Vascular smooth muscle cells derived from atherosclerotic human arteries exhibit greater adhesion, migration, and proliferation than venous cells

BACKGROUND: Phenotypic variation of vascular smooth muscle cells (VSMC) may result in altered biological behavior and responses. Within the vessel wall, arterial VSMC have a greater propensity to form atherosclerotic lesions as compared to venous VSMC. In this study the rates of proliferation, adhesion, and migration were compared between VSMC of atherosclerotic arterial and venous origin. MATERIALS AND METHODS: Human VSMC cultures were isolated from 18 infragenicular arteries at the time of below knee amputation and from 20 saphenous veins during lower extremity revascularization surgery. Cell cultures were isolated from the media of each specimen and maintained in distinct cell lines for all assays. Cells from passages 2 and 3 were assayed for their proliferative capacity using total DNA fluorescence photometry and for adhesion and migration using a modified Boyden chamber. RESULTS: Patient age and the incidence of atherosclerotic risk factors did not vary significantly between the arterial and the venous patient groups. VSMC of atherosclerotic arterial origin demonstrated greater proliferation (arterial, 162 +/- 59 absorption units, vs. venous, 106 +/- 56 absorption units, P < 0.001), adhesion (arterial, 74.1 +/- 22.6 cells/microscopic field, vs. venous, 41.3 +/- 12.8 cells/microscopic field, P < 0.001) and migration (arterial, 427 +/- 185 cells/microscopic field, vs venous, 119 +/- 101 cells/microscopic field, P < 0.001) than VSMC of venous origin. CONCLUSION: Human atherosclerotic arterial VSMC exhibit significantly increased rates of proliferation, adhesion, and migration as compared to human venous VSMC. These observations of VSMC in culture are consistent with the clinical predilection for the hyperplasic responses that result in the development of atherosclerosis in the arterial wall. Possible intrinsic differences in VSMC phenotype should be considered in designing methods to limit atherosclerosis.     

普伐他汀抑制肿瘤坏死因子α介导的人脐动脉血管平滑肌细胞成骨样钙化

目的 研究普伐他汀干预对肿瘤坏死因子α（TNF-α）介导的人脐动脉血管平滑肌细胞（hUASMC）成骨样分化标志蛋白骨特异性碱性磷酸酶（BAP）、骨桥蛋白（OPN）和骨形成蛋白2（BMP-2）表达以及细胞外钙盐沉积的影响，并探讨普伐他汀在血管钙化防治中的潜在作用。 方法 植块贴壁法原代培养人hUASMC。予以TNF-α刺激和普伐他汀干预，甲O-酚酞络合酮方法测定细胞外基质钙盐沉积；实时定量PCR法观察血管平滑肌细胞BAP和OPN mRNA表达水平；免疫印迹法观察血管平滑肌细胞BAP、OPN和BMP-2蛋白表达水平。 结果一定浓度范围的普伐他汀呈浓度依赖方式抑制TNF-α对hUASMC的促增殖作用（r = －0.946，P < 0.01）；一定浓度范围的普伐他汀呈浓度依赖方式下调TNF-α介导上调的BAP、OPN mRNA表达（r = －0.972，P < 0.01）与蛋白的表达水平（BAP蛋白，r = －0.820，P < 0.01；OPN蛋白，r = －0.972，P < 0.01；BMP-2蛋白，r = －0.928，P < 0.01），抑制血管平滑肌细胞成骨样分化，减少细胞外基质钙盐的沉积（r = －0.973，P < 0.01）。 结论 普伐他汀可抑制TNF-α对hUASMC的促增殖作用，抑制细胞成骨样分化，减少细胞外基质钙盐的沉积。     

ERK1/ERK2磷酸化在西地那非抑制猪肺动脉平滑肌细胞增殖中的作用

目的探讨丝裂原激活的蛋白激酶-44/蛋白激酶-42（ERK1/ERK2）磷酸化在西地那非抑制猪肺动脉平滑肌细胞增殖中的作用。方法体外原代培养猪肺动脉平滑肌细胞。细胞培养到3～5代后用于实验。随机分为4组：对照组（C组）、血小板衍生生长因子（PDGF）组（P组）、西地那非干预组（PS1组和PS2组）,P组细胞用20 ng/ml PDGF孵育,PS1组和PS2组在加入PDGF前20 min分别加入24、96μmol/L西地那非。于加入PDGF后1 h测定ERK1/ERK2磷酸化水平;于加入PDGF后24 h掺入5-溴脱氧尿嘧啶核苷（5-BrdU）,计算5-BrdU阳性细胞百分率,反映细胞DNA合成水平,并测定增殖细胞核抗原（PCNA）蛋白表达;于加入PDGF后3 d测定细胞增殖程度。结果与C组比较,P组、PS1组和PS2组ERK1/ERK2磷酸化水平、PCNA表达、5-BrdU阳性细胞百分率及细胞增殖率增加（P＜0．05或0．01）,PS1组和PS2组ERK1/ERK2磷酸化水平、PCNA表达、5-BrdU阳性细胞百分率及细胞增殖率低于P组（P＜0．05或0．01）,各组间总ERK1/ERK2水平差异无统计学意义（P＞0．05）。结论西地那非通过抑制ERK1/ERK2的磷酸化,下调PCNA的表达,抑制了DNA的合成,从而抑制了PDGF诱导猪肺动脉平滑肌细胞的增殖。     

Carbamylated low-density lipoprotein induces proliferation and increases adhesion molecule expression of human coronary artery smooth muscle cells

Aim: Presence of accelerated atherosclerosis in dialysis patients cannot be entirely explained by conventional risk factors. Exposure to urea, which is elevated in patients with kidney disease, leads to the carbamylation of proteins. We investigated the effects of carbamylated low-density lipoprotein (cLDL) on human coronary artery vascular smooth muscle cells (VSMC). Methods: Native LDL (nLDL) was carbamylated with potassium cyanate. Cells were incubated with different concentrations of cLDL carbamylated at different time points. Cytotoxicity, apoptosis, proliferation (bromodeoxyuridine incorporation), expression of adhesion molecules and extracellular matrix protein synthesis were studied. Results: Carbamylated low-density lipoprotein exposure leads to morphological alterations and presence of cellular debris. Neither nLDL nor cLDL caused apoptosis. Lactate dehydrogenase (LDH) release was not different between groups. Carbamylated low-density lipoprotein led to a striking proliferation in VSMC compared to nLDL. Carbamylated low-density lipoprotein significantly increased intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression compared to the control. The effects of cLDL on proliferation and adhesion molecule expression were dose-dependent and correlated with the degree of low-density lipoprotein carbamylation. cLDL had no effect on extracellular matrix protein synthesis. Conclusion: The results support the hypothesis that cLDL may contribute to the pathogenesis of atherosclerosis in uraemic patients.     

Platelet-derived growth factor-induced expression of c-fos in human vascular smooth muscle cells: implications for long-term graft patency

BACKGROUND: The internal mammary artery (IMA) has been shown to have a significantly superior long-term patency rate when compared with the saphenous vein (SV) graft. Cultured smooth muscle cells (SMCs) from the IMA are more resistant to the mitogenic effects of platelet-derived growth factor (PDGF), when compared with SMCs that are derived from the SV. The radial artery (RA) is currently being used as an alternative to the SV. However, no long-term patency data are available for the RA, and there is no information on the biological behavior of RA-derived SMCs in culture. METHODS: Smooth muscle cell cultures were taken from patients who underwent coronary artery bypass grafting with the IMA, RA, and SV. A quiescent state was induced by serum deprivation for 5 days. Thereafter cells were induced to proliferate by exposure to PDGF-BB. Levels of c-fos expression and 3H-thymidine incorporation were used as markers of cell proliferation. RESULTS: We found that even after serum deprivation, c-fos was still detectable; however, basal levels were higher in cells from the SV than cells from either the RA (p = 0.003) or IMA (p = 0.008). After stimulation with PDGF-BB, c-fos expression was greater in SMCs from the SV relative to the RA (p < 0.001) or the IMA (p = 0.02). Finally, relative to the SV, 3H-thymidine in the RA was 0.76 +/- 0.22 (p < 0.05) and 0.39 +/- 0.24 (p < 0.002) in the IMA, respectively. CONCLUSIONS: The data indicate that SMCs from arterial conduits are more resistant to the mitogenic effects of PDGF-BB than those from venous conduits. Our results offer a mechanistic explanation of why arterial conduits might demonstrate patency superior to that of the SV.     

Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells

BACKGROUND: Cyclophilin A (CypA) is a cytosolic protein which involves many biological functions including immune modulation, cell growth, tumorigenesis, and vascular disease. The objective of this study was to determine the effect of CypA on cell proliferation and several gene expressions in human endothelial cells and vascular smooth muscle cells. METHODS: Human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HMVEC-L), and human aorta smooth muscle cells (HAoSMC) were used in this study. Cells were treated with 10 nM CypA for 24 h. The cell proliferation was determined by [3H]thymidine incorporation. The mRNA levels of 13 genes including CD147 (receptor for CypA), PDGF-BB, endothelin-1 (ET-1), vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1), NRP-2, eNOS, iNOS, nNOS, ICAM-1, and PECAM-1 were semiquantitatively determined by real time RT-PCR as standardized with a house keeping gene beta-actin. RESULTS: CypA significantly increased cell proliferation of HAoSMC and HMVEC-L by 31% and 45%, respectively, as compared to controls, but had no effect on HCAEC. Blocking CD147 did not affect the mitogenic action of CypA. In addition, CypA also significantly increased the mRNA expression of CD147 by 43% and VEGFR-2 by 65% in HAoSMCs (P < 0.05, t test). HAoSMCs expressed much higher CD147 and neuropilin-1 (NRP-1) mRNA than HMVECs-L and HCAECs (P < 0.017, ANOVA). Furthermore, CypA increased ET-1 mRNA by 22% and VEGFR-1 mRNA by 23% in HMVECs-L, but had limited effects on HCAECs. HMVECs-L had much higher expressions of PDGF-BB, ET-1, VEGFR-2, VEGFR-1, VEGFR-3, and NRP-2 than HAoSMCs and HCAECs (P < 0.017, ANOVA). By contrast, HCAECs had much higher ICAM-1 mRNA levels than HMVECs-L and HAoSMCs (P < 0.017, ANOVA). CONCLUSIONS: These data demonstrate that CypA has a mitogenic effect on HAoSMCs and HMVECs-L, but not HCAECs. CD147 may not mediate the action of CypA. In addition, CypA substantially alters the mRNA levels of several key genes in human vascular cells, indicating potential multifunctional roles of CypA in vascular system. Furthermore, this study provides several new aspects of gene expressions in vascular cells.     

The effect of six different statins on the proliferation, migration, and invasion of human smooth muscle cells

Intimal hyperplasia (IH) can occur after any vascular injury and results from smooth muscle cells (SMC) proliferation, migration, and invasion into the subintimal space. The purpose of this study was to investigate the effect of six different statins on the proliferation, migration, and invasion of human venous SMC. The statins were all used at their Cmax concentrations. SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with the six statins. Migration and invasion experiments were performed using modified Boyden chambers. The invasion experiments were performed using Matrigel coated plates. We found that all of the statins significantly inhibited SMC proliferation compared to the platelet-derived growth factor control (ranging from fluvastatin 33% of control to pravastatin 72% of control, P = 0.03). SMC migration through uncoated polycarbonate membranes in presence of the six statins was significantly reduced (ranging from lovastatin 43% to pravastatin 57% of control, P = 0.006). All six statins also significantly reduced SMC invasion (ranging from fluvastatin 65% to simvastatin 87% of control, P = 0.002). We conclude that the inhibitory effect of statins on SMC proliferation, migration, and invasion is a class, rather than drug specific effect.     

Porcine complement regulators protect aortic smooth muscle cells poorly against human complement-induced lysis and proliferation: consequences for xenotransplantation

BACKGROUND: Accelerated atherosclerosis after transplantation has been observed and is characterized by smooth muscle cell proliferation in the graft. Porcine cells are frequently used in models of atherosclerosis and porcine organs are considered for use in transplantation. Complement (C) activation is known to play a major role in rejection of xenografts and is also considered to play a role in the development of atherosclerosis. The aim of this study was to investigate the expression and function of membrane bound regulators of complement (CReg) on porcine aortic smooth muscle cells (PASMC). METHODS: The PASMC were assessed for expression of CReg and susceptibility to lysis by human C by flow-cytometry. The effect of various cytokines on CReg expression and C-susceptibility was investigated. The ability of human C to induce cell proliferation was assessed using the Alamar blue assay. RESULTS: The PASMC only express the CReg membrane cofactor protein (MCP) and CD59 on their cell surface. MCP expression was increased by interleukin (IL)-4. In contrast to porcine aortic endothelial cells (PAEC), PASMC were found to be surprisingly sensitive to C-mediated lysis, mainly due to a low level of expression of CD59. Human C-induced proliferation of PASMC, which was dependent on complete membrane attack complex (MAC) formation. CONCLUSIONS: Endogenously expressed CReg on PASMC poorly protect these cells to human C. Human C can induce proliferation of PASMC. In order to prevent accelerated atherosclerosis in porcine xenografts, increased levels of CReg not only have to be obtained on the endothelial cells but also on the smooth muscle cells.     

重组人骨形态发生蛋白-2对体外培养的肺动脉平滑肌细胞增殖的影响及其机制

目的探讨重组人骨形态发生蛋白(rhBMP)-2对体外培养的肺动脉平滑肌细胞(PASMC)增殖的影响及机制。方法将用贴块法培养的PASMC传代后分成5组,Ⅰ组:含0.1?S培养基培养;Ⅱ组:含10?S培养基正常培养;Ⅲ组:10?S rhBMP-2 1μg/L;Ⅳ组:10?S rbBMP-2 10μg/L;Ⅴ组:10?S rhBMP-2 100μg/L。倒置相差显微镜及免疫荧光法观察并鉴定培养的PASMC;使用噻唑蓝(MTT)比色法测定PASMC增殖率;流式细胞仪分析PASMC细胞周期的变化;3H-TdR掺入实验检测DNA合成情况;RT-PCR检测细胞周期素(Cyclin)D1 mRNA的表达量;Western blot检测pSmad蛋白量及Cyclin D1蛋白表达。结果抗SM-α-actin免疫荧光染色鉴定所培养细胞为PASMC;Ⅱ组引起PASMC增殖率升高(90.26±30.12),S期细胞百分比增加(35.90±0.73),DNA合成增加(4890±372)的作用可以被Ⅲ、Ⅳ组的rhBMP-2抑制(66.29±27.19、15.90±0.61、3390±198,P<0.01),同时Ⅲ、Ⅳ组的rhBMP-2明显增加了pSmad蛋白量(1.37±0.26,1.68±0.31,P<0.01),并抑制了Cyclin D1 mRNA及蛋白表达(1.38±0.13,1.01±0.10,P<0.01)。结论rhBMP-2可以通过Smad信号转导途径抑制10?S培养的PASMC表达Cyclin D1,进而抑制血清刺激引起的PASMC增殖。     

高迁移率族蛋白B1对体外人肺动脉血管平滑肌细胞增殖、迁移和凋亡的影响

目的 评价高迁移率族蛋白B1(HMGB1)对体外培养人肺动脉血管平滑肌细胞(hPASMC)增殖、迁移和凋亡的影响.方法 体外培养hPASMC,调整细胞密度(2× 105个/ml)后接种到96孔板(100 μl/孔,2× 105个/ml)、6孔板(1ml/孔,2× 106个/ml)和改良24孔Boyden趋化小室(100μg/孔,5×103个/ml),采用随机数字表法,将其分为5组:对照组(C组)和不同浓度HMGB1组(H1组～H4组),分别在DMEM和含HMGB1 1、10、100、1000 ng/ml的DMEM培养液孵育.孵育24和48 h时,采用MTT法检测细胞增殖率,Boyden小室法检测透膜细胞数,TUNEL法检测hPASMC凋亡情况.结果 与C组比较,H1组～H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);与H1组比较,H2组～H4组细胞增殖率升高,H3组和H4组透膜细胞数增多(P＜0.05);与H2组比较,H3组和H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);H3组和H4组间各指标比较差异无统计学意义(P＞0.05);与孵育24h时比较,各组孵育48 h时细胞增殖率升高(P＜0.05).各组细胞凋亡率比较差异无统计学意义(P＞ 0.05).结论 HMGB1可促进hPASMC的增殖和透膜迁移,可能参与肺损伤肺血管重构的发生.     

Smooth excimer laser coronary angioplasty (SELCA) and conventional excimer laser angioplasty: Comparison of vascular injury and smooth muscle cell proliferation

Although the excimer laser, which utilizes ‘non-thermal ablation effects’, has achieved encouraging results in early clinical trials, the long-term results have failed to show any advantage over conventional percutaneous transluminal coronary angioplasty (PTCA). A new system, Smooth Excimer Laser Coronary Angioplasty (SELCA), has been developed to reduce the tissue damage in the vessel wall caused by shock waves and vapour bubbles.SELCA (wavelength 308 nm, pulse duration 115 ns, repetition rate 150 Hz and energy density 50 mJ mm^-2) lowers the amount of shock wave formation and pressure peak amplitude in the surrounding tissue by about eight times when compared to the conventional 308 nm excimer laser (ELCA). In this preclinical evaluation, this new system was compared to ELCA. Fifty New Zealand White rabbits were stimulated by repeated weak DC impulses for a period of 28 days in order to form an atherosclerotic plaque in the right carotid artery. The vessels were excised 3, 7,14 and 28 days after laser irradiation for immunohistochemical analysis. SELCA and ELCA laser treatment lead to a decrease in maximal intimal wall thickness 3 days after intervention (control: 177±4 μm; SELCA: 131±22μm; ELCA: 120 ±33μm). In the period between 3 and 28 days, a moderate increase in intimal wall thickness was observed after SELCA treatment compared to a significant increase after ELCA (28 days after intervention: SELCA: 157±22μm; ELCA: 274 ±28μm). Bromodeoxyuridine (BrdU) was applied 18 and 12 h before excision of the vessels in order to determine the percent of cells undergoing DNA synthesis. The percent of BrdU labelled SMC in the intima (control: 13 ± 2 cells mm_-2) increased in both groups after 3 days (SELCA: 248 ± 107 cells mm^-2; ELCA: 162 ± 41 cells mm^-2) and 7 days (SELCA: 162± 55 cells mm^-2; ELCA: 279 ± 119 cells mm^-2). The present results demonstrate that vascular wall injury and increase in intimal wall thickness following SELCA are reduced in comparison to the results achieved with the conventional technique. Further trials are necessary to assess whether these improvements will lead to more favourable long-term results after excimer laser angioplasty.     

Effect of cyclic hydrodynamic pressure‐induced proliferation of human bladder smooth muscle through Ras‐related C3 botulinum toxin substrate 1, mitogen‐activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2

Objectives: To examine the role of Ras‐related C3 botulinum toxin substrate 1, mitogen‐activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 in the cyclic hydrodynamic pressure‐induced proliferation of human bladder smooth muscle cells. Methods: Human bladder smooth muscle cells were exposed to cyclic hydrodynamic pressures in vitro with defined parameters (static, 100 cmH₂O, 200 cmH₂O and 300 cmH₂O pressure) for 24 h. The proliferation of cells was assessed by flow cytometry. Ras‐related C3 botulinum toxin substrate 1, mitogen‐activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 messenger ribonucleic acid, and protein expression was analyzed by real‐time polymerase chain reaction and Western blot. Specificity of the Rac1 was determined with real‐time polymerase chain reaction and Western blot technique with small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766). Results: The proliferation of human bladder smooth muscle cells was increased. Ras‐related C3 botulinum toxin substrate 1, mitogen‐activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 were activated by 200 and 300 cmH₂O cyclic hydrodynamic pressure compared with static and 100 cmH₂O pressure. The “knockdown” of activation of Rac1 using target small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766) decreased proliferation of human bladder smooth muscle cells, and downregulated mitogen‐activated protein kinase kinase 1/2, extracellular regulated protein kinases 1/2. Conclusion: The Rac1 pathway is activated in mechanotransduction and regulation of human bladder smooth muscle cell proliferation in response to cyclic hydrodynamic pressure.