Homocysteine induces connective tissue growth factor expression in vascular smooth muscle cells.

BACKGROUND: Increased homocysteine levels in blood might be an important risk factor for the development of cardiovascular diseases. Connective tissue growth factor (CTGF) was found to be involved in atherosclerotic plaque progression. So far, the possible connection between homocysteine and CTGF has not been studied. OBJECTIVE: This study was designed to test whether homocysteine could induce CTGF expression in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. CTGF mRNA and protein expression was detected in the aortas isolated from hyperhomocysteinemic rats, but not in the controls. The underlying mechanism of homocysteine-induced CTGF expression was investigated in cultured human umbilical vein smooth muscle cells (HUVSMC). CTGF mRNA expression was induced after treatment with dl-homocysteine (50 micromol L(-1)) for 1 h, which remained at the elevated level for up to 8 h. CTGF protein level increased after homocysteine treatment for 8 h, and the elevated status was maintained for up to 48 h. Several intracellular signals elicited by homocysteine are involved in CTGF synthesis, including protein kinase C (PKC) activation and reactive oxygen species (ROS). Transfection HUVSMCs with a CTGF small interference RNA (siRNA) plasmid, which specifically inhibited the expression of CTGF, decreased extracellular matrix (ECM) accumulation caused by homocysteine. CONCLUSION: Our results demonstrate that homocysteine could increase the expression of CTGF in VSMC both in vivo and in vitro. The novel findings suggest that homocysteine might contribute to accelerated progression of atherosclerotic lesions by inducing CTGF expression.     

Human interleukin 1 induces interleukin 1 gene expression in human vascular smooth muscle cells

The recognition that cells of the vascular wall can secrete cytokines such as IL-1 suggests new mechanisms for initiating or sustaining inflammatory responses in blood vessels. We report that purified human monocyte-derived IL-1 or recombinant human IL-1 (rIL-1 beta and rIL-1 alpha) induce cultured human smooth muscle cells derived from veins or arteries to synthesize IL-1 beta mRNA and produce and release biologically active IL-1. rIL-1 beta also stimulated the production of PGE2 by smooth muscle cells. Exposure to rIL-1 beta (1-100 ng/ml), or rIL-1 alpha (0.01-10 ng/ml) increased IL-1 beta mRNA levels within 30 min. Actinomycin D (1 microgram/ml) prevented the induction of IL-1 beta mRNA by rIL-1. IL-1 alpha mRNA was detected in SMC treated with cycloheximide (1 microgram/ml) and rIL-1 beta, or cycloheximide alone. rIL-1 alpha and rIL-1 beta produced maximal levels of IL-1 beta mRNA after 4 h, and intracellular IL-1 biological activity after 6 h of exposure. Release of IL-1 activity in the extracellular medium began after 1 h of incubation with rIL-1 beta or rIL-1 alpha, and continued for up to 24 h. Anti-TNF antiserum that neutralized the biological activity of rTNF did not affect rIL-1-induced production of IL-1 beta mRNA or IL-1 release, suggesting that the release of TNF does not mediate these processes. Several experimental approaches indicated that the release of IL-1 by smooth muscle cells was not due to endotoxin contamination of the IL-1 preparations. Anti-IL-1 antiserum blocked the induction of smooth muscle cell IL-1 gene expression by rIL-1 beta. Polymyxin B did not prevent IL-1-induced IL-1 expression by these cells, but blocked the effect of endotoxin. Heat treatment destroyed the stimulatory capacity of rIL-1 beta, but did not affect the ability of bacterial endotoxin to induce IL-1 expression. The production of IL-1 by human vascular smooth muscle cells was not due to contamination of the cell cultures with blood monocytes, inasmuch as treatment with an antimonocyte antibody (anti-Mo2) and complement did not alter IL-1 beta mRNA content or the amount of IL-1 released from the cells in response to endotoxin, rIL-1 alpha, or rIL-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric oxide synthase inhibitors inhibit interleukin-1 beta-induced depression of vascular smooth muscle.

Interleukin-1 beta (IL-1) reduces vascular smooth muscle contractility. The purpose of the present study was to investigate the role of nitric oxide synthesis in mediating this effect of IL-1. We studied the influence of inhibitors of nitric oxide synthesis on the depression of norepinephrine-induced contractions of rat aortic rings by IL-1. Also, we examined the ability of IL-1 to increase the production of nitric oxide by rat aortic smooth muscle cells in culture as determined indirectly by measuring nitrite concentrations. NG-amino-L-arginine blocked the effect of IL-1 on norepinephrine-induced contractions of rat aortic rings whereas NG-monomethyl-L-arginine and NG-nitro-L-arginine were considerably less effective. In addition, this effect of IL-1 was prevented by coincubation of the rings with cycloheximide. IL-1 greatly elevated nitrite production by rat aortic smooth muscle cells, and this effect could also be blocked completely by the arginine analogs. NG-amino-L-arginine was the most potent inhibitor of nitrite synthesis (IC50 = 1.7 microM) whereas NG-monomethyl-L-arginine and NG-nitro-L-arginine were about 10-fold less potent (IC50 = 16 and 22 microM, respectively). These results suggest that IL-1-induced depression of norepinephrine-induced vascular contraction is mediated by the increased synthesis of nitric oxide synthase by vascular smooth muscle cells. The relative potency of the arginine analogs for the inhibition of nitrite synthesis suggests that the synthase in vascular smooth muscle is similar to the synthase in macrophages.     

Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells. Role of Ca2+ mobilization and protein kinase C activation.

Tissue factor (TF) is a low molecular weight glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis. TF is not expressed in the intima or media of normal adult blood vessels. Accordingly, it has been hypothesized that the initiation of intravascular coagulation may require the "induced" expression of TF in the vessel wall. We report that TF mRNA and protein are rapidly and markedly induced in early and late passaged vascular smooth muscle cells (VSMC) by growth factors (serum, platelet-derived growth factor, epidermal growth factor), vasoactive agonists (angiotensin II), and a clotting factor (alpha-thrombin). The induction of TF mRNA by these agents is dependent upon mobilization of intracellular Ca2+ and is blocked by Ca2+ chelation. In contrast to other growth factor-responsive genes, such as KC and c-fos, downregulation of protein kinase C activity by prolonged treatment with phorbol esters fails to block agonist-mediated TF induction. This raises the possibility that protein kinase C activation may not be necessary for TF mRNA induction in VSMC. VSMC may play a role in the generation or propagation of thrombus through the induction of TF, particularly in settings, such as those associated with acute vessel injury, where the endothelium is denuded and the VSMC are exposed to circulating blood.     

Inducible interleukin-1 gene expression in human vascular smooth muscle cells.

Interleukin-1 (IL-1) mediates many components of generalized host response to injury and may also contribute to local vascular pathology during immune or inflammatory responses. Because altered function of smooth muscle cells (SMC) accompanies certain vascular diseases, we tested whether SMC themselves might produce this hormone. Unstimulated SMC contain little or no IL-1 mRNA. However, exposure to bacterial endotoxin caused accumulation of IL-1 mRNA in SMC cultured from human vessels. Endotoxin maximally increased IL-1 beta mRNA in SMC after 4-6 h. The lowest effective concentration of endotoxin was 10 pg/ml. 10 ng/ml produced maximal increases in IL-1 beta mRNA. Interleukin-1 alpha mRNA was detected when SMC were incubated with endotoxin under "superinduction" conditions with cycloheximide. Endotoxin-stimulated SMC also released biologically functional IL-1, measured as thymocyte costimulation activity inhibitable by anti-IL-1 antibody. Thus, human SMC can express IL-1 beta and IL-1 alpha genes, or very similar ones, and secrete biologically active product in response to a pathological stimulus. Endogenous local production of this inflammatory mediator by the blood vessel wall's major cell type could play an important early role in the pathogenesis of vasculitis and arteriosclerosis.     

氧化型低密度脂蛋白诱导大鼠血管平滑肌细胞增殖及周期素D1的表达

背景:氧化型低密度脂蛋白可通过Ras/Raf/丝裂原活化蛋白激酶激酶,丝裂原活化蛋白激酶途径诱导血管平滑肌细胞增殖及细胞周期蛋白的表达.目的:观察胞外信号调节激酶是否参与了氧化型低密度脂蛋白诱导的血管平滑肌细胞周期推进以及周期素D1的表达.设计、时间及地点:对照观察细胞学实验,于2008-02/10在广州医学院完成.材料:人血浆低密度脂蛋白来自健康志愿者血清.SD大鼠由广州医学院实验动物中心提供.方法:采用超速离心法分离人低密度脂蛋白进行氧化修饰及鉴定.以酶消化法培养大鼠血管平滑肌细胞,采用免疫组织化学染色法鉴定.主要观察指标:在静止的血管平滑肌细胞中加入MEK1/2选择性抑制剂10 μmol/L PD98059和,或50 mg/L氧化型低密度脂蛋白,以CCK-8和细胞计数法测定细胞增殖,流式细胞术观察细胞周期,蛋白印迹检测周期素D1蛋白表达及胞外信号调节激酶的活性.结果:CCK-8和细胞计数法显示,氧化型低密度脂蛋白能明显诱导血管平滑肌细胞增殖,PD98059抑制氧化型低密度脂蛋白诱导的血管平滑肌细胞增殖,并且呈时间依赖性.流式细胞术分析表明,经氧化型低密度脂蛋白处理24 h后,血管平滑肌细胞从G0/G1期进入S期,S期细胞百分比明显增高(P<0.01),加入PD98059处理24 h后,血管平滑肌细胞由 G0/G1期向S期的推进受到抑制.蛋白印迹结果显示,氧化型低密度脂蛋白能明显诱导周期素D1蛋白表达及胞外信号调节激酶的磷酸化,PD98059抑制氧化型低密度脂蛋所诱导的周期素D1蛋白表达以及胞外信号调节激酶的磷酸化.结论:氧化型低密度脂蛋白能诱导血管平滑肌细胞增殖,促进细胞由G0/G1期向S期转换以及周期素D1表达,其作用部分是由胞外信号调节激酶所介导.     

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.     

Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle.

Sodium nitroprusside (SNP) is thought to exert its vasodilating activity, at least in part, by vascular activation to nitric oxide (NO), but the activation mechanism has not been delineated. This study has examined the potential for vascular metabolism of SNP to NO in bovine coronary arterial smooth muscle subcellular fractions using a sensitive and specific redox-chemiluminescence assay for NO. SNP was readily metabolized to NO in subcellular fractions, and the dominant site of metabolism appeared to be located in the membrane fractions. NO-generating activity was significantly enhanced by, but did not absolutely require, the addition of a NADPH-regenerating system, NADPH per se, NADH or cysteine. A correlation analysis of NO-generating activity (in the presence of a NADPH-regenerating system) with marker enzyme activities indicated that the SNP-directed NO-generating activity was primarily membrane-associated. Radiation inactivation target-size analysis revealed that the microsomal SNP-directed NO-generating activity was relatively insensitive to inactivation by radiation exposure, suggesting that the functioning catalytic unit might be quite small. A molecular weight of 5 to 11 kDa was estimated. NO-generating activity could be solubilized from the crude microsomes with 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and the solubilized extract was subjected to gel filtration chromatography. NO-generating activity was eluted in two peaks: one peak corresponding to an approximate molecular weight of 4 kDa, thus confirming the existence of a small molecular weight NO-generating activity, and a second activity peak corresponding to a molecular weight of 112 to 169 kDa, the functional significance of which is unclear at present.(ABSTRACT TRUNCATED AT 250 WORDS)     

过氧化氢诱导人血管内皮细胞凋亡及HSP70和Bcl2 基因蛋白表达的研究

目的探讨过氧化氢（H     

Nitric oxide mediates cytotoxicity and basic fibroblast growth factor release in cultured vascular smooth muscle cells. A possible mechanism of neovascularization in atherosclerotic plaques.

To define the pathophysiological role of nitric oxide (NO) released from vascular smooth muscle cells (VSMC), we examined whether NO released from VSMC induces cytotoxicity in VSMC themselves and adjacent endothelial cells (EC) using a coculture system. Prolonged incubation with interleukin-1 (IL-1) induced large amounts of NO release and cytotoxicity in VSMC. NG-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited both NO release and cytotoxicity induced by IL-1. In contrast, DNA synthesis in cocultured EC was not inhibited but rather stimulated by prolonged incubation with IL-1 or sodium nitroprusside (SNP), a NO donor. However, IL-1 and SNP did not stimulate but inhibited DNA synthesis in EC alone. On the other hand, conditioned medium from VSMC incubated for a long period with IL-1 or SNP stimulated DNA synthesis in EC alone. Furthermore, the concentration of basic fibroblast growth factor in the conditioned medium was increased and correlated with the degree of cytotoxicity in VSMC. These results indicate that NO released from VSMC induces VSMC death, which results in release of basic fibroblast growth factor, which then stimulates adjacent EC proliferation. Thus, NO released from VSMC may participate in the mechanism of neovascularization in atherosclerotic plaques.     

Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages

Both chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis. We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages. We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6). We also tested whether either HSP 60 induces nuclear factor-kappaB (NF-kappaB), which contributes to the gene expression of these molecules. Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS). Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E. coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA). In ECs, either HSP 60 triggered activation of NF-kappaB complexes containing p65 and p50 Rel proteins. Heat treatment abolished all these effects, but did not alter the ability of E. coli LPS to induce these functions. Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications. These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.     

tMfn2基因诱导血管平滑肌细胞的凋亡

背景:线粒体融合素2蛋白,主要通过磷脂酰肌醇3激酶,蛋白激酶B诱导血管平滑肌细胞凋亡.去除穿膜区序列的线粒体融合素2蛋白,相对分子质量减小41%,诱导凋亡作用可能更强.目的:观察比较去除穿膜区序列的大鼠线粒体融合素基因2对大鼠血管平滑肌细胞凋亡的影响及其相关的信号通路.设计、时间及地点:基因水平的对比观察实验.于2008-01/10在华中科技大学同济医学院附属同济医院分子心脏病中心实验室完成.材料:大鼠血管平滑肌细胞及携带半乳糖甘酶基因、携带线粒体融合素2基因和携带去除穿膜区序列线粒体融合素2基因的重组腺病毒均由陈光慧教授惠赠.方法:将大鼠血管平滑肌细胞传代培养3～10代后随机分为4组,①空白对照组:不加干预.②携带半乳糖甘酶基因的对照组:感染携带半乳糖甘酶基因的重组腺病毒.③携带线粒体融合素2基因的实验组:感染携带线粒体融合素2基因的重组腺病毒.④携带去除穿膜区序列线粒体融合素2基因的实验组:感染携带去除穿膜区序列线粒体融合素2基因的重组腺病毒.主要观察指标:①重组腺病毒感染血管平滑肌细胞24 h后观察完整的和去除穿膜区序列的线粒体融合素2基因的表达情况.②感染后24,48和72 h采用流式细胞术、酶联免疫吸附法观察细胞凋亡情况.③免疫印迹分析病毒感染血管平滑肌细胞24 h后磷酸化蛋白激酶B表达变化.结果:①感染后完整的和去除穿膜区序列的线粒体融合素2基因在血管平滑肌细胞中均有表达.②去除穿膜区序列的线粒体融合素2基因诱导血管平滑肌细胞凋亡的作用显著增强,且呈时间依赖性(P<0.01).③两个实验组中磷酸化蛋白激酶B水平均明显降低,但去除穿膜区序列的线粒体融合素2基因实验组更显著(P<0.01).结论:去除穿膜区序列的线粒体融合素基因2诱导血管平滑肌细胞凋亡的作用更强,其机制与抑制蛋白激酶B磷酸化有关.     

Heat shock protein 70 binding protein 1 induces enhanced apoptotic response against anticancer drugs in tumor cells

HspBP1 was originally identified and characterized as a novel Hsp70/Hsc70-interacting protein that inhibited the Hsp70/Hsc70 chaperone activity. In this respect, Hsps have been shown to influence cell-death pathway through the interaction with key components of the apoptotic machinery. In this report, we have examined the effect of HspBP1 overexpression on the sensitivity of tumor cells to anticancer drug-induced apoptotic response. Analysis with HspBP1 deletion mutant genes showed that C-terminus 47 amino acid residues were essential for the specific interaction of HspBP1 with Hsp70/Hsc70. Overexpression of HspBP1 induced enhanced apoptotic response against anticancer drugs, but HspBP1 lacking C-terminus 47 amino acid residues did not. These results support the notion that inhibition of the function of Hsp70 enhances the therapeutic efficacy of chemotherapy.