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We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.

Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.

Due to its dependence on intracellular development Chlamydia pneumoniae has developed numerous strategies to create an adequate environment within its host cells ensuring both chlamydial reproduction and target cell survival. The bacterium that has been related to atherogenesis due to its presence in vascular tissue is able to enter a persistent state of chronic infection in the vasculature that escapes antibiotic targeting. Ingestion of the bacterium results in severe modifications and reprogramming of signalling pathways and the metabolism of the host cell. Processes range from the prevention of direct lysosomal destruction of chlamydial inclusions to the inhibition of host cell apoptosis and an enhanced cellular glucose uptake to maintain energy-consuming mechanisms. Furthermore, infection regularly causes the development of a proinflammatory and proproliferative phenotype in the host cell in vitro, ex vivo and in vivo and own new findings suggest a detrimental proliferative loop within vascular cells upon a modified endothelin-1 axis demonstrating a potential for proatherosclerotic processes in early and progressed atherosclerosis. This review displays crucial mechanisms of Chlamydia pneumoniae-induced interactions with vascular host cell signalling cascades with an emphasis on mitogenic and inflammatory processes as well as target cell activation.

Endothelin-1 (ET-1) is a vasoactive peptide that modifies vascular function via the G-protein coupled transmembrane receptors, Endothelin-A receptor (ETAR) and Endothelin-B receptor (ETBR). Dysregulation of the ET-1 axis plays a role in atherosclerotic development as it triggers cell proliferation, inflammation, and vasoconstriction. The respiratory pathogen Chlamydia pneumoniae (Cp) has been recovered from atherosclerotic lesions, and related to atherogenesis, via activation of vascular small GTPases and leukocyte recruitment. Cp effectively reprograms host cell signalling and is able to enter an intracellular persistent state in vascular cells that is refractory to antibiotics. Upon chlamydial infection, vascular smooth muscle cells, which do not produce significant ET-1 under physiological conditions were switched into a fundamental source of ET-1 mRNA and protein in a p38-MAP-kinase-dependent pathway. Endothelial cells did not overproduce ET-1 but showed upregulation of mitogenic ETAR mRNA and protein while the counterbalancing ETBR, which regulates ET-1 clearance, remained unaffected. This disruption of the ET-1 axis was confirmed in an ex vivo mouse aortic ring model, and resulted in endothelial cell proliferation that could be abrogated by ETAR-siRNA and the selective ETAR-antagonist BQ-123. Chronic chlamydial infection of the vascular wall might represent a permanent noxious stimulus linked to the endothelial cell proliferation characteristic of early atherosclerosis. Suppression of this deleterious paracrine loop by ETAR antagonism opens up a new option of preventing possible vascular sequelae of otherwise untreatable chronic chlamydial infection. In conclusion, this is the first study to demonstrate infection to dysregulate the ET-1 axis towards inducing a proatherogenic proliferative phenotype.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Atherosclerosis is characterized by inflammation and proliferation of vascular cells. The intracellular bacterium Chlamydia (Chlamydophila) pneumoniae uses blood monocytes [peripheral blood mononuclear cells (PBMCs)] for dissemination, has been found to persist in atherosclerotic lesions, and has been implicated in atherogenesis by small GTPase activation and T lymphocyte recruitment. Infection of human coronary artery smooth muscle cells with C. pneumoniae significantly induced mRNA and protein for the angiogenic transcription factor Egr-1, resulting in enhanced coronary artery smooth muscle cell proliferation, which was reduced by transfection with small interfering RNA duplexes targeted at Egr-1 mRNA. These effects required viable chlamydiae and depended on p44/42 mitogen-activated protein kinase activity but not on the p38 mitogen-activated protein kinase pathway. Postinfectious Egr-1 mRNA up-regulation in arterial vessels was confirmed ex vivo in a rat aortic ring model of focal vascular chlamydial infection. An in vivo model based on the injection of C. pneumoniae-infected PBMCs into mice confirmed Egr-1 mRNA up-regulation within 24 h of endovascular infection. Arterial injury from repeated direct chlamydial infections and cell-to-cell contact with C. pneumoniae-infected PBMCs might represent a chronic focus of proliferative activity linked to the media proliferation seen in advanced atherosclerosis. Overall, chlamydial infection induces a proliferative phenotype in vascular cells via transcription factor Egr-1 activation in vitro, ex vivo, and in vivo.

Chlamydia pneumoniae uses blood monocytes (PBMC) for systemic dissemination, persists in atherosclerotic lesions, and has been implicated in the pathogenesis of atherosclerosis. During transmigration in a newly developed transendothelial migration model (TEM) C. pneumoniae-infected PBMC spread their infection to endothelial cells. Transmigrated PBMC retained their infectivity and transmitted the pathogen to smooth muscle cells in the lower chamber of the TEM. Detection of chlamydial HSP60 mRNA proved pathogen viability and virulence. We conclude that PBMC can spread chlamydial infection to vascular wall cells and we suggest the TEM as a novel tool to analyze host-pathogen interactions in vascular chlamydial infections.

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay. Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05). Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation. Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis.

Since its description in 1986, Chlamydia pneumoniae has remained one of the most enigmatic pathogens. This intracellular bacterium is highly seroprevalent, but rarely recovered from cell culture, it can genetically switch between a proliferative and a nonreplicative state and has been linked to a vast number of chronic diseases, most notably to atherosclerosis, as it can be found in the plaques. It has become quite clear that persistent bacteria in atherosclerotic lesions cannot be eradicated by currently available antibiotic treatments and that attempts to do so without a better understanding of the pathobiology of chlamydial persistence are futile. However, there is growing knowledge on how vascular chlamydial infection may lead to the pathological reprogramming of the host cell signaling pathways. Chlamydia pneumoniae is now well known to induce, at least in vitro, the two pathogenetic main events that define atherosclerosis: angiogenesis and inflammation. In vivo a contribution of chlamydial infection to the progression of atherosclerosis remains unproven. This minireview provides a brief overview on the proproliferative and proinflammatory effects of vascular C. pneumoniae infection and their potential link to atherogenesis. [ABSTRACT FROM AUTHOR]

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1α regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1α stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1α degradation. HIF-1α was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1α stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen–host interaction in chlamydial diseases. [ABSTRACT FROM AUTHOR]