Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.     

Inhibition of vascular smooth muscle cell proliferation in vitro by genetically engineered marrow stromal cells secreting calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) has a beneficial effect in pulmonary hypertension and is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold promise for use in adult stem cell-based ex vivo gene therapy. To test the hypothesis that genetically engineered MSCs secreting CGRP can inhibit vascular smooth muscle cell proliferation, rat MSCs were isolated, ex vivo expanded, and transduced with adenovirus containing CGRP. Immunocytochemical analysis demonstrated that wild type rat MSCs express markers specific for stem cells, endothelial cells, and smooth muscle cells including Thy-1, c-Kit, von Willebrand Factor and alpha-smooth muscle actin. Immunocytochemistry confirmed the expression of CGRP by the transduced rat MSCs. The transduced rat MSCs released 10.3+/-1.3 pmol CGRP/1 x 10(6) cells/48 h (mean+/-S.E.M., n=3) into culture medium at MOI 300 and the CGRP-containing culture supernatant from the transduced cells inhibited the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and rat aortic smooth muscle cells (ASMCs) in culture. Co-culture of the transduced rat MSCs with rat PASMCs or rat ASMCs also inhibited smooth muscle cell proliferation. These findings suggest that this novel adult stem cell-based CGRP gene therapy has potential for the treatment of cardiovascular diseases including pulmonary hypertension.     

Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.     

Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Cytostatic drugs differentially affect phenotypic features of porcine coronary artery smooth muscle cell populations

We studied the effects of cytostatic drugs on porcine coronary artery spindle-shaped (S) and rhomboid (R) smooth muscle cell (SMC) biological activities related to intimal thickening (IT) formation. Imatinib, and to a lesser extent curcumin, decreased proliferation of S- and R-SMCs and migratory and urokinase activities of R-SMCs more efficiently compared with cyclosporine plus rapamycin. Imatinib increased the expression of alpha-smooth muscle actin in both SMC populations and that of smoothelin in S-SMCs. It decreased S100A4 expression in R-SMCs. By promoting SMC quiescence and differentiation imatinib and curcumin may represent valid candidates for restenosis preventive and therapeutic strategies.     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑肌细胞生长的影响

从动脉粥样硬化（ＡＳ）高（北京）、低（南宁）发区人正常胸主动脉内－中膜分离ＨＳＰＧ,观察其对体外培养的ＨＡＳＭＣ生长的影响,细胞计数、~３Ｈ－ＴｄＲ参入及形态观察均表明ＡＳ高、低发区人主动脉ＨＳＰＧ都能剂量依赖性地抑制ＨＡＳＭＣ增殖,但抑制百分数未见显著差异,结果提示,人动脉壁中ＨＳＰＧ的含量可能与ＡＳ发病有关．     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells.

Prostaglandins E1 and E2 are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE1 and PGE2 under various conditions and their growth was assessed. PGE1 and PGE2 did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 microgram/ml) increase in [3H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [3H]-thymidine uptake at 24 hours with PGE1 and PGE2 respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 microgram/ml PGE1). Furthermore, PGE1 could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE1 may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE1 on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE1 and PGE2 do not inhibit the proliferation of PA SMC but rather stimulate it.     

Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH‐SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or ?1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH‐SMC (fold change 1.5≤ or ?1.5≥, p < 0.05). HUASMC expressed increased amount of α‐smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH‐SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH‐SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH‐SMC. There was a trend toward reduced proliferation of PAH‐SMC with paxillin‐si‐RNA and increased proliferation with ELAVL1‐siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH‐SMC proliferation.     

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

小鼠肌母细胞的增殖与分化

通过对小鼠肌母细胞C2C12的培养,研究C2C12细胞的增殖与分化的关系以及胰岛素在细胞分化过程中的作用。在对照组中,C2C12细胞增殖占了明显的优势,细胞形态几乎没有发生变化;而在实验组中,C2C12细胞在换为分化培养基24小时后,就出现了部分细胞衰亡和死亡的现象,尤其是在48小时细胞的死亡率达到最高,存活细胞开始从增殖期进入分化期,72小时出现了少量肌管,在96小时细胞分化效果达到最好。而在添加了胰岛素的分化培养基中的细胞分化效果明显好于没有添加胰岛素的分化培养基中的细胞,结果表明,胰岛素促进C2C12细胞的分化。     

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha- smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha- smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm- 1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha- smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti- desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin- negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.