Chlamydia pneumoniae stimulates proliferation of vascular smooth muscle cells through induction of endogenous heat shock protein 60

Chlamydia pneumoniae infection has been linked with atherosclerosis. However, the mechanism responsible for the atherogenic effects of C pneumoniae remains unclear. Heat shock proteins (HSPs) have been found in atherosclerotic lesions. HSPs of HSP70 and HSP90 families are involved in the regulation of cell cycle progression and cell proliferation. We assessed the hypothesis that HSP60 is induced in vascular cells infected with C pneumoniae and stimulates cell proliferation. Rabbit vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were infected with C pneumoniae. Western blot analysis demonstrated the induction of endogenous HSP60 expression in C pneumoniae-infected VSMCs. C pneumoniae infection significantly increased the number of VSMCs, and the mitogenic effect correlated with the expression level of endogenous HSP60. In contrast to VSMCs, C pneumoniae infection had no effect on the expression level of HSP60 and did not stimulate cell proliferation in HUVECs. Exogenous addition of recombinant chlamydial HSP60 had no mitogenic effect on VSMCs and HUVECs. However, overexpression of HSP60 within VSMCs by infection with adenovirus encoding human HSP60 resulted in a significant increase in cell numbers compared with uninfected VSMCs. These results suggest that overexpression of endogenous HSP60 may be a central intracellular event responsible for the mitogenic effects induced by C pneumoniae infection. In addition to C pneumoniae, other infectious agents and atherogenic risk factors may also stimulate VSMC proliferation and contribute to the lesion formation through the induction of HSP60.     

p38MAPK表达与血管平滑肌细胞增殖关系的研究

目的探讨丝裂素活化蛋白激酶p38(p38MAPK)的表达与血管平滑肌细胞(VSMC)增殖的关系以及检测p38MAPK反义寡核苷酸(AODN)对VSMC增殖的抑制作用。方法将培养大鼠胸主动脉VSMC,随机分为对照组、p38MAPKAODN组、正义寡核苷酸(SODN)组。采用噻唑蓝比色分析法(MTT)和流式细胞仪检测VSMC,用蛋白免疫印迹法测定p38MAPK蛋白量。结果p38MAPKAODN能减少p38MAPK蛋白表达,明显抑制VSMC增殖,其抑制作用与p38MAPK蛋白表达相关,呈剂量依赖性。结论p38MAPKAODN能抑制大鼠VSMC增殖,该信号分子与VSMC增殖密切相关,可能是VSMC增殖的信号途径。     

雌激素膜快速效应对兔血管平滑肌细胞iNOS快速激活的影响

目的 探讨17β-雌二醇（17β-estradiol,E2）通过快速激活诱导型一氧化氮合成酶（iNOS）的活性,调节细胞外信号调节激酶（mitogen-activated protein kinase,p42/44 MAPK）表达抑制血管平滑肌细胞（vascular smooth muscle cells,VSMCs）增殖的机制。方法 在培养的VSMCs的基础上,采用放射免疫测定法(RIA)和Western blot检测E2预处理前后胎牛血清（fetal calf serum,FCS）对VSMCs中诱导型iNOS的活性及磷酸化p42/44 MAPK蛋白和p42/44 MAPK总蛋白表达的影响。采用比色法检测E2预处理后VSMCs中NO含量的变化。结果 E2预处理5 min,即可逆转FCS诱导的iNOS活性下降,此效应在E2预处理15 min时达到高峰,处理30 min后此效应减弱。基因转录抑制剂放线菌素D（actinomycin D,Act D）预处理后对此过程无影响,而三苯氧胺预处理可明显逆转E2诱导的iNOS活性增加。比色法检测上清液中NO的含量显示,E2预处理15 min,NO的含量明显增加。Western blot的结果显示,E2预处理15 min,可明显抑制磷酸化p42/44 MAPK蛋白的表达,而对总p42/44 MAPK蛋白的表达无影响。NO合酶(NOS)抑制剂L-硝基左旋精氨酸甲酯(L-NAME)预处理,可逆转E2诱导的磷酸化p42/44 MAPK的表达下降。结论 E2可通过快速激活iNOS增加NO释放,下调磷酸化p42/44 MAPK的活性,抑制VSMCs增殖。     

丝裂素活化蛋白激酶的激活和转核与大鼠 血管平滑肌细胞增殖关系的研究

目的 探讨丝裂素活化蛋白激酶（MAPK）激活、转核与血管紧张素Ⅱ（AngⅡ)刺激血管平滑肌细胞（VSMC）增殖间的关系。方法 本实验采用培养大鼠胸主动脉VSMC。用^3H-胸腺嘧啶核苷（^3H-TdR)掺入法测定DNA合成,用p43/p44磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白量,用免疫细胞化学技术观察MAPK活化并转位入细胞核的过程。结果 （1）AngⅡt和PD98059的上述作用都呈剂量依赖性。（2）AngⅡ对MAPK蛋白表达有显著增强作用。此作用同样被PD98059以剂量依赖方式抑制。（3）AngⅡ刺激5min后,MAPK出现在VSMC的细胞浆中,30min时MAPK进入细胞核,3h后MAPK染色从核内消失,上述MAPK转核过程被PD98059抑制。结论 本实验证实人细胞核,3h后MAPK染色从核人消失,上述MAPK转核过程被PD98059抑制。结论 本实验证实AngⅡ能激活培养大鼠主动脉VSMC的MAPK,活化的MAPK从细胞浆转位进入细胞核导致VSMC增殖。     

The inhibitory mechanisms of amlodipine in human vascular smooth muscle cell proliferation.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) is closely related to vascular diseases. There is growing evidence that calcium antagonists inhibit VSMC growth/proliferation, yet their molecular mechanisms remain to be determined. Recent reports suggest that p42/p44 mitogen-activated protein kinases (MAPKs) play an important role in cell growth and proliferation induced by growth factors. This study was designed to determine whether these MAPKs are involved in VSMC proliferation induced by basic fibroblast growth factor (bFGF) and to examine the inhibitory effect of amlodipine. Human VSMCs were obtained from inner mammary artery. p42/p44 MAPKs activity was measured by immunoblotting assay using anti-p42/p44 phospho-MAPK antibody. 1) bFGF (20 ng/ml) significantly activated p42/p44 MAPKs with a peak time of 5-15 min, which was maintained for 3 h. PD98059 (100 nM-10 microM), a specific inhibitor of MAPK kinase, inhibited bFGF-induced p42/p44 MAPKs activation in a dose-dependent manner. 2) Amlodipine (1-100 nM) dose-dependently inhibited p42/p44 MAPKs activation by bFGF. 3) Amlodipine (10 nM) could inhibit both short-term and long-term p42/p44 MAPKs activation by bFGF. Our results indicate that bFGF could activate p42/p44 MAPKs. Amlodipine, which could inhibit bFGF-induced human VSMC proliferation, inhibited both short-term and sustained p42/p44 MAPKs activation by bFGF, suggesting that bFGF-induced VSMC proliferation may be related to p42/p44 MAPKs activation, and that the antiproliferative effect of amlodipine may be related to its inhibition of p42/p44 MAPKs activation.     

Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.     

Involvement of p38 mitogen-activated protein kinase signaling in transformed growth of a cholangiocarcinoma cell line

Although mitogen-activated protein kinase (MAPK) pathways play a key role in cell growth, their role in mediating the altered growth phenotype of transformed cells remains unclear. The p44/p42 MAPK signaling cascades are activated by mitogenic stimulation of human cholangiocytes. In contrast, the p38 MAPK pathway is activated by mitogen stimulation of malignant, but not nonmalignant cholangiocytes. Thus, our aims were to determine the role of p38 MAPK signaling in mediating the growth phenotype of transformed cholangiocytes. KMCH-1 malignant human cholangiocytes required the presence of serum for proliferation, but were able to grow in reduced serum conditions. Inhibition of p38 MAPK decreased serum-dependent proliferation of KMCH-1 cells. Furthermore, inhibition of p38 MAPK, but not of p44/p42 MAPK, reduced anchorage-independent growth of KMCH-1 cells. Although both p38 and p44/p42 MAPK are activated in response to mitogens, they have divergent effects on anchorage-independent growth. Inhibition of p38 MAPK, but not of p44/p42 MAPK signaling, decreased cell cycle progression and increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIPl). However, expression of p27(KIP1) or p16(INK4A) was not altered by either pathway. Thus, mitogen activation of p38 MAPK decreases expression of p21(WAF1/CIP1) and mediates growth independent of anchorage signals, whereas mitogen activation of p44/p42 MAPK mediates an anchorage signal-dependent growth pathway. These data provide a link between aberrant stress-activated cell signaling and the altered growth phenotype of transformed cells that may be important for the development of therapies to limit transformed cell growth.     

The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.     

Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration

Cigarette smoke, specifically the nicotine contained within, has been shown to cause ultrastructural changes in vascular endothelium resulting in the development of atherosclerosis. Our study examines the effects of nicotine on vascular smooth muscle cell (VSMC) migration and attempts to eludicidate the cellular mechanisms governing those effects. Bovine aortic VSMC were cultured in 10% fetal bovine serum (FBS) growth media and exposed to 10(-8) nicotine for varying periods of time. Boyden chamber chemotaxis assays and a scrape injury model using confluent cells were used to assess cell motility. Activation of the mitogen-activated protein kinases (MAPK), p38 and p44/42, was assessed using Western blotting methods. Nicotine, itself, did not cause significant VSMC migration. However, augmented migration was seen in nicotine-treated VSMCs (16.6+/-3-fold) and media (17.0+/-4-fold) with 10% FBS as chemoattractant. Inhibitors of p38 and p44/42 diminished this migration by 48.5+/-6% and 29.4+/-2%, respectively. Immunoblotting verified p38 and p44/42 activation with nicotine and inhibition with inhibitors of p38 and p44/42. Nicotine-treated endothelial cell (EC) conditioned media (CM) was shown to increase migration 20.3+/-l.l-fold. This chemotactic effect was diminished both with heat treatment and serial dilution. In conclusion, nicotine enhances the chemoatactiveness of VSMC. This migration is mediated via the MAPKs p38 and p44/42. Nicotine causes EC production of a chemoattractant molecule that enhances VSMC migration.     

p38MAPK反义寡核苷酸对鼠血管平滑肌细胞增殖的影响

目的：探讨丝裂素活化蛋白激酶p38(p38mitogen—activated protein kinase，p38MAPK)反义寡聚脱氧核苷酸(antisense oliodeoxynucleotide，AODN)对血管平滑肌细胞增殖的影响。方法：培养大鼠胸主动脉平滑肌细胞。通过脂质体帮染p38MAPK AODN到血管平滑肌细胞(VMSC)。另设p38MAPK正义寡聚脱氧核苷酸(SODN)对照组和空白对照组。用流式细胞仪检测细胞增殖。结果：AODN明显抑制血管紧张素I刺激的血管平滑肌细胞增殖(P＜0.05～＜0．01)，其抑制作用呈剂量依赖性。结论：p38MAPK反义寡聚脱氧核苷酸能抑制大鼠的VSMC增殖，提示VSMC增殖与p38信号途径有关。     

Mediation of basic fibroblast growth factor-induced lactotropic cell proliferation by Src-Ras-mitogen-activated protein kinase p44/42 signaling

Basic fibroblast growth factor (bFGF), which is secreted from folliculostellate cells in the anterior pituitary, is known to be involved in the communication between folliculostellate cells and lactotropes during estradiol-induced lactotropic cell proliferation. We studied the role of MAPK p44/42 in bFGF-regulated cell proliferation using enriched lactotropes and the lactotrope-derived PR1 cell line. In cell cultures, bFGF increased cell proliferation of PR1 cells and enriched lactotropes. In both of these cell populations, bFGF also increased phosphorylation of MAPK p44/42. U0126, an inhibitor of MAPK p44/42, blocked the bFGF-induced activation of MAPK p44/42 as well as the bFGF-induced cell proliferation of enriched lactotropes and PR1 cells. Treatment of PR1 cells with bFGF increased the activity of Ras p21, whereas overexpression of a dominant negative mutant of Ras p21 abrogated the bFGF-induced activation of MAPK p44/42 in these cells. Furthermore, the Src kinase inhibitor PP1 suppressed bFGF-induced activation of MAPK p44/42 in both enriched lactotropes and PR1 cells. The Src kinase inhibitor PP1 also reduced bFGF activation of Ras p21 and cell proliferation in PR1 cells. On the other hand, the bFGF-induced activation of MAPK p44/42 in enriched lactotropes and PR1 cells was not affected by protein kinase C inhibitors. These data suggest that bFGF induction of lactotropic cell proliferation is possibly mediated by activation of Src kinase, Ras p21, and MAPK p44/42.     

Antibody response to the 60-kDa heat-shock protein of Chlamydia pneumoniae in patients with coronary artery disease

Serum specimens from 752 individuals undergoing coronary arteriography were examined for antibodies to Chlamydia pneumoniae. Patients with coronary artery disease (CAD) were more likely to have IgG antibodies to C. pneumoniae than were individuals without CAD (60% vs. 52%; P=.007; odds ratio, 1.8; 95% confidence interval, 1. 17-2.77). Antibodies to recombinant hsp60 of C. pneumoniae were found with nearly the same frequency in patients with CAD and individuals without CAD (29% vs. 30%; P=.751). There was no association between chlamydial hsp60 antibodies and the severity of CAD or a previous myocardial infarction. Patient sera reacted most frequently to C. pneumoniae proteins of 17, 38, 40, 58, and 60/62 kDa. Reactivity to these proteins was not different between patients with and without CAD. Study results indicate that neither antibodies to chlamydial hsp60 nor antibodies to other C. pneumoniae proteins are useful for discriminating between seropositive patients with and without CAD.