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。     

Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells.

Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte-derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions.     

LDL enhances monocyte adhesion to endothelial cells in vitro.

Monocyte adhesion to the arterial endothelium is an early event in diet-induced atherogenesis. The possibility that low-density lipoprotein (LDL) may influence this adhesion was investigated by using an in vitro monolayer collection assay. Postprandial and fasting LDL was isolated from 12 normal adult human donors (8 male and 4 female) and incubated with primary cultures of bovine aortic endothelial cells (BAEC) for 6 hours. 51Cr-labeled mononuclear leukocytes (MNLs) were then added and incubated an additional 30 minutes. When results were expressed as the ratio of adherent counts per minute in LDL-treated BAEC cultures to that in PBS-treated controls, 10 of the 16 LDL samples isolated from male donors induced a significant increase (P less than 0.05) in MNL adhesion (1.06-1.27) attributable to esterase-positive cells. This increase was dose-dependent and maximal at 100 micrograms LDL protein/ml. The magnitude of the response was significantly correlated with LDL composition (r = 0.857, P less than 0.01) such that LDL rich in cholesterol and triglyceride relative to protein enhanced MNL adhesion, whereas lipid-poor LDL (typically isolated from the women) reduced adhesion.     

Estradiol reduces basal and cytokine induced monocyte adhesion to endothelial cells

OBJECTIVE: To investigate the effect of 17beta-estradiol (E2) on binding of monocytes to human aortic endothelial cells (HAECs) with or without cytokine induction. METHODS: Confluent monolayers of HAECs were incubated with or without E2 for 48 h prior to the monocyte adhesion assay. In studies with cytokines, 1 ng/ml tumor necrosis factor-alpha (TNF-alpha), 20 U/ml interleukin-1beta (IL-1beta) or both were added to the culture medium for the final 24 or 4 h. For the measurement of monocyte adhesion, 3H-thymidine labeled human THP-1 monocytes (4 x 10(5) cells per well) were added to the confluent monolayer of HAECs and incubated at 37 degrees C for 90 min. The unbound THP-1 cells were removed by gentle washing, and bound cells were digested with NaOH and quantified by measuring radioactivity. RESULTS: When HAECs were pretreated for 48 h with E2 the basal adhesion of THP-1 cells was reduced by an average of 28%. Estrogen significantly reduced cytokine-induced adhesion by 30-35% when the cytokines were added for 4 h. When the cytokine treatment was prolonged to 24 h, pretreatment of HAECs with E2 had no effect on THP-1 cell adhesion. CONCLUSIONS: E2 reduces basal and short-term cytokine induced monocyte binding to HAECs. Since monocyte adhesion to vascular endothelial cells is one of the initial steps in the pathogenesis of atherosclerosis, E2 may mediate vascular protection by reducing monocyte-endothelial cell binding in the early stages of atherogenesis.     

Insulin-like growth factor-I modulates monocyte adhesion to EAhy 926 endothelial cells

IGF-I is a ubiquitous growth factor, found in platelets and elaborated by many other cell types. It is thought to be involved in several pathophysiological processes including embryonic development, angiogenesis and wound healing. We report that the adherence of human peripheral blood monocytes to an endothelial cell line (EAhy 926) is inhibited in a dose and time-dependent manner by pre-incubating the endothelial cells with IGF-I ( P  < 0.001). Monocyte adhesion was inhibited 17.9 ± 1.9% by IGF-I at a dose of 1000 ng/ml ( P  < 0.01). In contrast, IGF-I had no significant effect on monocyte adherence to plastic. The inhibitory effects of IGF-I were reversed by co-incubating the endothelial cells with the nitric oxide synthase inhibitor, L-NAME. These data suggest that the effects of IGF-I are mediated by the release of nitric oxide from the endothelial cells.     

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。     

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.     

The effect of Chlamydia pneumoniae on the expression of peroxisome proliferator-activated receptor-gamma in vascular smooth muscle cells

PURPOSE: This study was designed to investigate the change of peroxisome proliferator-activated receptor gamma (PPARgamma) after the infection of the human coronary artery smooth muscle cells (HCSMCs) with Chlamydia pneumoniae (C. pneumoniae) and the effect of PPARgamma agonist on the expression of PPARgamma of C. pneumoniae-infected HCSMCs. MATERIALS AND METHODS: To determine the effect of PPARgamma agonist on the proliferation of C. pneumoniae-infected HCSMCs, rosiglitazone at various concentrations was applied 1 hour before inoculation of HCSMCs. RESULTS: The expression of PPARgamma mRNA in HCSMCs increased from 3 hours after C. pneumoniae infection and reached that of noninfected HCSMCs at 24 hours (p<0.05). The expression of PPARgamma protein in HCSMCs also increased from 3 hours after C. pneumoniae and persisted until 24 hours as compared with that of noninfected HCSMCs (p<0.05). The pretreatment of HCSMCs with rosiglitazone followed by the infection with C. pneumoniae augmented the expression of PPARgamma mRNA and protein (p<0.05) and decreased cell proliferation. CONCLUSION: Our results showed that the expression of PPARgamma increases in response to C. pneumoniae infection and rosiglitazone further augmented the expression of PPARgamma. It is suggested that rosiglitazone could ameliorate the chronic inflammation in the vessel wall induced by C. pneumoniae by augmenting PPARgamma expression.     

Effects of magnetic cell separation on monocyte adhesion to endothelial cells under flow

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 degrees C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 degrees C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non-activated monocytes are needed for adhesion assays under flow.     

A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.     

Chlamydia pneumoniae decreases smooth muscle cell proliferation through induction of prostaglandin E2 synthesis

Chlamydia pneumoniae may modulate the proliferation of smooth muscle cells (SMC) in atherosclerotic plaques. Conditioned medium from C. pneumoniae-infected SMC decreased the proliferation of uninfected SMC. Treatment of infected cells with the cyclooxygenase-2 inhibitor NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide] suppressed the up-regulation of prostaglandin E(2) (PGE(2)) and abolished the antimitogenic effect of conditioned medium, suggesting that C. pneumoniae can decrease SMC proliferation via stimulation of PGE(2) synthesis.     

Chlamydia pneumoniae inhibits apoptosis in human epithelial and monocyte cell lines

Chlamydia pneumoniae is an obligate intracellular pathogen with a tendency to cause persistent infections that has been associated with many chronic conditions such as asthma and coronary artery disease. However, its immunopathogenic mechanisms are poorly understood. When aiming to study the impact of C. pneumoniae infection on host cell apoptosis, we found that epithelial infected (HL) cells and macrophages (U937-line) were resistant to staurosporine and tumour necrosis factor (TNF)-alpha-induced physiological apoptosis 48, 72 or 120 h post-infection, as determined by flow cytometry, DNA fragmentation assay and fluorescence microscopy. The antiapoptotic influence was observed even at a late stage of the chlamydial life cycle and was dependent on the chlamydial protein synthesis. The mechanisms involved blockage of mitochondrial cytochrome c release and caspase 3 activation. We also found that during a persistent C. pneumoniae infection induced in vitro by penicillin treatment of cell cultures, the inhibition of apoptosis was extended for up to 120 h of follow-up post-infection and was restricted to the cells carrying chlamydial inclusions. Our findings suggest that inhibition of apoptosis may be one of the pathogenetic mechanisms by which C. pneumoniae infection can mediate the development of chronic diseases.     

Human vascular adhesion protein-1 in smooth muscle cells

Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.     

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 micro g/ml for 0-60 min at 37 degrees C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 micro g/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.     

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.     

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.