Journal of Vascular Surgery
Volume 43, Issue 6 , Pages 1301-1307, June 2006

Chlamydia pneumoniae and vascular disease: An update

  • Firas F. Mussa, MD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • ,
  • Hong Chai, MD, PhD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • ,
  • Xinwen Wang, MD, PhD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • ,
  • Qizhi Yao, MD, PhD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • ,
  • Alan B. Lumsden, MD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • Texas Heart Institute at St Luke’s Episcopal Hospital, Houston, Texas
    • ,
  • Changyi Chen, MD, PhD

      Affiliations

    • Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas
    • Corresponding Author InformationReprint requests: Changyi (Johnny) Chen, MD, PhD, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Mail Stop: NAB-2010 Houston, TX 77030

Received 16 January 2006; accepted 26 February 2006.

Article Outline

Exposure to Chlamydia pneumoniae is extremely common, and its incidence increases with age. C pneumoniae infection is strongly associated with coronary artery disease, as well as with atherosclerosis of the carotid artery, aorta, and peripheral arteries. This association has been shown in seroepidemiologic studies and by direct detection of the organism in atherosclerotic lesions by immunohistochemistry, polymerase chain reaction, electron microscopy, and tissue culture. Animal models of atherosclerosis have been used to study the role of C pneumoniae in the initiation and progression of atherosclerotic disease. The association of this organism with cardiovascular complications has inspired many human trials of antibiotics for the secondary prevention of atherosclerosis. C pneumoniae can infect several types of cells, including circulating macrophages, arterial smooth muscle cells, and vascular endothelial cells, causing the secretion of proinflammatory cytokines and procoagulants by endothelial cells and foam cell formation by infected macrophages. This report reviews the role of C pneumoniae in atherogenesis in light of recent, large antibiotic treatment trials, animal studies, and in vitro studies. The role of Chlamydia heat shock protein as a potential mediator of this harmful effect is also reviewed.

 

The notion that atherosclerosis is linked to a chronic inflammatory state is not new.1 This state may result from chronic infection that can initiate or promote atherogenesis. Recent seroepidemiologic studies suggest that Chlamydia pneumoniae infection predicts the development of vascular disease, just as smoking, hypertension, and elevated low-density lipoprotein (LDL) cholesterol do.2 C pneumoniae is an obligatory intracellular Gram-negative bacterium that was first isolated as a respiratory pathogen two decades ago.3 This organism has also been isolated from the coronary arteries of patients with acute coronary syndrome,4 as well as from carotid arteries,5, 6 the aorta,7 and peripheral arteries.8

On the cellular level, C pneumoniae antigen may trigger an early signal transduction cascade in target cells, leading to endothelial activation, inflammation, and thrombosis. Reviews of the role of C pneumoniae in cardiovascular disease have focused on the organism’s pathogenic mechanisms and on methods of testing for C pneumoniae infection. For vascular surgeons, C pneumoniae is not well characterized as a risk factor for atherosclerosis outside of the coronary circulation. The purpose of this report is to examine the role of C pneumoniae in vascular disease by reviewing human and animal laboratory studies, clinical trials, and in vitro studies of the interactions between C pneumoniae and key inflammatory cells.

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C pneumoniae and vascular disease 

The presence of C pneumoniae in atheroma has been detected by serology by using specific antibodies against C pneumoniae, including immunoglobulin (Ig) G, IgM, and IgA,9, 10 and confirmed by immunohistochemistry,7, 11 polymerase chain reaction (PCR),6, 7, 11, 12 Southern hybridization, in situ hybridization, electron microscopy, and electron-microscopic immunohistochemistry.13 In addition, viable organisms have been found in the atherosclerotic lesion in the patient with coronary artery disease.14

In general, the prevalence of C pneumoniae infection increases with age; positive antibody titers are found in 80% of men and 70% of women aged ≥65 years compared with 50% in age 20.2 This age-related difference in prevalence, combined with the lack of standardized methods to document this age relationship, have caused various studies to produce conflicting findings about the association of circulating C pneumoniae DNA and antibody titers with C pneumoniae infections in atherosclerotic patients. Another possible explanation for the lack of coherence among study findings is that C pneumoniae in atheromas exists in a noncultivatable, persistent form that makes isolating viable organisms more difficult.15

Saikku et al4 found that patients with acute myocardial infarction (MI) have higher antibody titers against C pneumoniae than matched normal individuals. The ultrastructural presence of C pneumoniae has also been detected during autopsy in the coronary arterial fatty streaks and intimal smooth muscle cells (SMCs) of deceased patients with coronary atherosclerosis.16 These findings were reproduced by Kuo et al,11 who found histologic and PCR-based evidence of C pneumoniae in 86% of atheromas in the coronary arteries of young adults (ie, 15 to 34 years old). In addition, most of these patients with coronary atherosclerosis tested positive for C pneumoniae by PCR, immunocytochemistry, electron microscopy, or in situ hybridization.14

In 1996, Muhlestein et al17 used immunofluorescence to detect the presence of C pneumoniae in 71 (79%) of 90 specimens of atherosclerotic tissue from symptomatic patients undergoing coronary atherectomy. In contrast, the organism was detected in only one specimen (4%) of nonatherosclerotic coronary tissue. More recently, Arcari et al18 found that high titers to C pneumoniae IgG and IgA were significantly associated with acute MI in a cohort of 30- to 50-year-old men in the United States military and suggested that recent or chronic active infections could be associated with an increased risk of acute MI.

The link between C pneumoniae and coronary atherosclerosis is also substantiated by studies in which antibodies against members of the heat shock protein (HSP) family were detected in individuals with persistent C pneumoniae infections.19, 20, 21, 22, 23, 24 Elevated levels of these Chlamydia heat shock proteins (cHSPs) produce a local inflammatory state. This persistent intracellular infection may explain how C pneumoniae can resist antibiotic treatment, and it may be the mechanism linking atherogenesis with chronic latent infections.15

The detection of C pneumoniae by using immunocytochemistry and PCR in atheromatous plaque tissue removed during carotid endarterectomy6 inspired many more studies of the possible association between C pneumoniae infection and carotid atherosclerosis. In one such study, viable C pneumoniae bacteria were detected in 18 (38%) of 48 samples of carotid plaque after endarterectomy. The presence of the organism was associated with elevated levels of the acute phase protein and proinflammatory markers such as C-reactive protein.25

In another study, Sessa et al5 found serologic evidence supporting the association between C pneumoniae DNA in peripheral blood mononuclear cells and symptomatic carotid atherosclerotic disease. The organism was detected in 72.2% of patients with symptomatic carotid atherosclerotic disease but only in 30.3% of patients with asymptomatic disease. The authors suggested using C pneumoniae DNA in peripheral blood mononuclear cells as a surrogate marker of risk for endovascular Chlamydia infection.

The association between atherogenic risk and seropositivity to C pneumoniae, Helicobacter pylori, and cytomegalovirus was further verified in a prospective, population-based study by Mayr et al.26 They found that the prevalence and severity of atherosclerosis in carotid and femoral arteries was significantly associated with the presence of IgA antibodies to C pneumoniae, regardless of whether the patient had other risk factors. However, the presence of C pneumoniae alone did not appear sufficient to explain the occurrence of cerebrovascular symptoms. Correlations between the presence of IgG antibodies against H pylori and vascular disease were limited. Also, antibody titers against cytomegalovirus do not appear to be markers for atherosclerosis in this population.27

Early reports suggested a link between C pneumoniae infection and abdominal aortic aneurysm (AAA) expansion, risk of rupture,28 and the timing of elective repair.29 However, the lack of standardized methods of testing for C pneumoniae infection led to conflicting findings.30 C pneumoniae may induce immunologic activation, causing chronic endothelial cell damage that can mediate a proteolytic process in the wall of the abdominal aorta.

It has been proposed that C pneumoniae outer membrane protein triggers an autoimmune reaction in the aortic wall, as shown by the strong cross-reaction between C pneumoniae outer membrane protein and human immunoglobulin. These reactions have been demonstrated by two-dimensional polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometric protein identification.31 An electron microscopy study found elementary bodies of C pneumoniae in macrophage-like cells in the intima of the atherosclerotic aorta.13 Furthermore, Cheuk et al32 performed PCR-enzyme immunoassay in 16 patients with ruptured AAA and found C pneumoniae DNA in all of them. In the Blasi et al33 study of 41 patients with AAA, C pneumoniae DNA was detected in both the peripheral blood mononuclear cells and the AAA tissues of 39% of the patients.

The possibility of potential nonspecific findings on immunostaining was assessed by Vammen et al34 in 20 surgical candidates with infrarenal AAA. Analysis was performed using polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometric protein identification. C pneumoniae antigen was not found in any of samples of excised AAA tissues, but a cross-reacting protein was present in all AAA samples. Therefore, the direct detection of C pneumoniae by immunohistostaining should be interpreted with caution because of potential cross-reaction with non-Chlamydia proteins.

In 1997, Kuo et al8 reported finding C pneumoniae in surgically excised vascular tissues from patients undergoing lower-extremity bypass for claudication. Arterial biopsy specimens obtained from femoral and popliteal arteries were examined by using immunocytochemistry and PCR. The pathogen was detected in 11 (48%) of 23 patients. In another study, PCR was positive for C pneumoniae in 50 (59%) of 85 arterial tissue samples taken from patients who underwent surgery for peripheral vascular disease.35 Furthermore, azithromycin, an oral macrolide with an expanded spectrum of activity and few adverse effects, taken in doses of 300 mg/day for 28 days, has been shown to prevent progression of peripheral arterial occlusive disease in C pneumoniae seropositive men for 2.7 years.35

Taken together, the evidence that C pneumoniae DNA is present in peripheral artery atheromas suggests that C pneumoniae infection is important in the pathogenesis of peripheral vascular disease. It should be noted, however, that the presence of C pneumoniae in atherosclerotic vascular tissues merely suggests an association; it does not establish an etiologic relationship. Instead, C pneumoniae infection and atherosclerosis may both be secondary to inflammation or some other process.

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Clinical treatment trials 

Treatment trials of antibiotics for the prevention of untoward cardiovascular events in animal models of atherosclerosis36, 37 have produced results that were sufficiently encouraging to inspire multiple, small-scale secondary prevention trials in patients. In one such study, Gupta et al38 administered 500 mg/day of azithromycin or placebo for 3 to 6 days to acute MI survivors who had high antibody titers to C pneumoniae. During the 18 months after this treatment, patients with high antibody titers who received placebo had four times as many adverse cardiovascular events as patients with low antibody titers. Antibiotic treatment significantly reduced the frequency of these events in the high-titer patients.

In the Randomised Trial of Roxithromycin in non–Q-wave Coronary Syndromes (ROXIS) trial,39 in which 202 patients with unstable angina or non–Q-wave MI were randomly assigned placebo or roxithromycin treatment (150 mg twice daily for 30 days), roxithromycin significantly reduced the incidence of recurrent angina, MI, and death ≤31 days of the start of treatment. The final report of the study showed that patients still had reduced rates of death and reinfarction for at least 6 months after treatment with roxithromycin.40

In contrast, the Azithromycin in Coronary Artery Disease: Elimination of Myocardial Infection with Chlamydia (ACADEMIC) trial,41, 42 in which patients with coronary artery disease were randomly assigned to 3 months of treatment with placebo or azithromycin, found only a nonsignificant trend towards a treatment benefit 12 to 18 months later. No treatment benefit for secondary prevention of coronary heart disease was seen in the Weekly Intervention with Zithromax [Azithromycin] for Atherosclerosis and its Related Disorders (WIZARD) study.43 The patients in that study, however, had advanced atherosclerosis.

In 2005, Grayston et al44 published the results of the Azithromycin and Coronary Event Study (ACES) trial sponsored by the National Institutes of Health. In this randomized, multicenter, prospective trial, 4012 patients with documented stable coronary artery disease received either 600 mg of azithromycin or placebo weekly for 1 year. Mean follow-up was 3.9 years. At 1 year, no significant risk reduction was noted in the azithromycin group with regard to the primary end points, which was a composite of death due to coronary heart disease, nonfatal MI, coronary revascularization, or hospitalization for unstable angina. Azithromycin treatment also did not significantly reduce the risk of stroke or of death from any cause.

Similar findings were reported by Cannon et al,45 who performed a double-blind, randomized, placebo-controlled trial of long-term fluoroquinolone therapy (gatifloxacin 400 mg/day for a 2-week period that began 2 weeks after randomization, followed by a 10-day course every month for a mean duration of 2 years) in patients with acute coronary syndrome. At 2 years, the primary end point event—a composite of death from all causes, MI, documented unstable angina requiring rehospitalization, revascularization (performed at ≥30 days after randomization), and stroke—had occurred in 23.7% of the gatifloxacin patients and 25.1% of the placebo patients (P = .41). These results show the need for better understanding of the pathogenic mechanisms of C pneumonia in order to target specific Chlamydia antigens and design trials with new effective antibiotic regimens.

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C pneumoniae interactions in vitro 

C pneumoniae accesses the vasculature during local inflammation in lower respiratory tract infection. The infected alveolar macrophages transmigrate through the mucosal barrier and give the pathogen access to the lymphatic system, systemic circulation, and atheromas.46, 47, 48 C pneumoniae can infect a variety of cells commonly found in atheromas, including coronary artery endothelial cells, macrophages, and aortic SMCs.49, 50, 51, 52, 53 Infected cells upregulate adhesion molecule expression and produce inflammatory cytokines, thereby promoting leucocyte adherence, leucocyte migration, and intimal inflammation.54 Infection of human endothelial cells also triggers transendothelial migration of neutrophils and monocytes55 and the secretion of interleukin (IL)-8, procoagulant tissue factor, IL-6, and plasminogen activator inhibitor-1.50, 52 The infection and proliferation of SMCs may result from endothelial infection with C pneumoniae or may be caused by direct infection leading to the release of IL-6 and basic fibroblast growth factor (bFGF).56, 57, 58

C pneumoniae infects human macrophages, which secrete enhanced levels of inflammatory cytokines such as tumor necrosis factor-α, IL-1β, IL-6, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-α, and IL-12, which may promote lesion progression.57, 59 C pneumoniae also enhances the secretion of IL-10, which may prevent apoptosis and thereby perpetuate inflammation.60, 61

Immunocytochemical studies show that atheromas contain activated CD4+ helper T cells, CD8+ cytotoxic T cells, and monocyte-derived macrophages.62 C pneumoniae may trigger specific cell-mediated immunity within plaques, as evidenced by the detection of Chlamydia-specific T lymphocytes in atherosclerotic lesions.63, 64, 65 These cells contribute to plaque destabilization by producing cytokines. C pneumoniae can also enhance the production of matrix-degrading metalloproteinases by infected macrophages.66, 67

Another way in which C pneumoniae may influence atheroma biology is by modulating macrophage-lipoprotein interactions. Infected macrophages may block cholesterol efflux, an important component of the mechanism that transports cholesterol from the periphery towards the liver. Additionally, infected macrophages may ingest excess lipoprotein to become foam cells, the hallmark of newly formed atherosclerotic lesions.68, 69, 70, 71

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Role of heat shock proteins 

HSPs belong to a family of approximately two dozen proteins that are important in regulating the molecular response to the vessel wall under both normal and stressed conditions. HSPs are highly conserved across species, and their expression in vascular tissues can be triggered by several different stimuli. Upregulation of HSPs is mediated by heat shock transcription factors binding to the regulatory elements of the HSP gene promoters.72, 73 C pneumoniae secrets cHSPs, and they are expressed in abundance by cellular constituents of atherosclerotic lesions. A persistent Chlamydia infection is accompanied by an increased production of cHSP60, which may induce antigenic mimicry and chronic immunostimulation in the vascular wall and may lead to the release of local and systemic cascades of inflammatory cytokines. These augmented immune responses target bacterial peptides and host homologous peptides. Purified anti-cHSP60 antibodies also stain heat-shocked macrophages and confer both antibody-dependent and complement-mediated cytotoxicity.74 Vaccines containing cHSP have been shown to cause endothelial dysfunction in cholesterol-fed rabbits.75 Thus, immune responses in C pneumoniae infection may be involved in vascular lesion formation.

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Animal models of C pneumoniae infection 

Animal models have been used to define further the role of C pneumoniae as a risk factor in atherosclerosis. The first models tested76, 77 involved the use of either the hyperlipidemic mouse strain (ApoE−/− mice) or a strain (LDL receptor−/ − mice) that develops lesions when fed an atherogenic diet. C pneumoniae was detected in atheromas taken from mice of either strain after they were infected intranasally with C pneumoniae. These mice all had high levels of cholesterol-rich LDL.

Blessing et al,78 however, showed that chronic C pneumoniae infection can induce inflammatory changes in the heart and aorta of normocholesterolemic C57BL/6J mice but does not initiate the formation of atherosclerotic lesions. This may be because C pneumoniae infection could accelerate the development of vascular disease in hyperlipidemic animals.

Another animal model, which uses New Zealand white rabbits that are fed a hyperlipidemic diet, has also been used to examine the relationship between C pneumoniae infection and atherosclerosis.79, 80, 81 When infected with C pneumoniae, these rabbits develop atherosclerotic changes in the aorta. Moreover, weekly treatment with azithromycin prevents the accelerated intimal thickening normally seen in this rabbit model.36

A study in a normocholesterolemic rabbit model showed that infection with C pneumoniae induces SMC growth factor production, SMC proliferation, and aortic intimal thickening through increased platelet-derived growth factor-B messenger RNA expression.82 Similarly, acute infection with C pneumoniae has been shown to induce endothelial dysfunction in a porcine model83 of atherosclerosis by promoting a procoagulant status through impaired vasodilatation, induced vasospasm, elevated fibrinogen levels, and involvement of the nitric oxide pathway.83, 84 Taken together, these animal studies strongly suggest that C pneumoniae can target the vasculature, induce inflammation, and initiate or promote lesion development in atherosclerotic individuals.

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Conclusion and future directions 

Evidence suggests that immune mechanisms are crucial in atherosclerosis. Chronic infection with C pneumoniae has been identified in atheromas of the coronary, carotid, and iliac arteries and of the aorta. Evidence for the presence of the organism in these atherosclerotic lesions has emerged from nearly 40 studies conducted by several different groups of investigators. Data have accumulated from serologic studies, pathologic analysis of plaque, in vitro examinations, and animal models. The direct link between infection and atherosclerosis has yet to be identified, however. This may be partly due to the lack of standardized testing methods.

Despite the considerable laboratory and clinical research that has been done on the role of C pneumoniae in the progression of atherosclerosis in arterial tissue and on the cellular interactions of C pneumoniae with vascular and inflammatory cells, several important questions remain unanswered. For example, we do not yet know the true prevalence of C pneumoniae in either atheromatous tissues or normal, carefully matched vascular tissues. We also do not know whether the high rate of C pneumoniae detection by immunohistochemistry, compared with other techniques such as PCR,6, 11, 85 results from higher sensitivity or lower specificity on the part of the immunohistochemical tests. Most importantly, we do not know whether the C pneumoniae bacterium is an innocent passenger aboard atheromas or whether it is actively involved in the initiation or progression of atherosclerotic disease. To answer this question, well-planned studies are needed to further characterize the molecular mechanisms that link C pneumoniae to vascular disease. In particular, cHSP60 needs to be explored further as a potential culprit and therapeutic target.

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We thank Stephen N. Palmer, PhD, ELS, scientific medical writer at the Texas Heart Institute of St. Luke’s Episcopal Hospital, for his assistance in the manuscript preparation.

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 This work was supported by National Institutes of Health Grants R01 HL65916, R01 HL72716, R01 EB-002436 and R01 HL083471 (C. C.); R01 DE15543 and R21 AT003094 (Q. Y.); R01 HL75824 (A. B. L.).

 Competition of interest: none.

PII: S0741-5214(06)00413-7

doi:10.1016/j.jvs.2006.02.050

Journal of Vascular Surgery
Volume 43, Issue 6 , Pages 1301-1307, June 2006

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