Chlamydia pneumoniae and Atherosclerosis

Atherosclerosis is a major cause of stroke, coronary heart disease, peripheral vascular disease, and aortic aneurysm. Because of the prevalence and importance of these diseases, atherosclerotic lesions within arteries have been extensively studied.^{1 - 2} While many risk factors have been identified,^{1 - 2} the mechanism by which the lesions are formed remains unknown. The most popular concept is that the endothelium lining the lumen of the artery becomes damaged. This damage alters the properties of the endothelium and leads to a cascade of events culminating in fibrosis, necrosis, lipid accumulation, and eventually calcification. There have been several candidates forwarded as putative initiators of endothelial injury including microorganisms.¹ Recent studies have shown an association between an obligate intracellular bacterium, Chlamydia pneumoniae, and atherosclerosis.^{3 - 4}

Chlamydia pneumoniae was first described by Grayston et al⁵ in 1986. Seroepidemiological studies have concluded that the majority of the population has been exposed to the organism.⁶ While C pneumoniae appears to cause 10% of community-acquired pneumonia, infection is typically mild or asymptomatic.⁷ The association of the organism with heart disease was first demonstrated, serologically, in a Finnish population.⁸ This association has been shown in subsequent studies.^{9 - 10} A meta-analysis of seroepidemiological studies demonstrated that overall, having antibodies to C pneumoniae conferred a 2-fold relative risk for heart disease.¹¹ The strength of the serological association and heart disease varies from study to study and some of the variation may be due to methods.¹² The association of C pneumoniae and atherosclerotic lesions itself was first shown in 1992 in a South African population.¹³ This association has been established in several countries, in a wide range of arteries, using a variety of techniques, such as polymerase chain reaction (PCR), immunocytochemistry, immunofluorescence, in situ hybridization, enzyme immunoassay, and electron microscopy.^{14 - 15} Using these methods, on average, C pneumoniae is detected in 59% of atheromatous arteries and in only 3.1% of control arterial tissue.¹⁴ Although PCR has been the most common method used, it does not appear to give the most consistent results¹⁶ and has been associated with detection rates in atherosclerotic lesions from 0%¹⁷ to 100%.¹⁸ The standardization of the PCR technique has been called for.^{12 ,19}

Chlamydia pneumoniae has been cultured from atherosclerotic arteries, attesting to the viability of the organism in the lesion,^{20 - 22} with some laboratories reporting a 16% recovery rate for atherosclerotic lesions.²³ Reverse transcriptase PCR has also indicated that the organisms are viable in atherosclerotic plaques of the carotid artery.²⁴

There is much speculation as to the role C pneumoniae may play in initiating or contributing to atherosclerosis.^{15 ,25} A MEDLINE search of the terms C pneumoniae and atherosclerosis from 1991 to August 1999 produced 155 references, of which only 70 (45%) present original data and the remaining 85 (55%) offer an opinion, present a hypothesis, or review the subject. Opinion varies from the organism being an innocent bystander to a causative agent.^{7 ,14}

However strong the association, causality cannot simply be implied. Criteria that determine whether an association is causal have been examined for C pneumoniae and atherosclerosis and many of the criteria are fulfilled.⁴ To test the hypothesis of causality, clinical trials using antibiotics to eradicate C pneumoniae have been initiated.²⁶ The reported findings from these early trials^{27 - 30} suggest antibiotics may confer some benefit; however, the trials were small and the results by no means conclusive. It has been suggested that the benefits might be ascribed to the anti-inflammatory properties of the antibiotics used rather than their antichlamydial properties.¹² Larger antibiotic trials are in progress but not complete.²⁶ Animal studies indicate that C pneumoniae can accelerate the development of atherosclerosis and antibiotic administration can prevent it.³¹

Another line of investigation is to examine the pathology of the lesion with respect to the presence of C pneumoniae. Such investigations have shown the organism to be present in the earliest stages of the lesion, adding weight to the argument that it plays an initiating role rather than being a secondary invader.⁴ To date, most of the studies that demonstrate C pneumoniae in atherosclerotic lesions have largely dealt with the question of presence of the organism. Some of the techniques used such as PCR do not allow visualization of C pneumoniae, and information about which cell types are affected by the organism come from in situ hybridization, immunocytochemistry, and electron microscopy. Apart from 1 report of weak positive staining using immunocytochemistry,³² C pneumoniae has not been demonstrated in human arterial endothelium in vivo. In atherosclerotic arteries, however, it has been reported in smooth muscle cells and macrophages of the intima. The intima of the artery is the innermost layer and includes the endothelium, underlying connective tissue, and smooth muscle on the luminal side of the internal elastic lamina. This intimal layer has been referred to as the battleground of the atherosclerotic process.³³ Using in situ hybridization, heavy staining for C pneumoniae has been reported in the intimal layer of an atherosclerotic coronary artery.²⁰ Immunoperoxidase double-labeling techniques have localized chlamydial antigens to both smooth muscle cells and infiltrating macrophages.^{32 ,34 - 36} Transmission electron microscopy has identified structures with the morphologic features of C pneumoniae in smooth muscle cells and macrophages of the intima of atherosclerotic arteries that are positive for C pneumoniae by PCR or immunocytochemical techniques.^{3 - 4 ,13 ,37 - 39} While only 1 study to date has examined the pathology of the atherosclerotic lesion with respect to the presence of C pneumoniae,³⁸ previous studies have noted that the presence of the organism in smooth muscle cells is associated with cellular damage.^{3 - 4 ,13 ,37 ,39} The damage takes the form of vacuolation of the smooth muscle cells (Figure 1 and Figure 2) with a concomitant loss of intracellular myofilaments and an accumulation of intracellular lipid. We also have noted that fragmented smooth muscle cells are often associated with C pneumoniae and these fragments, with their accompanying organisms, are engulfed by macrophages (Figure 2). The presence of C pneumoniae in macrophages in association with fragmented extracellular matrix adjacent to apoptotic and necrotic cells of the intima has recently been reported elsewhere.³⁷ It has been suggested that macrophages carry C pneumoniae from the lungs to arteries.^{25 ,34 ,40} An alternative mechanism to account for the presence of the organism in macrophages may be through engulfing damaged, infected, smooth muscle cells.³⁸

In summary, the presence of C pneumoniae in many atherosclerotic lesions can no longer be disputed.¹⁴ The organisms have been demonstrated to be viable^{20 - 24} and present in early lesions.^{4 ,13} They have been identified in the smooth muscle cells of the intima and are associated with pathological changes.^{4 ,37 - 39} Determining the exact nature of the association between C pneumoniae and atherosclerosis is extremely important. If C pneumoniae is found to be causal or to significantly contribute to the lesion, some of the world's major diseases become amenable to new regimens for treatment or prevention. Chlamydia pneumoniae fulfills many of the criteria for causality.⁴ Attempts to test the causality hypothesis have revolved around antibiotic intervention. So far studies in animal and human subjects have not been conclusive.^{27 - 31 ,41} Larger trials are needed and the lesion itself needs to be examined to determine the effects of antibiotic treatment.⁴² Future researchers should be cognizant of the fact that the lesion is not sterile. If C pneumoniae plays a role in atherosclerosis, the underlying pathological process needs to be elucidated.