Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

Background

All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.

Methods

We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[¹⁵N₂]arginine conversion to L-[¹⁵N]citrulline respectively.

Results

Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.

Conclusion

Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.     

3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells

Aims

The 3D geometry of individual vascular smooth muscle cells (VSMCs), which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation.

Methods and Results

A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI) selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell’s initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations) was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9μm, 4.6±0.6μm and 6.2±1.8μm (mean±SD). In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle) was found to be 8±7.6° with median as 5.7°.

Conclusions

A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function.     

脂多糖对血管平滑肌细胞一氧化氮合酶基因表达及细胞增殖的影响

应用ＲＮＡ印迹分析和亚硝酸盐含量测定检查脂多糖（ＬＰＳ）对大鼠血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）基因表达及ＮＯ合成的影响,用３Ｈ－ＴｄＲ参入实验观察ＬＰＳ对细胞ＤＮＡ合成的影响．结果表明,ＬＰＳ在诱导ＶＳＭＣｉＮＯＳｍＲＮＡ表达和促进ＮＯ合成的同时,抑制ＶＳＭＣＤＮＡ合成．证明ＬＰＳ的作用与其浓度和作用时间有关     

Effects of Trimethoxystilbene on Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells

Proliferation of pulmonary artery smooth muscle cells (PASMC) is an important contributor to the progress of pulmonary arterial hypertension (PAH). Anti-inflammatory therapies may have therapeutic applicability for PAH. Resveratrol (RES) has prominent anti-inflammatory effects in vitro, but in vivo its beneficial effects are limited by short systemic half life and poor lipotrophy. A derivative of RES, trimethoxystilbene (TMS), has higher lipotropy and extended half life compared with RES, and can potentially overcome the limitations of RES. In the present study, we studied the effects of TMS and RES on proliferation and apoptosis of PASMC stimulated with Tumor Necrosis Factor-??. The effects on PASMC proliferation were quantified by MTT, while apoptosis was assessed by flow cytometry (Annexin V/propidium iodide assay). We observed strong inhibitory effects of TMS on the growth of PASMC, and these effects were 20 times more potent than those of RES. We further documented induction of apoptosis in PASMC treated with TMS, again, to a higher degree than with RES. In conclusion, TMS is more potent than RES in the inhibition of proliferation and induction of apoptosis of PASMC, demonstrating its potentially beneficial role for treating PAH.     

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background

Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).

Methodology/Principal Findings

The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10⁻⁸ to 10⁻⁵ M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.

Conclusion

GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.     

Critical Role of Exogenous Nitric Oxide in ROCK Activity in Vascular Smooth Muscle Cells

Objective

Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo.

Methods

VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor.

Results

Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP.

Conclusions

These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.     

MicroRNA-18a通过HIF-1-alpha调控缺氧引起的人肺动脉平滑肌细胞增殖

目的：研究microRNA-18a (miR-18a) 对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, hPASMCs)增殖的调控作用及其可能机制。方法：体外培养hPASMCs,分为未转染组、miR-18a 模拟物对照组、miR-18a 模拟物组、 miR-18a 抑制剂对照组、miR-18a 抑制剂组、siRNAcontrol组、siHIF-1-alpha组、miR-18a 抑制剂和siHIF-1-alpha共转染组。分别于常氧(21% O2)和低氧(3%O2)作用24小时。采用CCK-8 法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1-alpha(HIF-1alpha)是 否为miR-18a 的靶基因,并通过western-blot 以及实时荧光定量PCR 技术检测相关蛋白和基因的表达。结果：缺氧可促进 hPASMCs 增殖,使miR-18a 表达减少;miR-18a 模拟物可抑制hPASMCs 增殖,而miR-18a 抑制剂可促进hPASMCs 增殖;抑制 miR-18a 可使HIF-1-alpha的表达上调。同时抑制miR-18a 和HIF-1-alpha,可使miR-18a 对hPASMCs 增殖调控的能力消失。结论：缺氧通 过抑制miR-18a,上调HIF-1-alpha的表达,促进hPASMCs增殖。     

Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

反义凝血酶受体基因的表达对人ASMC增殖的影响

介入治疗后再狭窄的发生严重降低了该治疗手段的最终疗效．为探讨再狭窄的发生机理、寻找有效的预防手段,利用反义ＲＮＡ技术构建了含有部分反义凝血酶受体（ＡＴＲ）ｃＤＮＡ片段的真核表达质粒ｐｃＤＮＡ３／ＡＴＲ,并观察了其对培养的人主动脉平滑肌细胞（ＡＳＭＣ）增殖的影响．结果表明ｐｃＤＮＡ３／ＡＴＲ的瞬时转染即能显著抑制人ＡＳＭＣ的３Ｈ－ＴｄＲ参入量,且该作用与导入的ＤＮＡ量呈剂量依赖性．说明部分反义凝血酶受体基因的表达可以抑制ＡＳＭＣ的增殖     

Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum’s stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.     

氧化修饰HDL剌激培养人主动脉平滑肌细胞增殖

平滑肌细胞（smooth muscle cell, SMC）增殖在动脉粥样硬化（atherosclerosis, AS）形成中起着重要作用.氧化修饰HDL（oxidized HDL, OX-HDL）可刺激 ³H-TdR掺入培养人动脉SMC的DNA,促进SMC增殖.以四甲基偶氮唑盐(MTT)法直接观察OX-HDL对培养人动脉SMC增殖细胞数的影响.结果显示,天然HDL（native HDL N-HDL）对SMC增殖没有影响,而OX-HDL则显著刺激SMC增殖（P＜0.01）,N-HDL显著抑制OX-LDL刺激SMC增殖作用（P＜0.01）,而OX-HDL则显著增加OX-LDL刺激SMC增殖作用（P＜0.01）     

麻黄碱抑制支气管平滑肌细胞增殖的机制

BK通道(large-conductance Ca2 -activated K channel)是一种大电导、电压和钙离子依赖的钾通道,存在于哺乳动物几乎所有的组织中.β1亚基是首次从平滑肌细胞中分离到的一种BK通道辅助亚基.麻黄作为一种β肾上腺素受体激动剂,是一味治疗哮喘的常用中药.利用RT-PCR和膜片钳等技术,研究麻黄治疗哮喘的作用机制.结果表明:麻黄碱(ephedrine,Eph)作为麻黄的一种主要成分,显著抑制支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)的增殖和BK通道β1亚基的表达,但是对BK通道的电流没有显著影响.     

同型半胱氨酸对大鼠血管平滑肌细胞增殖的作用

血中同型半胱氨酸（ｈｏｍｏｃｙｓｔｅｉｎｅ,ＨＣＹ）浓度的升高已成为动脉粥样硬化发生的一个独立危险因子．为进一步阐明ＨＣＹ促进血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌｓ,ＶＳＭＣｓ）增殖,从而引起动脉粥样硬化发生的机理．本实验采用细胞计数、３Ｈ－ＴｄＲ参入、细胞周期分析、Ｎｏｒｔｈｅｒｎ杂交方法证明,一定剂量的ＨＣＹ可促进离体培养的ＷＫＹ大鼠血管平滑肌细胞增殖,使其ＤＮＡ合成增加,细胞周期中Ｓ期细胞所占比例增加４３％,并促进ｃ－ｍｙｃ与ｃ－ｆｏｓ原癌基因ｍＲＮＡ表达增加．提示ＨＣＹ可能通过促进ＶＳＭＣｓ增殖而诱发动脉粥样硬化     

Ca/CaM与缺氧肺动脉平滑肌细胞增殖的关系研究

目的:探讨缺氧时钙内流、钙调素、NF□κB与肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)增殖的关系。方法:组织块贴壁法培养成年大鼠PASMCs,细胞分组处理后,分别应用四甲基偶氮唑(MTT)检测细胞数量、流式细胞术检测细胞周期分布和增殖指数、免疫荧光检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达。结果:2%O_2条件下培养12h显著刺激PASMCs增殖,与常氧组相比,细胞活力增加40%,增殖指数(proliferation index,PI)是常氧组的1．46倍, PCNA表达增强。L-型钙通道阻滞剂硝苯吡啶(nifedipine,NFD,10μM)、钙调素拮抗剂三氟拉嗪(trifluoperazine,TFP,10μM)、核转录因子NF□κB抑制剂PDTC(20μM)均抑制了缺氧诱导的PASMCs的增殖,其抑制作用的强度依次为NFD＜PDTC＜TFP。结论:钙信号转导参与缺氧诱导的PASMCs增殖,涉及通过L-型钙通道的钙内流、钙调素以及转录因子NF-κB激活等环节。