Chlamydia pneumoniae facilitates monocyte adhesion to endothelial and smooth muscle cells

Chlamydia pneumoniae has been linked to atherosclerotic heart disease. However, there is a limited knowledge by which C. pneumoniae gain access to atheromatous lesions. The adhesion of C. pneumoniae -infected circulatory component(s) to endothelium and smooth muscle cells represents the first step in an inflammatory response. We examined the ability of viable as well as heat inactivated C. pneumoniae to infect human monocytes and subsequently the ability of infected monocytes to adhere to human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC). Our results demonstrate susceptibility of monocytes to in vitro chlamydial infection. Inclusions of varying sizes and intensities were observed 3-5 days after inoculation with viable C. pneumoniae. Monocytes infected with heat inactivated organisms revealed no inclusions, in keeping with the observations of uninfected monocytes. Moreover, monocytes infected with viable C. pneumoniae adhered preferentially to HCAEC and HCSMC, as compared to uninfected monocytes or monocytes harbouring heat inactivated Chlamydia.     

Chlamydia pneumoniae multiplies in human endothelial cells in vitro

The ability of three C. pneumoniae isolates, Kajaani 6, Helsinki 12 and TW-183, to grow in human umbilical vein endothelial cells (HUVEC) and in an immortalized endothelial cell line EA.hy 926 was studied. All C. pneumoniae isolates were capable of multiplying in endothelial cells. EA.hy 926 cells could support the growth of C. pneumoniae better than HUVEC, yet less efficiently than HL and HEp-2 cells that are conventionally used in C. pneumoniae culturing. Although centrifugation of the inoculum greatly increased the inclusion yields, it was not necessary for infectivity. In addition, a persistent infection of C. pneumoniae in EA.hy 926 and HL cells ensued and it was followed up for two months. The fact that endothelial cells can serve as hosts to C. pneumoniae might be a significant contributing factor in the pathogenesis of atherosclerosis, a disease which recent studies show to be associated with chronic C. pneumoniae infection.     

Inhibition of Chlamydia pneumoniae replication in human aortic smooth muscle cells by gamma interferon-induced indoleamine 2, 3-dioxygenase activity

Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis, possibly because the pathogen can exist in a persistent form similar to that described for Chlamydia trachomatis. The present study investigated whether gamma interferon (IFN-gamma) can induce indoleamine 2,3-dioxygenase (IDO) activity in aortic smooth muscle cells, leading to a marked inhibition of C. pneumoniae growth. Our data indicate a stimulation of IDO mRNA expression and dose-dependent enzymatic activity following IFN-gamma treatment. IDO-mediated increase in tryptophan catabolism resulted in a dose-dependent marked inhibition of C. pneumoniae replication.     

培养的人主动脉平滑肌细胞异常表达HLA—DR,DP抗原

用γ-IFN、淋巴细胞条件培养液(LCM)对培养的人主动脉平滑肌细胞(SMC)进行诱导,观察其是否异常表达DR、DP抗原,以探讨在动脉粥样硬化(AS)发病中的意义。结果表明,γ-IFN组DR、DP阳性率分别为80%和73%;LCM组DR、DP阳性率为74%和70%。两组均明显高于正常对照组DR、DP的阳性率(11%,7%)。提示激活的T淋巴细胞通过分泌γ-IFN(或合并其它因子)诱导SMC异常表达DR、DP抗原,可能在AS的发生发展中起一定作用。     

Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

Chlamydia pneumoniae infection increases adherence of mouse macrophages to mouse endothelial cells in vitro and to aortas ex vivo

Interactions between monocytes/macrophages and endothelial cells play an important role in the pathogenesis of atherosclerosis, and the adherence of monocytes to the arterial endothelium is one of the early events in atherogenesis. In the present study, peritoneal macrophages harvested from green fluorescent protein (GFP) transgenic mice were used to analyze how Chlamydia pneumoniae infection affects the adherence of GFP-macrophages to mouse endothelial cells in vitro and to the aorta from normolipidemic and hyperlipidemic mice ex vivo. In vitro studies showed that C. pneumoniae-infected GFP-macrophages adhered better than uninfected macrophages to endothelial cells and GFP-macrophages adhered better to infected than uninfected endothelial cells. The ex vivo studies showed that C. pneumoniae-infected macrophages adhered better than uninfected macrophages to aortas from both normolipidemic and hyperlipidemic C57BL/6J mice and apolipoprotein E (ApoE)-deficient mice. In contrast, adherence of C. pneumoniae-infected macrophages to the aortas of intercellular adhesion molecule 1 (ICAM-1) knockout mice was not enhanced, suggesting that ICAM-1 is crucial for activation of the adherence of C. pneumoniae-infected macrophages to the endothelium. In conclusion, the present study defined a homing mechanism by which C. pneumoniae promotes the adherence of mononuclear phagocytes to the endothelium at the site of atherosclerotic lesion formation to promote the progression of atherosclerosis.     

Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal.

To study the mechanism(s) of vascular smooth muscle cell proliferation in vivo, mRNA levels of c-fos, c-jun, and c-myc were determined by Northern blot analysis following vascular balloon de-endothelialization (BDE). Medial smooth muscle cells (SMC) were separated and studied by enzymatic digestion of the vessel wall. mRNA levels of c-fos and c-jun from aortic smooth muscle cells (SMC) were simultaneously induced within 30 minutes of BDE and declined to baseline by 1.5 hours, c-myc mRNA did not begin to increase until 1 hour after vascular injury. Levels of c-myc peaked at 2 hours and were sustained for an additional 4 hours before gradually declining. Smooth muscle cells derived from enzyme-treated control aortae that did not undergo BDE expressed c-fos and c-jun, but showed no evidence of c-myc message. In contrast, nonenzymatically treated, non-BDE whole aortae (containing both media and adventitia) demonstrated a prominent c-myc signal, but failed to express c-fos and c-jun. Corresponding examination of adventitia derived from enzyme-treated aortae showed this tissue to be a source of all three proto-oncogenes. The results of this study demonstrate the earliest in vivo molecular markers of vascular injury reported to date and implicate SMC proto-oncogene expression in the initiation of SMC proliferation. Furthermore these findings suggest two avenues for proto-oncogene induction, that are due to (1) vessel wall manipulation and (2) humoral stimulation.     

肺炎衣原体感染对血管平滑肌细胞黏附和迁移的影响

目的:观察肺炎衣原体(C.pn)感染对血管平滑肌细胞(VSMCs)黏附和迁移的影响。方法:C.pn在人喉癌细胞系HEp-2细胞中增殖培养后感染大鼠VSMCs,吖啶橙(AO)荧光染色观察细胞内C.pn包涵体的形态特点;聚合酶链反应(PCR)检测C.pn特异性DNA片段;细胞黏附实验观察C.pn感染对VSMCs黏附能力的影响;wound-healing assay和Transwell assay检测C.pn感染VSMCs后细胞迁移能力的改变。结果:C.pn感染VSMCs后,少数细胞内出现空泡状结构即包涵体,但在多数细胞内以点状感染灶形式存在,包涵体较大,但数量较少。利用PCR可在感染的VSMCs内检测到437 bp的C.pn特异性DNA片段。细胞黏附实验结果显示,C.pn感染VSMCs 2 h后,细胞黏附能力明显增强,其吸光度值明显高于正常对照组(P0.01),细胞黏附率为134.38%。Wound-healing assay结果显示,C.pn感染VSMCs 24 h后,细胞向划痕中央迁移的距离明显大于正常对照组(P0.05)。Transwell assay结果显示,C.pn感染VSMCs 24 h后,C.pn感染组的细胞迁移数明显多于正常对照组(P0.01)。结论:C.pn感染能够显著增强VSMCs的黏附和迁移能力。     

Differences in the requirements for cryopreservation of porcine aortic smooth muscle and endothelial cells.

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me(2)SO) in these cells at 2-4 degrees and 22 degrees C. The following results were obtained: Table unavailable in HTML format. Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me(2)SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me(2)SO. Following cooling at 0.3, 1, or 10 degrees C/min and storage at less than -160 degrees C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3 degrees C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10 degrees C/min.     

Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells

Collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) implanted in the abdominal walls of immunodeficient mice form mature microvessels invested by host-derived smooth muscle cells (SMC) by 8 weeks. We tested the hypothesis that coengraftment of human aortic SMC (HASMC) could accelerate vessel maturation. To prevent SMC-mediated gel contraction, we polymerized the gel within a nonwoven poly(glycolic acid) (PGA) scaffold. Implanted grafts were evaluated at 15, 30, and 60 days. Acellular PGA-supported protein gels elicited a macrophage-rich foreign body reaction and transient host angiogenic response. When transplanted alone, HASMC tightly associated with the fibers of the scaffold and incorporated into the walls of angiogenic mouse microvessels, preventing their regression. When transplanted alone in PGA-supported gels, Bcl-2-HUVEC retained the ability to form microvessels invested by mouse SMC. Interestingly, grafts containing both Bcl-2-HUVEC and HASMC displayed greater numbers of smooth muscle alpha-actin-expressing cells associated with human EC-lined arteriole-like microvessels at all times examined and showed a significant increase in the number of larger caliber microvessels at 60 days. We conclude that SMC coengraftment can accelerate vessel development by EC and promote arteriolization. This strategy of EC-SMC coengraftment in PGA-supported protein gels may have broader application for perfusing bioengineered tissues.     

牛主动脉平滑肌细胞体外钙型的制备

目的利用β-甘油磷酸盐处理牛主动脉平滑肌细胞(BASMC)制备体外血管钙化模型。方法移植块法原代培养牛主动脉中膜平滑肌细胞,8代内的传代细胞添加10 mmol/Lβ-甘油磷酸盐培养10 d,Von Kossa染色、茜素红S染色和电镜检查鉴定钙化,同时测量细胞层钙沉积、碱性磷酸酶活性及培养上清的骨钙素含量。结果10 d后细胞层出现大量钙盐沉积,并且β-甘油磷酸盐时间依赖性增加钙沉积,碱性磷酸酶活性及骨钙素含量各时间点均较正常培养细胞显著增加(P<0.01)。结论甘油磷酸盐能在短期内诱导出BASMC广泛钙化,用此方法制备的BASMC是一种良好的研究血管钙化的体外模型。     

肺炎衣原体感染致血管平滑肌细胞的改变

肺炎衣原体感染是动脉粥样硬化发生的重要危险因素。肺炎衣原体可以感染血管壁内皮细胞、平滑肌细胞、单核细胞/巨噬细胞,并与这些细胞之间相互作用,影响这些细胞的形态与功能,从而参与动脉粥样硬化发生发展。在动脉粥样硬化发病机制中,平滑肌细胞形态功能的变化与动脉粥样硬化密切相关,肺炎衣原体能够感染平滑肌细胞,影响平滑肌细胞在血管内的生理生化特征,促进动脉粥样硬化的发展过程。     

In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is a common respiratory pathogen. Recent studies have demonstrated the presence of C. pneumoniae in coronary and aortic atherosclerotic lesions. To study the role of C. pneumoniae in atherosclerosis, we investigated the susceptibilities of three different cells of the human vascular wall to infection with C. pneumoniae AR-39. These cell types were endothelial cells, smooth muscle cells, and macrophages derived from peripheral blood monocytes. Infection was assessed by using a direct fluorescent antibody to assess inclusion counts. Duplicate cell samples were harvested 3 days postinfection and were passed in HL cells, a susceptible human epithelial cell line, to determine if infectious organisms were produced. Endothelial cells, smooth muscle cells, and macrophages were capable of supporting C. pneumoniae growth in vitro. These results showed that three different cell types known to be important in atherogenesis are susceptible to infection with C. pneumoniae.     

漏芦蜕皮甾酮和维生素E对培养主动脉平滑肌细胞的作用

漏芦蜕皮甾酮(RUE)是从祁州漏芦根中提取的一种植源性蜕皮激素。本文以2％高脂血清培养家兔主动脉平滑肌细胞,形成细胞损伤;采用TBA萤光法,放射自显影和电镜,观察RUE与维生素E对受损细胞的影响。结果发现二种药物作用相似,均使细胞增生,细胞内过氧化脂含量降低,胞浆脂滴缩小、减少,未发现细胞死亡。由此认为RUE可能因具有抗脂质过氧化作用,保护了平滑肌细胞结构完整,从而显示出抗动脉粥样硬化的作用。     

Replication of endothelial cells and smooth muscle cells induced in vivo by hypercholesterolaemia and materials released from platelet-rich white thrombus.

Endothelial cell injury is considered to be the primary event in atherogenesis. In this study, we investigated the effects of hypercholesterolaemia, and substances released from platelet-rich thrombi, individually and together on endothelial cells and the wall of the rabbit in vivo. We divided 24 rabbits into four groups: I was a control group on a normal diet; II was a tubing group on a normal diet, in which polyethylene tubing was inserted into the ascending aorta; III was a group being fed a cholesterol diet; and IV was a combined group being fed a cholesterol diet with polyethylene tubing in the ascending aorta. Segments from the descending thoracic and abdominal aortas which were not injured directly by tubing were examined morphologically and for [3H] thymidine incorporation into endothelial cells and smooth muscle cells. The descending aortas of groups II, III, and IV showed various degrees of endothelial cell damage. [3H]Thymidine incorporation into endothelial cells and aortic wall was increased in groups II, III, and IV (most in group IV) as compared with group I. These data indicate that hypercholesterolaemia and substances released from activated platelets and/or white mural thrombi can cause endothelial damage which may result in endothelial and smooth muscle cell proliferation. In addition, a combination of these two factors showed an additive effect on the endothelial injury and regeneration in vivo.     

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.     

Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies

Sections of human atherosclerotic plaques were stained by the indirect immunoperoxidase technique using three rat anti-human monoclonal antibodies: YAML 501.4 (anti-'leukocyte-common' (T200) antigen; YTH 8.18 (antimacrophage cytoplasm); and YPC 1/3 . 12 (anti-smooth muscle cell). The cells of diffuse intimal thickening were almost all smooth muscle cells but there were rare subendothelial macrophages. Focal lesions, in contrast, contained numerous macrophages as well as smooth muscle cells. Macrophage 'foam cells' were particularly numerous in fatty streaks and in advanced fibro-fatty plaques, but were less conspicuous in focal fibro-elastic lesions. The results confirm that macrophages are an important component of atherosclerotic plaques and suggest that they may have a significant role in atherogenesis in man.