醇抑制内皮素诱导的血管平滑肌细胞增殖作用

目的和方法：利用组织块贴壁法进行大鼠VSMC培养,胰蛋白酶分散细胞法传代。实验采用第4-6代细胞。采用氚-胸腺嘧啶核苷（[^3H]-TdR)掺入和细胞计数来作为VSMC增殖的指标,以RT-PCR的方法检测ETAR的表达,观察17β-雌二醇（E2）对内皮素-I（endothelin-l,ET-1)介导的血管平滑肌细胞（VSMC）增殖反应以及对内皮素A型受体（ETAR）表达的影响。结果：ETAR特异性拮抗剂BQ123能完全阻断ET-1介导的VSMC增殖反应;E2可明显抑制ET-1促进VSMC增殖的作用,RT-PCR结果显示E2能抑制ETAR的表达,12h时抑制作用最为明显;E2受体阻断剂Tamoxifen亦能部分抑制ET-1对VSMC的增殖及ETAR的mRNA的表达。结论：ET-1促进VSMC增殖作用主要通过ETAR介导的,雌激素可通过抑制ETARmRNA表达来发挥对ET-1促进VSMC增殖的抑制作用。     

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.