Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall

Summary Smooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the sample yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density=1.0×10⁴ cells/cm², 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the “hill and valley” configuration typical of SMC. Plaquederived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age of sex, with method of culture, or with whether the cells were plaque derived or not. This study was supported in part by National Institutes of Health Grant HL-17269     

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

培养大鼠主动脉血管平滑肌细胞产生巨噬细胞集落刺激因子及其受体的表达

本研究用培养大鼠主动脉血管平滑肌细胞（ＶＳＭＣｓ）,结果如下：（１）用生物活性检测法发现ＶＳＭＣｓ无血清条件培养液可刺激巨噬细胞集落形成,其作用能被抗巨噬细胞集落刺激因子（ＭＣＳＦ）抗体抑制;（２）用免疫细胞化学技术证明ＶＳＭＣｓ存在ＭＣＳＦ受体;（３）用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ技术证明ＶＳＭＣｓ有ＭＣＳＦ及其受本的ｍＲＮＡ表达,血清刺激使两者表达明显增强。本研究首次报道了培养大鼠主动脉ＶＳＭＣ     

A VSV-G pseudotyped HIV vector mediates efficient transduction of human pulmonary artery smooth muscle cells

Attempts were made to infect human vascular smooth muscle cells derived from the pulmonary artery (hPASMC) with two different human immunodeficiency virus (HIV) vector systems. ADA/Luc or HXB2/Luc were generated by cotransfection of luciferase reporter gene vector, pNL4-3-Luc-E- R-, and one of two envelope expressing vectors, pSMADA (R5) or pSMHXB2 (X4). The VSV-G/Luc or VSV-G/GFP were produced by a three-plasmid expression system which consisted of vesicular stomatitis virus G protein (VSV-G) expressing vector, packaging plasmid, and one of two reporter genes (pHR'-CMV-Luc or pHR'-CMV-GFP). We used hPASMC, U87.CD4.CCR5 and U87.CD4.CXCR4 for infection. Neither ADA/Luc nor HXB2/Luc could infect hPASMC, though they could infect U87.CD4 with corresponding coreceptors. On the other hand, the transduction of both VSV-G/Luc and VSV-G/GFP to hPASMC was remarkable. At day 3, the relative proportion of positive cells of hPASMC infected with VSV-G/GFP was 15%. The above finding indicates a direct role of HIV-1 infection in pulmonary hypertension 'a rare complication of HIV-1 infection' and HIV-based vectors could introduce foreign genes into hPASMC for gene therapy of pulmonary hypertension.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

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.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

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.     

Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes

Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced ³H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

A single-cell transcriptomic inventory of murine smooth muscle cells

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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.     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.