Airway smooth muscle cell proliferation is increased in asthma

Increased airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely to be the result of increased muscle proliferation. We have for the first time been able to culture ASM cells from asthmatic patients and to compare their proliferation rate with that of nonasthmatic patients. Asthmatic ASM cell cultures (n = 12) were established from explanted lungs and endobronchial biopsies. Nonasthmatic ASM cells (n = 10) were obtained from explanted tissue from patients with no airway disease, emphysema, carcinoma, and fibrosing alveolitis. Cell counts, tritiated thymidine incorporation, and cell cycle analysis were conducted over 7 d. Asthmatic ASM cell numbers at Days 3, 5, and 7 were significantly higher than corresponding values for nonasthmatic cells (p < 0.05). Tritiated thymidine incorporation was increased 3.2-fold in asthmatic cells compared with nonasthmatic cells within the first 24 h (p = 0.026). Flow cytometric analysis of DNA content on Days 1 and 2 revealed that a significantly greater percentage of asthmatic ASM cells were in the G2 + M phase (p < 0.05). This study shows for the first time that proliferation of ASM cells is increased in patients with asthma and provides evidence for an intrinsic abnormality in the ASM cell in this disease. Keywords: asthma; human airway smooth muscle; cell culture; cell proliferation; hyperplasia     

Galectin 1 is involved in vascular smooth muscle cell proliferation

OBJECTIVE: Smooth muscle cell (SMC) migration and proliferation are the key steps in the development of atherosclerosis and restenosis. Matricellular proteins have been implicated in cell adhesion, migration and proliferation. Here we investigated the role of the matricellular protein galectin-1 (Gal-1), a beta-galactoside-binding lectin, in SMC proliferation in atheroma and DNA synthesis in cell culture. METHODS: Protein expression was visualised by tissue section immunostaining. RNA expression was analysed using Northern blot analysis. DNA synthesis of human vascular SMCs was determined by 3H-thymidine incorporation. Recombinant glutathione S-transferase-galectin-1 fusion protein (Gal FP) binding to extracellular matrix (ECM) proteins was measured by ELISA. Gal-1 binding to cells and ECM was estimated using 125I-labelled Gal FP. RESULTS: Prominent Gal-1 staining coincided with SMC proliferation in human coronary endarterectomy samples in organoid culture. In cell culture, Gal-1 mRNA was upregulated in growing SMCs. Gal FP increased serum-induced DNA synthesis of human SMCs on plastic or endogenous ECM, but not of a rat PAC1 SM cell line. Also, Gal FP slightly increased SMC adhesion to ECM. SMCs exhibited a complex pattern of receptor-ligand interactions with Gal FP. The Gal-1 binding to SMCs was much stronger than to ECM, produced by these SMCs. We identified new ECM proteins: thrombospondin, vitronectin and osteopontin, which bound to Gal FP in a dose- and beta-galactoside-dependent manner in ELISA. CONCLUSIONS: Gal-1 binding to SMCs was stronger than to ECM, although ECM of atherosclerotic blood vessels contained additional ECM proteins which bound to Gal-1. Gal-1 was upregulated during SMC growth and Gal FP enhanced serum-induced DNA synthesis in SMCs. Overall, Gal-1 upregulation is likely to provide a reinforcement of serum-induced events during vascular injury.     

Rabbit ear model of injury-induced arterial smooth muscle cell proliferation. Kinetics, reproducibility, and implications

Recently, considerable interest has focused on the vascular smooth muscle cell (SMC) response to injury, particularly as it relates to restenosis after angioplasty. In an effort to find an optimal experimental model of arterial SMC proliferation after injury, we examined the effects of external injury to the central artery of the rabbit ear and assessed the reproducibility, morphological changes, and time course of cellular proliferation after such an injury. With rabbits under general anesthesia, direct pressure was applied at two sites along the central artery of the ears of 19 New Zealand White rabbits. Rabbits were maintained on a diet of 2.4% fat and 0.001% cholesterol throughout the experiment. In seven rabbits examined after 21 days, marked SMC proliferation with neointimal formation was observed at all 28 sites (100%). Mean neointimal area, expressed as a percentage of the area of the tunica media, was 82 +/- 40% (range, 21-203%). Compared with the uninvolved artery displaced 2 mm from the injury site, mechanical crush caused a 38% increase in total vessel area (p less than 0.001), a 40% decrease in luminal area (p less than 0.002), and no change in the area of the media. Serial histological studies were performed 1-42 days after injury, using light and electron microscopy and bromodeoxyuridine immunohistochemistry. Beginning at day 3, activated medial SMCs were noted to migrate through defects in the internal elastic membrane, with a gradual increase in neointimal area between days 5 and 12. Peak DNA synthesis was identified in the media 5 days after injury, with proliferative activity shifting almost exclusively to the neointima thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

紫杉醇抑制大鼠血管平滑肌细胞增殖的实验研究

目的 以体外培养的大鼠血管平滑肌细胞为研究对象,探讨紫杉醇对平滑肌细胞增殖的作用.方法 SD大鼠腹腔注射麻醉后,解剖、分离其主动脉,仔细分离血管的平滑肌层并剪成小块置于培养瓶中培养.采用倒置像筹显微镜下观察及α平滑肌肌动蛋白免疫荧光检测对传4代～6代的细胞进行鉴定.根据加入的干预药物将平滑肌细胞分为无血清M199培养基组(A组)、1 nmol/L紫杉醇组(B组)、10 nmol/L紫杉醇组(C组)及100 nmol/L紫杉醇组(D组)共4组,每组均为5个样本.采用流式细胞仪检测不同干预组平滑肌细胞的生长周期,用增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)检测方法 检测细胞的PCNA阳性百分比.结果 培养约2周后出现大量细胞生长,可见致密的细胞层.倒置像差显微镜下观察,细胞呈梭形;随着细胞融合度的增加,可以看到平滑肌细胞特有的"峰-谷"生长现象.培养的血管平滑肌细胞α平滑肌肌动蛋白免疫荧光检测呈阳性,总阳性率大于90%.随着紫杉醇用量的增加,细胞的PCNA阳性百分比呈逐渐减少的趋势,与对照组(A组)比较,10 nmol/L(C组)和100 nmol/L(D组)两种浓度下的紫杉醇引起的PCNA阳性率明显减低.流式细胞仪检测显示,随着紫杉醇用量的增加,细胞S期百分比旱逐渐减少的趋势.与对照组相比,10 nmol/L(C组)和100 nmol/L(D组)两种浓度下的紫杉醇引起的S期百分比明显减低.结论 紫杉醇能够抑制大鼠血管平滑肌细胞的增殖.     

Aortic smooth muscle cell proliferation and endothelial nitric oxide synthase activity in fructose-fed rats

The aim of this study was to evaluate the proliferative behavior of vascular smooth muscle cells in primary culture (pC-SMC) and the endothelial nitric oxide synthase (eNOS) activity in the endothelial lining of the aorta of fructose-fed rats (FFR). This is an experimental model of syndrome X, a cluster of cardiovascular risk factors including hyperinsulinemia, insulin resistance, and hypertension that has been suggested to be of pathophysiologic importance for the development of atherosclerosis.Male Wistar rats were used: Control (n = 12) and FFR (n = 12). After receiving fructose in drinking water (10% w/v) during 8 weeks, biochemical parameters, systolic blood pressure (SBP) and relative heart weight (RHW) were determined. The proliferative effect of 10% fetal calf serum (FCS) was examined in aortic pC-SMC by [³H]thymidine incorporation and by cell counting. Ca²⁺/calmodulin-dependent NOS activity was estimated in aortic endothelial lining and in heart tissue homogenates by conversion of [³H]arginine into [³H]citrulline.Fructose-fed rats showed hyperinsulinemia (P = .0263), altered glucose tolerance test (P < .001), higher SBP (P < .0001), and RHW (P = .0145), compared to control rats. These animals also showed an increase of 10% FCS-induced [³H]thymidine incorporation (P < .0001) and cell number of aortic pC-SMC (P = .0049) and decreased eNOS activity in both aortic endothelium (P = .0147) and cardiac tissue (P < .0001).These data support the hypothesis that syndrome X is associated to changes in SMC proliferation and endothelial dysfunction, which could be involved in the onset or progression of the atherogenic process.     

紫杉醇抑制大鼠血管平滑肌细胞增殖的实验研究

目的以体外培养的大鼠血管平滑肌细胞为研究对象,探讨紫杉醇对平滑肌细胞增殖的作用。方法SD大鼠腹腔注射麻醉后,解剖、分离其主动脉,仔细分离血管的平滑肌层并剪成小块置于培养瓶中培养。采用倒置像差显微镜下观察及α平滑肌肌动蛋白免疫荧光检测对传4代～6代的细胞进行鉴定。根据加入的干预药物将平滑肌细胞分为无血清M199培养基组（A组）、1nmol／L紫杉醇组（B组）、10nmol／L紫杉醇组（c组）及100nmol／L紫杉醇组（D组）共4组,每组均为5个样本。采用流式细胞仪检测不同干预组平滑肌细胞的生长周期,用增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）检测方法检测细胞的PCNA阳性百分比。结果培养约2周后出现大量细胞生长,可见致密的细胞层。倒置像差显微镜下观察．细胞呈梭形：随着细胞融合度的增加,可以看到平滑肌细胞特有的“峰-谷”生长现象。培养的血管平滑肌细胞α平滑肌肌动蛋白免疫荧光检测呈阳性,总阳性率大于90％。随着紫杉醇用量的增加,细胞的PCNA阳性百分比呈逐渐减少的趋势,与对照组（A组）比较,10nmol／L（C组）和100nmol／L（D组）两种浓度下的紫杉醇引起的PCNA阳性率明显减低。流式细胞仪检测显示,随着紫杉醇用量的增加,细胞S期百分比呈逐渐减少的趋势。与对照组相比,10nmol／L（C组）和100nmol／L（D组）两种浓度下的紫杉醇引起的S期百分比明显减低。结论紫杉醇能够抑制大鼠向管平滑肌细朐的增殖．     

Statins effect on smooth muscle cell proliferation

Clinical trials have firmly established that 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitors (statins) can induce regression of vascular atherosclerosis as well as reduction of cardiovascular-related morbidity and death in patients with and without coronary artery disease. These beneficial effects of statins are usually assumed to result from their ability to reduce cholesterol synthesis. However, because mevalonic acid is the precursor not only of cholesterol but also of many nonsteroidal isoprenoid compounds, inhibition of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase may result in pleiotropic effects. Indeed, statins can interfere with major events involved in the formation and the evolution of atherosclerotic lesions, such as arterial myocyte migration and proliferation and cholesterol accumulation, independent of their hypolipidemic properties. The aim of this article is to focus on clinical and experimental data that show that statins possess effects beyond cholesterol lowering, particularly on arterial smooth muscle cell proliferation. The contribution of these direct vascular effects to the reduction of cardiovascular events observed in clinical trials with statins represents one of the major challenges for future studies to understand the antiatherosclerotic benefits of these agents.     

Vascular smooth muscle cell proliferation is effectively suppressed by the non-specific growth factor inhibitor suramin

The purpose of this study was to investigate the effects of the non-specific growth factor inhibitor suramin on smooth muscle cell proliferation in vitro and in vivo. Cultured vascular smooth muscle cells (VSMC) were stimulated by platelet-derived growth factor (PDGF) and cellular DNA synthesis assessed by [3H]-thymidine uptake. Suramin dose-dependently inhibited DNA synthesis in VSMC, and 100 microM of suramin completely suppressed the PDGF-AB-induced cellular DNA synthesis. Rabbit carotid arteries were injured by the balloon catheter, and then suramin locally delivered using a porous balloon catheter over ten minutes. Three weeks after the vascular injury, the extent of intimal thickening was compared between the suramin-treated and control rabbits. The neointimal formation triggered by balloon-mediated vascular injury was suppressed significantly and dose-dependently by locally infused suramin, and the intima to media area ratios of the control and 1 mM suramin-treated animals were 48.8+/-14.9 and 12.2+/-6.0%, respectively (p < 0.01. n = 6 for each group). These results suggest that one time local administration of suramin was sufficient to suppress neointimal formation after balloon-mediated vascular injury, and that pharmacological intervention targeting the growth factor's signaling pathways could be a promising approach to prevent smooth muscle cell proliferation in various proliferative vascular diseases.     

Inhibition of tracheal smooth muscle cell proliferation by phosphodiesterase inhibitors

Agents that increase intracellular cyclic 3',5'-adenosine monophosphate (cAMP), such as forskolin, prostaglandin (PG)E₂, salbutamol and 8-bromo-cAMP, have been shownto inhibit the proliferation of airway smooth-muscle (ASM) cells in vitro. However, it has not yet been determined whether selective inhibitors of phosphodiesterase (PDE) isoenzymes III and IV that catalyze cAMPto 5'-adenosine monophosphate have the ability to inhibit ASM cell proliferation. To evaluate the effectsof PDE inhibitors on ASM cell proliferation, ASM cells isolated from bovine tracheae were cultured in the presence of fetal bovine serum (FBS), with or without a non-selective PDE inhibitor (theophylline), a selective PDE III inhibitor (cilostazol), and a selective PDE IV inhibitor (rolipram). The number of ASM cells cultured with 5% FBS was significantly reduced by the presence of theophylline at 10^− 3 and 3 × 10^− 4 mol/L, cilostazol at 10^− 5, 10^− 6 and 10^− 7 mol/L, and rolipram at 10^− 4 and 10^− 5 mol/L. The release of lactic dehydrogenase from ASM cells cultured with any concentration of these agents was not significantly different from that with medium alone. Inhibitors of PDE III and IV were demonstrated to have an inhibitory effect on ASM cell proliferation induced by FBS. Our results suggest the value of the further development of PDE inhibitors for the treatment of hyperplasia of ASM cells characteristic of airway remodeling, in addition to bronchospasm and airway inflammation, in bronchial asthma.     

A greater stimulation of vascular smooth muscle cell proliferation by serum from spontaneously hypertensive rats.

In an attempt to delineate the differential characteristics of serum from hypertensives on the proliferation of vascular smooth muscle cells (VSMC), we compare the effect of serum from spontaneously hypertensive rats (SHR) with that from normotensive Wistar-Kyoto rats (WKY) in its ability to stimulate proliferation in VSMC. Cell number determination showed that 10% serum from either of the two strains substantially stimulated proliferation of serially passed cultured VSMC, the effect being significantly greater with SHR serum than with WKY serum. 3H-thymidine incorporation into newly synthetized DNA was also tested after treatment with 10% SHR or WKY serum. A substantial increase in the uptake was noted in response to the rat serum, the effect tending to be greater with SHR serum than with WKY serum. No difference was found between the two strains in cell size determined as intracellular water volume. These data provide the evidence of an increased growth stimulating activity of SHR serum. This abnormality may be superimposed on the already-known innate hyperproliferative capacity of VSMC of SHR, leading to an increase in the deterioration of vascular complications seen in this model.     

Stimulation of smooth muscle cell glycosaminoglycan synthesis by cultured endothelial cells is dependent on endothelial cell density

Smooth muscle cells (SMC) cultured in the presence of endothelial cells (EC), or in EC-conditioned medium, show increased synthesis of glycosaminoglycans (GAG). We have found that both the amount and type of GAG produced by the SMC are dependent on the density of the EC. EC (porcine) at a low density (0.1-0.5 X 10(6) cells/25 cm2), or their conditioned media, where the most active per cell in stimulating GAG. All GAG were stimulated but the increase was due mostly to hyaluronic acid (HA). At intermediate densities (1.0 X 10(6)/25 cm2) stimulation was markedly reduced, but still present, and both HA and sulphated GAG were similarly increased. At high densities (1.5-3 X 10(6)/25 cm2) where EC were confluent there was very little stimulation of HA but continued stimulation of sulphated GAG synthesis. The shift in stimulation from HA to sulphated GAG with increasing density was most clearly demonstrated by the decrease in the HA to the chondroitin sulphate ratio. These findings provide support for the general concept that SMC metabolism may be affected by changes in the state of the endothelium.     

Vascular smooth muscle cell outgrowth, proliferation, and apoptosis in young and old rats.

We attempted to identify the effects of aging on the response of vascular smooth muscle cells (VSMCs) to injury. Rat aortas were injured by ballooning, and cell outgrowth of the aortic explants, as well as cell proliferation and apoptosis induced by 7-ketocholesterol in the culture system were compared in young (6-7 weeks) and old (40-50 weeks) rats. Explant outgrowth in uninjured rats did not differ between young and old rats. However, 14 days after balloon injury, the number of explants with outgrowth increased by a factor of four (P<0.01) in young rats, while that of the old rats did not change. Cell proliferation in cultured VSMCs also did not differ between young and old uninjured rats, but, in the injured group, proliferation doubled in young rats (P<0.01), but did not change in the older group. The rate of unadhered cells in the presence of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured rats, however, that of young rats was lower (P<0.01) while that of older rats was higher (P<0.01). The extent of DNA fragmentation (%) after the addition of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured groups, DNA fragmentation was lower in the young rats (P<0.01), and higher in the old ones (P<0.01), when compared to their respective controls. These results indicate that the VSMC injury responses of cell outgrowth and proliferation were more pronounced in the young, and that 7-ketocholesterol-induced apoptosis was more extensive in old rats.     

Platelet membranes induce airway smooth muscle cell proliferation

The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localized in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS assay and the results confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GP-ASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.     

Inhibition of vascular smooth muscle cell proliferation by endothelium-dependent vasodilators.

This review considers the hypothesis that the endothelium-derived vasodilator agents, prostacyclin and nitric oxide, also function physiologically to inhibit vascular smooth muscle cell (VSMC) proliferation. The underlying biochemical mechanisms are also discussed. Prostacyclin and other agents that increase intracellular cAMP concentration are potent and effective inhibitors of the proliferation of isolated VSMC in culture. Such agents inhibit the initiation of proliferation in quiescent cells and the proliferation of logarithmically growing cells from a variety of sources, including man. The data implicate prostacyclin as an important regulator of VSMC proliferation, although there is little direct in vivo evidence. Nitric oxide-releasing drugs (and atriopeptins which increase intracellular cGMP concentration by a different mechanism) also inhibit proliferation of cultured VSMC. The effects are, however, partial and obtained at higher concentrations than those required for vasodilatation. Even allowing for the instability of the agents under tissue culture conditions, cGMP-elevating agents appear to be poorer at inhibiting proliferation than cAMP-elevating agents, despite similar or greater vasodilator potency. These data imply that nitric oxide is less likely than prostacyclin to be a physiological regulator of VSMC proliferation, although definitive experiments in vivo are again lacking. It also follows that nitrovasodilators are less attractive as therapy for VSMC proliferation than prostacyclin analogues or other cAMP-elevating agents, such as phosphodiesterase inhibitors. By analogy with the mechanisms of vasodilatation, inhibition of calcium mobilization and the subsequent activation of protein kinase C are considered as possible mechanisms underlying inhibition of proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gremlin promotes vascular smooth muscle cell proliferation and migration

Recent reports highlight the importance of BMP in the vasculature. We investigated the expression pattern and role of the BMP antagonist gremlin in VSMC. We detected gremlin mRNA constitutive expression in adult and embryonic rat aortic VSMC, and in rat carotids. In vitro analysis demonstrated that angiotensin II, TGF-β1 and PDGF induced significant changes in gremlin mRNA expression. Gremlin stable overexpression in A7r5 cells blocked BMP signaling. BMP-induced reduction in VSMC DNA synthesis was markedly inhibited by gremlin overexpression. In fact, gremlin overexpression increased DNA synthesis and cell counts, and accelerated cell cycle progression of VSMC, through mechanisms that include p27^kip1 down-regulation. Gremlin also led to marked increments in VSMC migration. In addition, gremlin gene silencing promoted a significant blockade on cell proliferation and migration. In vivo studies disclosed increased gremlin protein expression in the neointima of balloon-injured carotid arteries. In summary, the BMP antagonist gremlin is constitutively expressed in the normal vasculature. Gremlin induces VSMC proliferation and migration and is significantly regulated by growth factors and injury. We postulate that gremlin plays a part in the development of pathological phenotypic changes of adult VSMC.     

PKA and Epac synergistically inhibit smooth muscle cell proliferation

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2′-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2′-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.     

Inhibition of smooth muscle cell proliferation by a chemically modified tetracycline

INTRODUCTION: Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial derivatives of tetracycline, which exert antiproliferative and anticollagenolytic properties. The molecular mechanisms, however, are poorly understood. MATERIALS AND METHODS: The effect of CMT-3 on cultured, subconfluent rat aortic smooth muscle cells (SMCs) was analyzed by [(3)H]-thymidine incorporation, counting cell numbers, and flow cytometry analysis. RESULTS: CMT-3 inhibited the incorporation of [(3)H]-thymidine and reduced the cell number dose-dependently, with approximately 60% inhibition at the maximal CMT-3 concentration used (20 mumol/l). CMT-3 decreased the SMC proportion in S-phase and gradually increased the proportion at G2/M. Initially, the proportion of cells in G1-phase increased and then gradually decreased back to baseline as the CMT-3 concentration increased. CMT-3 treatment of confluent SMCs for 24 h did not induce apoptosis. CONCLUSIONS: CMT-3 inhibited SMC proliferation by inducing cell cycle arrest at the G2/M restriction point. Nonetheless, CMT-3 did not induce SMC apoptosis.