Effects of caffeic acid phenethyl ester on proliferation of vascular smooth muscle cells in rats

Objective： To investigate the inhibitory effect of caffeic acid phenethyl ester（CAPE） on the proliferation of vascular smooth muscle cells （VSMC） activated by lipopolysaccharide （LPS） and to clarify its mechanism. Methods： VSMC activated by LPS （1 mg-L^-1） were treated with CAPE at different concentrations. The inhibitory effecfs of CAPE on the proliferation of VSMC were determined by methabenzthiazuron（MTT） colorimetry. The effects of CAPE on the expression of proliferating cell nuclear antigen （PCNA） and Survivin protein in VSMC were evaluated by immunocytochemistry staining technique （SABC method）. Cell cycle was analyzed by flow cytometry（FCM） with propidiumiodide （PI） labeling method. The relative expression level of Survivin mRNA was measured with real-time quantified RT-PCR technique. Results. CAPE exerted significant inhibitory effects on. proliferation of VSMC at concentrations ranging from 5 mg·L^-1 to 80 mg·L^-1, decreased the rate of cells positive for PCNA and Survivin protein and repressed the expressioh of Survivin mRNA in a dose- and time-dependent manner （P 〈 0.05）. FCM analysis displayed that CAPE up-regulated the ratio of G0/G1 stages and reduced the percentage of VSMC in S stage （P 〈 0.05）. Conclusion： CAPE can significantly inhibit the proliferation of VSMC activated by LPS in a dose- and time-dependent manner, which may be carded out through regulating cell cycle and repressing the expression of PCNA and Survivin.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Effects of estradiol on cell cycle and cyclin proteins of vascular smooth muscle cells in rats

To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0～100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.

Different Responses of Cell Cycle between Rat Vascular Smooth Muscle Cells and Vascular Endothelial Cells to Paclitaxel

Summary： Although previous reports showed dmg-eluting stent （DES） could effectively inhibit neointima formation, in-stent restenosis （ISR） remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells （VSMCs） and vascular endothelial cells （VECs） of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, prolif- eration of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1 S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.

Batroxobin reduces intracellular calcium concentration and inhibits proliferation of vascular smooth muscle cells

Background Batroxobin (BX),a serine protease used in defibrinogenation and thrombolysis,also has an effect on c-fos gene and growth factor. This study attempted to determine the effects of BX on the proliferation of vascular smooth muscle cells (VSMCs) and calcium metabolism. Methods VSMCs were treated with BX at concentrations of 0.1,0.3,or 1.0 mmol/L and cell numbers were determined at 0,24,48,and 72 hours. Intracellular calcium concentration ([Ca 2+ ]_i) was measured using direct fluorescence methods. Results BX was found to suppress proliferation of VSMCs in a dose-dependent fashion with inhibition rates of 18% and 31% by 48 and 72 hours,respectively. In addition,BX decreases basal [Ca 2+ ]_i significantly. The basal level in untreated cells was 162.7±33.8 nmol/L,and decreased to 131.5±27.7 nmol/L,128.3±28.5 nmol/L,and 125.6±34.3 nmol/L with the three concentrations of BX,respectively. Noradrenaline (NE)-induced [Ca 2+ ]_i stimulation was also attenuated by BX (0.1 mmol/L BX,20%±8% inhibition; 0.3 mmol/L BX,54%±11% inhibition; 1.0 mmol/L BX,62%±15% inhibition). The ability of NE to stimulate [Ca 2+ ]_i was attenuated in cultures in Ca 2+ -free medium,as was the ability of BX to blunt NE-induced stimulation. Conclusion These findings demonstrate that BX can effectively inhibit proliferation of VSMCs,probably by blocking the release and uptake of Ca 2+ ,thus influencing [Ca 2+ ]_i.

Effect of Aloe Emodin on Proliferation of Vascular Smooth Muscle Cells after Arterial Injury

Objective: To study the effect of Aloe emodin (AE), an active ingredient of Rhubarb,on the kinetics of proliferation of smooth muscular cells (SMCs) cultured in vitro after rabbit iliac arterial injury. Methods: Forty-eight hours after de-endothelialization (balloon endothelial denudation), the iliac arteries of the Japanese white rabbits were isolated and the smooth muscle cells were cultured primarily.AE was added to culture medium containing 10% fetal calf serum (FCS ). The cultures were pulse-labeled with 3H-TdR and TdR uptake into VSMC were measured and the cell cycle of the cultures were analyzed by using flow cytometer. Results: Compared with control, when the concentration gradient ranged from 10 - 1 to 10-5 g/L, the amount (cpm,count per minute) of 3H-TdR uptake into SMCs has significant differences (P < 0. 05 )and 10 -1 and 10 -2 g/L AE showed strong inhibitory effects on TdR uptake into VSMC and the percentage of inhibition [% inhibition =(cpm without AE-cpm with AE)/cpm without AE] was more than 90%. AE displayed concentration dependent inhibitory effects. The percentage of cells in G0/G1 phase was increased, but the percentage of cells in S phase was decreased in AE group, the transition of SMC cycle phase from G0 to S was blocked.Conclusion: AE is a strong inhibitor to the proliferation of SMCs and the pharmacological action of AE may reduce SMC proliferation in vivo and decrease intimal hyperplasia of restenosis.Original article on CJIM(Chin) 1998; 18(7): 420

Activated focal adhesion kinase involved in adhesion and migration of vascular smooth muscle cells stimulated by fibronectin

OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively. RESULTS: FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P

Endothelin-1, an important mitogen of smooth muscle cells of spontaneously hypertensive rats

OBJECTIVE: To study the features of vascular smooth muscle cell (VSMC) proliferation induced by endothelin-1 (ET-1). METHODS: VSMCs of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats were cultured and treated with ET-1. Basic fibroblast growth factor (bFGF) gene expression was measured using both Northern blot and an enzyme-linked immunoassay. RESULTS: ET-1 resulted in an increase in bFGF transcripts at 8 - 24 h; bFGF levels were significantly higher in VSMCs treated with ET-1 than in those not treated. However, VSMCs growth responses in SHR and WKY were different. Smooth muscle cells of SHR were hyper-responsive to ET-1. Maximal bFGF mRNA levels were elevated 3.5-fold at 4 h of stimulation in WKY and 8-fold at 8h in SHR4. Moreover, the proliferation of VSMCs induced by ET-1 was inhibited by antisense phosphorothioate oligodeoxynucleotides (10 micromol/L AS-bFGF) but not sense bFGF oligomers at the same concentrations, being reduced by 80% in SHR and 40% in WKY vs control, respectively. Furthermore, the effect of AS-bFGF oligomers on SHR SMC proliferation is significantly greater than on WKY SMC proliferation. CONCLUSION: ET-1 may be required for exaggerated vascular growth responses in SHR and bFGF may be involved.

A new cerebroside and its anti-proliferation effect on VSMCs from the radix of Cyperus rotundus L.

A new cerebroside,1-O-(β-D-glucopyranosyloxy)-(2S,3R,4E,8Z)-2-[(2′R)-2’-hydroxylignoceranoylamino]-4,8-tetradecene-3- diol was isolated from the 60%EtOH extract of traditional Chinese medical plant Cyperus rotundus L.Its structure was determined on the basis of spectroscopic data.This new compound showed anti-proliferation effect on vascular smooth muscle cells(VSMCs).

钾离子通道在低氧性肺动脉高压中的作用及药物干预研究进展

钾离子(K^(+))通道是位于细胞膜上的一种跨膜蛋白,血管平滑肌细胞K^(+)通道通过膜电位在血管张力、细胞兴奋性和细胞增殖等方面发挥重要调控作用。肺动脉平滑肌细胞K^(+)通道功能障碍与低氧性肺动脉高压(HPH)的病理进程密切相关,K^(+)通道有望成为HPH的治疗靶点。对肺动脉平滑肌细胞K^(+)通道的种类以及在HPH中的研究进展、相关干预药物进行综述,旨在为HPH的发病机制研究和药物研发提供新思路。

血管支架植入对血管平滑肌细胞的影响

支架植入是冠心病的一种重要治疗方式,植入过程伴随着血管壁的损伤和机械性能的改变,进而导致血管平滑肌细胞进行一系列复杂的表型变化,包括从中膜向内膜的迁移和增殖,以及从收缩表型转变为合成表型等,从而导致新生内膜的增殖和支架内再狭窄的发生。恢复血管生理稳态、保持血管平滑肌细胞表型稳定是经皮冠状动脉介入治疗的最终理想,故探究血管支架植入后血管平滑肌细胞发生的生物反应,将有助于新一代心血管器械研发,帮助临床决策的制定。现对血管支架植入对血管平滑肌细胞的生物学影响进行综述。

硫化氢作为心血管信号分子的研究

To explore the possibility of hydrogen sulfide (H 2S) as a messenger molecule in cardiovascular system, the authors discovered that H 2S (5×10 -5 -5×10 -4 mol·L -1 )exerted an effect on inhibiting endothelin 1 induced proliferation of cultured vascular smooth muscle cells (VSMCs) of rats in vitro . The 3H TdR incorporation decreased by 16.8%～37.4% in H 2S treated VSMCs as compared with the controls ( P <0.01). The inhibitory effect was found to be associated with reduced activity of MAPK. The authors also observed that endogenous H 2S levels markedly increased in vessels of rats with either endotoxic shock or septic shock [H 2S level (pmol·min -1 ·mg -1 ):tail artery (16.18±2.06) vs (8.12±0.55);mesenteric artery (10.17±1.11) vs (6.19 ±0.55);pulmonary artery(11.38±1.24) vs (5.27±0.51); aorta(6.21±0.48) vs (4.10± 0.28), P < 0.01 ]. The above findings suggested that H 2S might play an important role in the regulation of cardiovascular pathophysiologic events.

理脾化痰方对食饵性动脉粥样硬化症家兔血管平滑肌细胞增殖和凋亡的调控

【目的】探讨由法半夏、白术、天麻、橘红、丹参等组成的理脾化痰方抗早期动脉粥样硬化症(atherosclerosis,AS)的可能性和作用机制。【方法】通过组织形态学 (包括光镜和电镜 )、免疫组化和TUNEL等方法观察了该方对食饵性动脉粥样硬化症家兔脂纹脂斑期主动脉内膜形态和相对厚度、血管平滑肌细胞 (vascularsmoothmusclecells ,VSMCs)ki- 6 7和凋亡细胞阳性率的影响。【结果】理脾化痰方确能阻抑早期AS的形成 ,观察组主动脉内膜相对厚度小于模型组 (P <0 .0 5) ,VSMCski- 6 7的表达率和凋亡的VSMCs阳性率均低于模型组 (P <0 .0 5)。【结论】该方抗早期AS的机制与其同时抑制VSMCs的增殖和过高水平的凋亡有关。

L-精氨酸调节大鼠低氧性肺血管结构重建与肺动脉平滑肌细胞凋亡

目的 :探讨L 精氨酸 (L arginine ,L Arg)对低氧性肺血管结构重建大鼠肺动脉平滑肌细胞凋亡的影响及其机制。方法 :将 17只Wistar大鼠随机分为对照组 (n =7)、低氧组 (n =5 )和低氧 +L Arg组 (n =5 )。对大鼠肺血管进行显微形态学观测。通过末端转移酶介导的dUTP切口末端标记法 (TUNEL)检测肺动脉平滑肌细胞凋亡 ,并以免疫组织化学方法研究肺动脉平滑肌细胞Fas表达。结果 :低氧 2周后出现大鼠肺血管结构重建。同时 ,肺中、小型动脉凋亡平滑肌细胞百分数均明显低于对照组 (P均 <0 .0 5 ) ,平滑肌细胞Fas表达明显降低。然而 ,L Arg缓解了低氧性肺血管结构重建的形成。低氧 +L Arg组大鼠肺中、小型动脉凋亡平滑肌细胞百分数均明显高于低氧组 (P均 <0 .0 5 )。L Arg使低氧大鼠肺动脉平滑肌细胞Fas表达明显增强。 结论 :L Arg通过使肺动脉平滑肌细胞Fas表达上调 ,促进肺动脉平滑肌细胞的凋亡 。

杭白菊对小牛血管平滑肌细胞凋亡及其抗氧化性研究

目的 :探讨杭白菊提取液对小牛主动脉血管平滑肌细胞 (VSMC)凋亡的影响及可能的作用机制。方法 :取小牛主动脉进行体外 VSMC培养 ,加入杭白菊提取液干预 ,用膜联蛋白 Annexin检测法 ,在流式细胞仪下观察VSMC的凋亡现象 ,同时取上清液在分光光度仪下测定超氧化物歧化酶 (SOD)、丙二醛 (MDA)。结果 :1在流式细胞仪下观察到加入杭白菊提取液后 VSMC凋亡数量减少 ,随着杭白菊提取液浓度的加大 ,细胞凋亡从 (4.4 2 5±0 .6 2 4 ) %降至 (2 .875± 0 .6 4 0 ) % ;2随着杭白菊提取液浓度的增加 ,SOD值由 (1.6 83± 0 .149)× 10 4 U/ L升高到(2 .2 97± 0 .2 30 )× 10 4 U/ L、MDA值由 (16 6 .4 5 4± 5 6 .80 5 ) μmol/ L下降至 (73.0 6 8± 2 7.2 0 3) μmol/ L。结论 :杭白菊提取液具有抑制小牛 VSMC凋亡及抗氧化作用 ,并呈浓度依赖性。

钙信号在尾加压素-Ⅱ促血管平滑肌增殖中的作用

目的 :探讨钙信号在尾加压素 Ⅱ (urotensin Ⅱ ,UⅡ )促血管平滑肌细胞 (vascularsmoothmusclecell,VSMC)增殖中的作用。方法 :在离体培养的VSMC上 ,用流式细胞仪和3 H TdR参入的方法探讨UⅡ的促增殖效应 ;在离体孵育的大鼠主动脉平滑肌组织上观察UⅡ对平滑肌45Ca2 + 摄入的影响 ;应用激光共聚焦显微镜检测UⅡ作用后细胞内游离钙的改变。结果 :UⅡ (10 -9～ 10 -7mol·L-1)浓度依赖地刺激VSMC的DNA合成 ,并诱导细胞由静止向增殖状态转化。与对照组相比 10 -8mol·L-1UⅡ使细胞的增殖指数 [(G2 M +S) % ]增加 192 % (P <0 .0 1)。钙通道阻断剂尼卡地平和Ca2 + 螯合剂EDTA均可阻断UⅡ对VSMC的促增殖效应 ,其中UⅡ (10 -8mol·L-1)与尼卡地平 (10 -5mol·L-1)共同孵育的VSMCDNA合成量仅为单纯UⅡ (10 -8mol·L-1)组的 5 9.0 %。UⅡ (10 -10 ～ 10 -8mol·L-1)可浓度依赖地使胸主动脉对45Ca2 + 的摄入增加 ,尼卡地平 (10 -5mol·L-1)也可显著对抗UⅡ (10 -8mol·L-1)对45Ca2 + 摄入的诱导作用 (P值均小于 0 .0 1)。用激光共聚焦显微镜观察发现UⅡ作用后细胞内Ca2 + 浓度迅速升高 ,持续约 3min ,形成钙波。结论 :UⅡ可通过促进细胞Ca2 + 摄取和增加细胞内Ca2 + 浓度 。

灯盏花素对大鼠主动脉肌环的松弛作用

用苯肾上腺素及氯化钡引起血管环收缩，并测定五种血管扩张药对这两种激动剂缩血管作用的拮抗作用的比值。结果表明，灯盏花素的作用与三氟拉嗪相似，是以抑制细胞内钙释放为主。在去除血管内皮的实验中发现，灯盏花素的松弛血管平滑肌的作用，与三氟拉嗪、脑益嗪及硝吡啶均相似，即并不依赖血管内皮的完整性，但从量效曲线来看，灯盏花素与前两者（尤其是与三氟拉嗪）相平行。然而，灯盏花素对五羟色胺的缩血管作用无任何拮抗作用，三氟拉嗪在低浓度时未显示拮抗作用，在高浓度时，可使量效曲线明显右移。这些结果说明灯盏花素的作用可能与拮抗钙调素的作用有关，但确切的机理仍有待进一步探讨。

莪术组分涂层支架预防猪冠状动脉再狭窄的研究

目的通过中华实验用小型猪冠状动脉再狭窄模型,观察中药莪术组分涂层支架抑制内膜增殖的有效性。方法将18只小型猪随机分为莪术组分涂层支架组(ZES组)、雷帕霉素涂层支架组(SES组)及金属裸支架组(BMS组),每组6只,分别在左前降支、左回旋支及右冠状动脉置入同一种支架各1枚。术后30 d冠状动脉造影后将猪处死,观察支架血管段的病理形态及影像学变化。结果 30 d时,与BMS组比较,ZES组和SES组平均管腔直径和平均管腔面积均明显增大(P<0.05),直径狭窄率和面积狭窄率明显减小(P<0.05);与SES组比较,ZES组和BMS组炎症积分明显降低,内皮化积分明显升高(P<0.05);3组损伤积分比较,差异无统计学意义(P>0.05);光学相干断层扫描及扫描电镜观察,SES支架组30 d时可见部分支架节段内皮化不全及炎性细胞浸润。结论 ZES支架可有效地抑制血管内膜增殖,具有良好的生物相容性。

丝裂素活化蛋白激酶介导同型半胱氨酸诱导的血管平滑肌细胞增生

目的：研究同型半胱氨酸（ＨＣＹ）促进血管平滑肌细胞（ＶＳＭＣ）的增殖及其机制。方法：采用同位素技术测定３ＨＴｄＲ参入和丝裂素活化蛋白激酶（ＭＡＰＫ）活性。结果：ＨＣＹ促进ＶＳＭＣ增殖，同时ＨＣＹ增加ＭＡＰＫ活性，二者均具有剂量效应关系，而且二者之间呈显著正相关。结论：ＨＣＹ促进ＶＳＭＣ增殖。