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(Circulation. 1997;96:4095-4103.)
© 1997 American Heart Association, Inc.

Articles

Roles of Infectious Agents in Atherosclerosis and Restenosis¹

An Assessment of the Evidence and Need for Future Research

Peter Libby, MD; Debra Egan, MSc, MPH; ; Sonia Skarlatos, PhD

From Brigham and Women's Hospital, Harvard Medical School, Boston, Mass (P.L.), and the National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md (D.E., S.S.).

Correspondence to Peter Libby, MD, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail plibby{at}bustoff.bwh.harvard.edu

Key Words: endothelium • leukocytes • muscle, smooth • risk factors • viruses

	Introduction

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
The past few years have witnessed a resurgence of interest in the possibility that infectious processes may contribute to arterial diseases, including atherosclerosis and restenosis after arterial intervention. The recent recognition that Helicobacter pylori contributes to peptic ulcers illustrates how a disease previously believed to result primarily from excessive gastric acid production often involves a microbial pathogen. Although hypercholesterolemia clearly predisposes to development of coronary heart disease, many patients with arterial pathology have serum cholesterol levels within or below the average range. Coronary heart disease frequently affects individuals who lack other traditional risk factors as well. These findings suggest that heretofore unrecognized risk factors may also contribute to the pathogenesis of coronary heart disease.

At a recent Special Emphasis Panel convened by the National Heart, Lung, and Blood Institute, a number of experts reviewed the evidence for links between infectious processes and atherosclerosis and restenosis. This report will summarize and examine critically the evidence for involvement of infectious agents in arterial diseases based on the deliberations of this expert panel. It will also highlight some of the unanswered questions in this area that may warrant further investigation.

Many reports of associations between a wide variety of infectious agents and atherosclerosis have recently appeared. An exhaustive evaluation of each of these purported associations lies beyond the scope of this survey. Rather, we will concentrate particularly on two particular infectious agents, one viral and one bacterial, currently linked with atherogenesis and supported by emerging evidence. Specifically, this report will focus primarily on herpesviruses, particularly cytomegalovirus (CMV), as an example of a viral agent and Chlamydia pneumoniae (C pneumoniae, also known as the TWAR strain on the basis of its original laboratory designation) as an example of a bacterial pathogen. Although the preponderance of recent studies have examined CMV and Chlamydia, the focus of this report on these more fully developed examples in no way excludes the possibility that similar principles may apply to other infectious agents.

	Evidence Supporting Involvement of Infectious Agents in Atherosclerosis and Restenosis

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
Seroepidemiological Evidence (Table)
Herpesviridae, Including CMV
The herpesvirus family includes three groups of human pathogens, the herpesvirus hominis family, currently known to contain more than half a dozen members; CMV; and the Epstein-Barr virus. Systematic studies have linked antibodies to CMV and atherosclerosis for many years. Pioneering examples include the studies from Melnick's group.^1,2 These authors performed case-control studies on patients undergoing cardiovascular surgery and those lacking evidence of atherosclerotic disease. High titers of CMV antibodies were more frequent in patients undergoing surgery for atherosclerotic disease (70%) than in matched control patients (43%).² In that study, there was no significant difference in the titers of antibodies directed against the herpesvirus hominis type I or type II. In addition, a case-control study performed on a subset of the Atherosclerosis Risk in Communities (ARIC) study documented a significant difference between carotid artery intimal thickening (determined by B-mode ultrasound) and the level of anti-CMV antibodies.³ There was no correlation with antibodies directed against Herpes simplex virus type 1 or type 2 antigens. The association with CMV antibody titers did not depend on traditional cardiovascular risk factors. A subsequent prospective study in the ARIC cohort confirmed this relationship.⁴

View this table: [in this window] [in a new window]   Table 1. Evidence Supporting Involvement of Infectious Agents in Atherosclerosis and Restenosis

In the realm of restenosis after coronary interventional therapy, recent evidence suggests an association between previous CMV infection and restenosis. Quantitative coronary arteriography documented a greater reduction in minimum luminal diameter at the sites of coronary atherectomy in patients seropositive for CMV than in seronegative patients.⁵ According to the criteria established by these investigators, 43% of seropositive versus 8% of seronegative patients developed angiographically definable restenosis (P<.002). Interestingly, the risk for restenosis correlated with IgG antibody rather than IgM antibody, and IgG levels did not vary between the primary intervention and follow-up angiogram, indicating prior infection rather than acute CMV infection as the risk factor for restenosis.

In the special case of accelerated arteriosclerosis developing in the coronary arteries of transplanted hearts, some seroepidemiological evidence also suggests an association with CMV infection. In the Stanford experience, cardiac transplant recipients who developed CMV infection, determined by a fourfold rise in IgG antibody, more frequently developed angiographically detectable graft arteriosclerosis than did patients without serological evidence of CMV infection.⁶ At 6 years after transplantation, the actuarial rate of development of >70% luminal narrowing was <10% in the patients without evidence for CMV infection versus 30% for those with evidence for CMV infection (P<.05). Independent studies from other centers have also linked CMV infection with transplantation arteriosclerosis.^7–9

The foregoing examples provide evidence for a potential association between CMV infection and arterial disease. However, not all such studies support an association between CMV and allograft arteriosclerosis.¹⁰ Such discrepancies point to some of the challenges of interpreting such seroepidemiological studies. Because infections with Herpesviridae, including CMV, are very common, many individuals in the general population will exhibit antibody titers suggestive of prior infection with these agents, including CMV. Conversely, many patients with documented arterial diseases will lack high titers of antibodies directed against these viruses. Thus, these data do not prove a causal relationship between infection with these viruses and arterial diseases.

Other limitations of these seroepidemiological studies include the relatively small numbers of patients enrolled in studies at any one center. Issues regarding the specificity and sensitivity of the antibody techniques used apply in such surveys. Often, the agents and methods used vary from center to center, making it difficult to compile the results obtained in various studies to increase the power of the analysis. Moreover, a bias exists against publication of negative studies, which may favor appearance in the literature of positive reports.

	Chlamydia

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
Chlamydia are Gram-negative bacteria. Three species cause human disease: (1) C trachomatis, which causes sexually transmitted disease and eye disease such as trachoma; (2) C psittaci, which infects birds and causes a human pneumonitis; and (3) C pneumoniae, which causes upper respiratory infections. Recent seroepidemiological studies support a relationship between prior infection with C pneumoniae and atherosclerosis. Originally, small observational studies established that patients with acute myocardial infarction or chronic coronary artery disease had higher titers of chlamydial antigens than did control patients.¹¹ Blood samples that had been collected prospectively in the course of the Helsinki Heart Study hinted at a relationship between antichlamydial IgA or immune complexes containing lipopolysaccharide from Chlamydia and the development of acute myocardial infarction.¹² These studies prompted independent efforts to correlate seroepidemiological manifestations of chlamydial disease with atherosclerosis by use of a nested case-control design.^13,14

The various seroepidemiological studies correlating various serological indexes of chlamydial infection with atherosclerosis have been for the most part descriptive (eg, cross-sectional) rather than analytical (eg, with a prospective cohort or case-control design). For this reason, the literature to date suffices to suggest hypotheses but cannot establish a causal relationship. A publication bias favoring positive studies must be taken into account when the existing database is evaluated. Moreover, descriptive studies generally do not control for certain confounding variables. For example, smoking, a risk factor for coronary heart disease events, may predispose to C pneumoniae infection and contribute to elevated titers of antibody to this organism.¹⁵

Studies Localizing Infectious Agents to Human Arterial Lesions (Table)

Herpesviridae, Including CMV
Despite attempts over many decades, it has been difficult to culture Herpesviridae from human atheroma.² However, failure to culture infectious virus particles from the lesions does not preclude their involvement in aspects of the pathogenesis of the disease. There are well-documented instances of "hit-and-run" mechanisms of herpes-mediated disease in which the viruses trigger the disease without persisting in an infectious form in the affected tissue.¹⁶ Indeed, a variety of subsequent studies from many centers have documented the presence of CMV or herpes simplex virus, nucleic acid, and/or antigen in human atheroma (reviewed in Reference 2² ). Landmark studies along these lines include demonstration of herpes simplex virus nucleic acid sequences in human intimal lesions by Benditt et al¹⁷ and work from Bruggeman's laboratory that showed more frequent detection of CMV genomic DNA by polymerase chain reaction amplification in atherosclerotic specimens than in nonatherosclerotic arteries.¹⁸ Interestingly, the prevalence of detection of CMV genome by this technique exceeded 50% in the uninvolved arteries, compared with 90% in atheromatous vessels, showing considerable overlap in nonatherosclerotic and atherosclerotic specimens. Direct DNA blotting analysis of these same specimens showed CMV DNA in 50% of either atherosclerotic or uninvolved arteries. CMV nucleic acids have also been localized in coronary arteriosclerotic lesions in transplanted hearts.¹⁹ Attempts to recover CMV RNA transcripts from human atherosclerotic specimens, however, have proved unsuccessful.²⁰ In contrast, CMV DNA is commonly found in the human arterial tree.^21,22

	Chlamydia

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
Several recent studies have furnished evidence for the presence of Chlamydia in human atherosclerotic specimens.^23,24 A variety of techniques, including immunocytochemistry, polymerase chain reaction detection of nucleic acid, electron microscopy, and culture support the presence of Chlamydia in various stages of atheromatous disease in a variety of human arteries.^25–28 A recent prospective study from the Utah group using immunofluorescence detected chlamydial antigen in 79% of 90 coronary atherectomy specimens but found antigen in only 1 of 24 nonatherosclerotic coronary specimens (4%).²⁹ The recovery of these organisms by culture, however, has been much less successful.³⁰

Evidence for Presence of Other Infectious Agents in Human Arterial Lesions
As noted above, over the years many individual potential pathogens have been implicated in the pathogenesis of atherosclerosis. Sporadic reports of the presence of such organisms in atheroma exist. A notable recent addition to the list of potential pathogens includes Helicobacter species.³¹

Animal Studies Implicating Infectious Agents in Atherosclerosis

	Herpesviridae, Including CMV (Table)

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
The firmest scientific foundation linking an infectious agent with chronic arterial disease is the case of Marek's disease in chickens. This avian herpesvirus causes epidemic lymphoproliferative disease in chickens. Infection of specific-pathogen–free chickens with the Marek's disease virus produced arterial lesions.³² Moreover, with concomitant consumption of a cholesterol-enriched diet, the Marek's disease-infected birds developed fibrofatty lesions sharing certain characteristics of human atherosclerosis. Subsequent studies in Japanese quail demonstrated the presence of sequences related to Marek's disease virus genome in the lesions of Japanese quail genetically susceptible to atherosclerosis, whereas relatively resistant strains lacked such sequences.^33,34 No infectious virions were recovered from these quail lesions, however.

CMV infection enhances the neointimal response to balloon injury in rats. Moreover, in experimental heart transplantation in rats, infection with rat CMV causes subendothelial inflammation, endothelial activation (defined by class II major histocompatibility antigen expression), and increased intimal thickening and promotes the development of allograft arteriosclerosis.^35,36

Animal Evidence Linking Chlamydia With Arterial Disease (Table)
The published literature contains only minimal data supporting a role for C pneumoniae infection in animal models of atherosclerosis. Repeated inoculation of genetically hyperlipidemic rabbits (the Watanabe strain) has resulted in lung abnormalities but no alteration in the aortic atherosclerosis that develops in this strain.³⁷ Moreover, attempts to isolate C pneumoniae and immunocytochemical detection of chlamydial antigen or polymerase chain reaction evidence for chlamydial nucleic acid in the aorta were unrevealing. After intranasal infection of mice with C pneumoniae, detection of organisms was much more frequent in the atheromatous aortas of atherosclerosis-susceptible apolipoprotein E–deficient mice than in the aortas of wild type strains.³⁸

Possible Pathophysiological Mechanisms of Infectious Agents in Atherosclerosis and Restenosis Based on In Vitro Observations

	Direct Infection of Cultured Cells

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
Herpesviridae, including CMV. A large body of experimental evidence shows that human vascular wall cells, including smooth muscle cells and endothelium, can be infected by Herpesviridae, including CMV.^39,40 CMV can also infect human endothelial cells and alter functions related to atherosclerosis, such as the procoagulant balance of the endothelium.^41–43

Chlamydial infection of cells involved in atherogenesis.C pneumoniae, an obligate intracellular parasite, commonly infects mononuclear phagocytes. Macrophages, derived from monocytes, characteristically localize in human atherosclerotic plaques. Data regarding the biology of Chlamydia infection of vascular smooth muscle and endothelial cells are scant.⁴⁴ However, Chlamydia species can infect epithelial cells persistently under certain conditions. Under normal circumstances, the life cycle of Chlamydia involves two developmental forms, the elementary body and the replicative form, the reticulate body. Treatment of the continuous cell line HeLa 229 with the cytokine gamma interferon renders these cells susceptible to a persistent infection with C trachomatis.^45–47 Thus, infection with Chlamydia need not result in a lethal infection but may cause a chronic persistent and nonlytic infection of cells. The susceptibility of macrophages and vascular wall cells to persistent infection by C pneumoniae remains unknown.

	Effects of Infection and Microbial Products on Vascular Cell Functions

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
The ability of various infectious agents and their secreted products to alter vascular cell functions in a manner that might promote vascular disease constitutes one of the most intellectually tantalizing aspects of this subject (Fig 1).

View larger version (37K): [in this window] [in a new window]   Figure 1. Direct effects of infectious agents on intrinsic vascular wall cells. Microbial agents can infect and, in the case of viruses, transform vascular wall cells. Lethal lytic damage may result (not shown here). Alternatively, the infected cells may survive but display deleterious functions such as those listed that could promote arterial lesion formation. Note potential cross talk between intrinsic vascular wall cells (endothelium and smooth muscle) and among vascular cells and leukocytes.

Effects on blood coagulation and fibrinolysis. Numerous studies have addressed changes in procoagulant and anticoagulant functions of endothelial cells infected by viruses.^48–53 For example, herpes simplex virus infection of endothelial cells augments the surface expression of tissue factor, a potent procoagulant.⁵⁴ This and other effects of herpesvirus do not require a productive infection. CMV infection likewise induces endothelial cell procoagulant activity.^41,49,55 Infections with viruses can alter other aspects of the ability of endothelial cells to maintain blood in an unclotted state. These effects include inhibition of plasminogen activator, reduced levels of endothelial thrombomodulin, and loss of heparin sulfate proteoglycan, among other factors that regulate thrombosis.⁵⁶ Little information exists regarding the effects of Chlamydia infection on the regulation of endothelial cell functions related to blood clotting. Like other Gram-negative bacteria, Chlamydia species release a lipopolysaccharide endotoxin. The ability of C pneumonia–derived lipopolysaccharide to activate endothelial functions that promote blood clotting, as in the case of Escherichia coli–derived endotoxin, remains unknown.

Ability of infectious agents to alter growth of vascular smooth muscle cells. The quest for Herpesviridae in human atheroma emerged from the concept that proliferation of a single virally transformed smooth muscle cell could account for the apparently monoclonal expansion of these cells in arteriosclerotic lesions.^57,58 Indeed, herpesvirus can transform rabbit aortic smooth muscle cells. Such cell lines can become "immortalized," in the cell biologist's parlance, and thus escape usual growth control mechanisms.⁵⁹

Recent evidence has suggested another specific manner in which CMV might influence arterial lesions. CMV infection can result in cytoplasmic sequestration of the tumor suppressor p53, perhaps because of complex formation with the viral immediate early gene product IE84.⁶⁰ Infection with human CMV protects human endothelial cells from apoptosis resulting from serum lack.⁶¹ In this manner, CMV-infected vascular cells might evade a usual check on proliferation.

Few or no data in the literature support a role for bacterial infection, including C pneumoniae, in the modulation of vascular cell growth or transformation.

Effects of infectious agents on lipid metabolism. Infection of chickens with Marek's disease virus promotes cholesterol accumulation in the arterial wall. Herpes simplex virus infection decreases lysosomal hydrolysis of cholesterol esters by the acidic cholesteryl ester hydrolase in vascular smooth muscle cells.^39,62,63 Herpesvirus infection also decreases the production of prostacyclin and other arachidonic acid metabolites. The consequent reduction in adenyl cyclase activity and cAMP concentration lowers the activity of protein kinase A, decreases the activity of the cytoplasmic cholesteryl ester hydrolase, and promotes accumulation of intracellular cholesteryl esters.⁶³ Little evidence supports a role for infection by bacteria, including C pneumoniae, in the modulation of lipid metabolism in vascular cells.

Alteration of cytokine and other mediator production by infectious agents. Vascular cells, including smooth muscle, endothelium, and leukocytes, commonly found in atherosclerotic lesions can produce as well as respond to cytokines.⁶⁴ These protein mediators of inflammation and immunity can alter many vascular functions related to atherosclerosis and restenosis. CMV infection in vitro can augment macrophage production of messenger RNAs encoding the cytokines interleukin-1ß, tumor necrosis factor-, and macrophage-colony–stimulating factor.⁶⁵ Likewise, infection of mononuclear cells by Herpes simplex or Epstein-Barr virus augments cytokine production.⁶⁶ CMV can induce tumor necrosis factor- expression in human monocytes.⁶⁷ CMV infection can also alter cytokine production by endothelial cells.⁶⁸

Bacterial lipopolysaccharides are classic activators of cytokine production from mononuclear phagocytes as well as intrinsic vascular wall cells. Ample evidence supports the role of bacterial endotoxins as regulators of production of such cytokines as interleukin-1 and tumor necrosis factor from human vascular endothelial and smooth muscle cells.^69–71 Most of these studies have used endotoxins derived from various strains of E coli. The effects of lipopolysaccharides derived from Chlamydia species has not been tested in this regard. This is an important issue, because the potent effects of E coli–derived endotoxins on cellular activation cannot necessarily be extrapolated to lipopolysaccharides derived from other bacterial species.⁷²

Induction of leukocyte adhesion molecules by infectious agents and their products. Interactions between leukocytes and the vascular endothelium participate in many forms of vascular disease, including aspects of atherogenesis. E coli endotoxin and cytokines typically released from endotoxin-stimulated cells augment the expression by endothelial cells of many of the adhesion molecules implicated in atherogenesis, including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and P-selectin. Similarly, infection of endothelial cells with CMV can induce the expression of endothelial-leukocyte adhesion molecules such as intercellular adhesion molecule-1.⁷³ Endothelial cells infected with herpesvirus hominis express a monocyte receptor.⁷⁴ By augmenting the expression of such leukocyte adhesion molecules, bacterial and viral infection could influence this crucial aspect of atherogenesis.

Systemic and Indirect Effects of Infectious Agents in Arterial Diseases
In addition to the direct effects of infection and microbial products on vascular functions, various systemic effects of infection may influence atherogenesis and its manifestations (Fig 2).

View larger version (40K): [in this window] [in a new window]   Figure 2. Indirect effects of infectious agents on intrinsic vascular wall cells. Microbial agents may infect leukocytes, including macrophages and lymphocytes, already present in evolving atheroma. Infected leukocytes may be activated to express maladaptive functions such as those depicted, which may promote lesion evolution. Note potential cross talk between leukocytes and intrinsic endothelium and smooth muscle cells.

Potential effects of infections on atherogenesis mediated by the acute-phase response. The host response to infectious agents usually involves a change in the program of hepatic protein synthesis. The cytokine interleukin 6 may mediate much of this switch from production of "housekeeping" proteins, such as albumin, to greater synthesis of proteins collectively known as "acute-phase reactants," some of which may influence atherogenesis. Augmented production of fibrinogen and plasminogen activator inhibitor during the acute-phase response could promote thrombosis. Increased production of serum amyloid A protein can alter the potential function of HDL in cholesterol export from atheromatous lesions and in coronary risk.⁷⁵ Also, systemic infections can decrease HDL cholesterol concentrations.⁷⁶

Systemic infection and triggering of acute coronary events. An ample literature links infectious processes and acute myocardial infarction.⁷⁷ The foregoing section examined ways in which the acute-phase response to such infections might promote atherogenesis or thrombosis. This section will examine how other aspects of the pathophysiology of acute infection might trigger acute coronary events. Physical disruption of atherosclerotic plaques commonly precipitates the acute coronary syndromes.⁷⁸ Plaque disruption in turn depends on a balance of the anatomic substrate (the so-called "vulnerable" plaque) and the biomechanical forces brought to bear on the atheroma. Acute infections might alter these biomechanical variables. For example, the tachycardia and increased cardiac output that accompany many acute infections and febrile illnesses could increase the wall stress experienced by an atheromatous plaque. This could trigger coronary events by promoting disruption of vulnerable plaques.

Systemic endotoxemia and atherogenesis and acute coronary events. Bacterial endotoxins profoundly influence local functions of vascular wall cells. During Gram-negative bacterial sepsis, circulating endotoxin provokes profound alterations in physiology. In addition to hypotension and decreased systemic vascular resistance, endotoxemia diffusely activates the endothelium, promoting the development of the disseminated intravascular coagulation that commonly accompanies Gram-negative sepsis.^79,80 Reduced cardiac perfusion due to the hypotension associated with sepsis and/or an augmented susceptibility to clot formation due to endothelial activation could also precipitate acute coronary events.⁸⁰

Even if episodes of endotoxemia or systemic infection do not provoke acute coronary events, they may influence local vascular functions related to atheroma formation and evolution. We hypothesized that local "echos" of systemic endotoxemia or cytokinemia may evoke a wave of heightened local cytokine production in the atheroma by resident cells and macrophages.^81,82 Experimental support for this notion derives from studies in which systemic administration of endotoxin to rabbits with diet-induced atheroma yielded increased cytokine gene expression in the aorta in relation to the amount of preexisting atheroma.⁸¹ In this manner, episodes of infectious illness could lead to a crisis in the evolution of atheroma-enhancing lesion development.

Indeed, serial angiographic studies indicate that human atheromas do not progress linearly in time.⁸³ Rather, these lesions appear to undergo periodic spurts of growth, as determined by luminal encroachment on angiograms. Many of these transient episodes of plaque enlargement may result from disruption and healing. In some cases, systemic infections might promote these episodes of plaque disruption by inflammatory or infectious activation of vascular wall cells or lesional leukocytes (Figs 1 and 2). In other cases, the locally induced burst of cytokine production due to infection might promote plaque growth even in the absence of physical disruption.

	The Weight of the Evidence: Are Koch's Postulates Fulfilled?

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
More than a century ago, Robert Koch proposed three criteria that should be met to link an infectious agent causally with production of a disease. Koch used these postulates to implicate the tubercle bacillus as the culpable microbe in tuberculosis. Does the present state of knowledge of infectious agents in relation to atherosclerosis and restenosis fulfill these criteria? Let us consider Koch's original postulates in turn.

The Causative Organism Can Be Isolated From the Affected Host
Attempts to culture infectious viruses from atheroma have generally failed. Likewise, the propagation of C pneumoniae from human atherosclerotic lesions is anecdotal. The presence of antigens associated with these infectious agents and/or nucleic acid characteristic of these agents is not an explicit criterion of Koch's postulates. Although human atheromas often contain such evidence of the presence of infectious agents, many cases lack these indirect indications of the presence of the agents. Moreover, even if infectious particles are present within the lesions, a pathogenic role is far from established.

The Infectious Agent Can Be Identified by Culture or Directly by Microscopy
In the case of CMV and Chlamydia, the agents are extremely well characterized with a good deal of information about their biology. This criterion is met not only in vitro but often in situ within lesions, as noted above.

	On Transfer to a Susceptible Host, the Infectious Agent Must Produce the Disease

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
In the case of Marek's disease in chickens, this criterion has been approached. At present, however, Koch's postulates in regard to an infectious cause of atherosclerosis or restenosis remain unfulfilled in humans.

It could be argued that Koch's postulates are irrelevant to a chronic disease that usually develops over decades. For example, previous infection with CMV or another virus could alter the function of cells permanently without persistence of the agents. Such a hit-and-run mechanism seems to apply to certain experimental tumors associated with herpesvirus infection. In contrast with the clarity of the epidemiological links between traditional risk factors for coronary heart disease (eg, hypercholesterolemia and hypertension), however, the evidence regarding involvement of Chlamydia and Herpesviridae in atherosclerosis remains rudimentary at present.

A possible instance in which an infectious agent could cause a disease without meeting Koch's postulates would be if it induced an immune response by antigenic mimicry. Another complexity not envisaged by Koch would be the dependence of the histocompatibility haplotype of an individual for susceptibility to infection. Particularly with viral agents, it is conceivable that only a subgroup of the population at large that had certain HLA haplotypes would be susceptible to the viral infection. This could explain lack of disease in some individuals exposed to the putative pathogen. Conversely, an equally vigorous argument to the contrary could be made. One could hold that herpesviruses, including CMV, are virtually ubiquitous. Their presence in atherosclerotic lesions may have no relationship whatsoever to disease production. Likewise, one could conceive that Chlamydia localizes more commonly in atherosclerotic lesions than in uninvolved arteries merely because of the presence of macrophages, which could harbor an innocent persistence of this common microbial pathogen.

Indeed, some combination of these contrasting situations could pertain to human atherosclerosis. In some cases, a viral arteritis could furnish the substrate for an atheroma, decisively setting the stage for its subsequent development, perhaps in combination with traditional risk factors such as hypercholesterolemia. The potentiating effect of an atherogenic diet from the development of arterial disease in chickens infected with the Marek's disease virus provides an example of such a mechanism. In the case of Chlamydia, persistent infection of lesional macrophages might potentiate atherogenesis by heightening cytokine and growth factor release from the phagocytic cells within the atheroma. Thus, the Chlamydia infection, although not a primary pathogenic stimulus, could hasten development of atheroma.

Although we must conclude that no infectious agent meets Koch's original postulates at present, we need not therefore discard the notion that infectious agents play a pivotal role in atherogenesis in some situations in which infections, when present, may promote the atherogenic process.⁸⁴ Indeed, H pylori does not meet Koch's postulates as a causative organism for duodenal ulcer.⁸⁵ Various workers have argued persuasively that Koch's postulates may lack sufficient sensitivity to reject a causal link between an infectious agent and a given disease.⁸⁶

	Unanswered Questions and Areas for Future Research

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 

Laboratory Investigations of the Vascular Biology of Infectious Agents in Relation to Atherogenesis and Restenosis
A great deal of in vitro work has probed potential mechanisms whereby Herpesviridae, including herpesvirus hominis and CMV, can alter the functions of vascular wall cells in ways that may promote atherosclerosis or restenosis. Yet a number of fundamental issues remain relatively unexplored. It is unknown how herpesviruses enter the arterial wall. A potential mechanism could involve entry into the artery wall, with blood monocytes known to accumulate in these lesions. Under ordinary circumstances, monocytes are minimally permissive for herpesvirus infection. However, differentiation of mononuclear phagocytes toward the macrophage phenotype by such agents as vitamin D₃ can augment virus yield from infected cells. The general inability to culture herpesvirus from atheromatous lesions may result from limited permissivity of vascular cells for virus replication. However, in some otherwise nonpermissive cell types, exposure to growth factors, such as fibroblast growth factor, can enhance viral gene expression. Smooth muscle cells in atherosclerotic lesions and in injured arteries may encounter fibroblast growth factors. The interactions between growth factors and smooth muscle cells might thus influence their interaction with herpesviruses in ways that might be important to the evolution of vascular lesions. Systemic factors might also influence the activity of latent herpesviruses in atherosclerotic lesions. For example, glucocorticoids, hormones released during stress, can enhance transcription of herpesvirus latent in cells.⁸⁷ Thus, a quiescent herpesvirus infection might be reactivated by such a mechanism, much as glucocorticoids or stress can reactivate latent Herpes zoster.

Although the interaction of herpesviruses, including CMV, in the artery wall has received much attention in the past, study of the vascular effects of Chlamydia has barely begun. Just as in the case of viruses, it is important to know how Chlamydia enter the artery wall. Could these agents enter in mononuclear phagocytes? Does the degree of differentiation of the mononuclear phagocyte or the stimuli that it may encounter in the vessel wall influence the latency of productivity of the infection? Specifically, would gamma interferon, a cytokine found within atheroma, favor a persistent infection of macrophages as in the case of certain transformed cell lines? What effects do the lipopolysaccharides produced by Chlamydia have on vessel wall cells and macrophages? Could major chlamydial proteins, such as the major outer membrane protein or heat shock protein, serve as antigens for a cellular or humoral immune response and thus add to the chronic immune response ongoing in atherosclerotic lesions?

Need for In Vivo Animal Models to Study the Interaction Between Infectious Agents and Atherosclerosis
As described above, the scientific foundation of the relationship between infection and atherosclerosis rests on the convincing data emerging from the avian herpesvirus infection, Marek's disease in chickens. Short of satisfying Koch's postulates in humans, the notion that infectious agents can provoke or potentiate arterial disease could benefit enormously from development of further animal models, particularly in mammalian species.

The advent of genetically modified mice susceptible to atherosclerosis, such as those lacking apolipoprotein E or the low-density lipoprotein receptor, has ushered in a new era of animal investigation of atherosclerosis. These new animal models provide lesions that share many more features of human atherosclerosis than previously achievable in rodents. Although differences in species permissivity require consideration, attempts to study the interaction of infectious agents with atheroma formation in these novel animal preparations seems worthy. The ability to infect apolipoprotein E–deficient mice with C pneumoniae provides support for this particular avenue toward development of relevant animal models.³⁸ Doubtless, other species would be appropriate for testing various hypotheses regarding the interaction of infectious agents and atherosclerosis and restenosis, including rabbits, swine, and nonhuman primates.

Clinical Studies Regarding Infectious Causes of Atherosclerosis and Restenosis
A large database of seroepidemiological studies, reviewed above, has established grounds for a clinically relevant link between Chlamydia and CMV with atherosclerosis and, in the case of CMV, with restenosis after angioplasty. In the realm of seroepidemiological studies, increasing the number of surveys of relatively small numbers of patients at a single center is not likely to provide further illumination. Retrospective studies are subject to well-known biases. Moreover, it is difficult to compare results across various seroepidemiological studies because of differences in reagents and assay protocols and in patient selection. A standardized set of reagents, protocols, and perhaps a core testing laboratory would facilitate future seroepidemiological studies. In particular, studies of geographically diverse populations with differing risks for atherosclerosis could provide new insight in testing of the relationship between infectious diseases and atherosclerosis. Ultimately, a well-designed and sufficiently powered prospective cohort study relating evidence for infection to future risk of cardiovascular disease and controlling for confounding variables (eg, smoking) would help to strengthen the seroepidemiological evidence for a causal role of infectious agents in aggravating atherosclerotic disease. Use of stored samples from previous clinical studies provides another potential source of materials for further study in this context.

Some have proposed consideration of a clinical trial of antibiotic therapy after coronary events as end points. Such studies would test the hypothesis that bacterial agents such as Chlamydia, for which effective antibiotic therapies exist, contribute to coronary events. A number of outstanding issues require resolution before we embark on such therapeutic trials. Criteria for defining a subset of patients with evidence of Chlamydia infection would prove helpful in targeting individuals who enter into a secondary prevention trial. Questions regarding the dose, duration, and choice of antibiotic agent would require considerable deliberation. Likewise, vaccination might provide a way to limit infection with microbial or viral agents implicated in atherogenesis.

However, a therapeutic trial of antibiotics still would not establish a causal relationship between any particular infectious agent, eg, Chlamydia, and atherosclerosis. Nonspecific effects of the antibiotic agent used in such a trial might influence the outcome. For example, tetracyclines can inhibit metalloproteinases, which may contribute to acute coronary syndromes. Other unexpected or unknown effects of antibiotics might confound conclusions regarding causality even in a successful therapeutic trial. Moreover, most antibiotics target a number of microorganisms, rendering conclusions about the causal contribution of any one susceptible organism difficult. Yet, if antibiotic treatment or vaccination could reduce atherosclerotic events, the public health implications could be enormous; hence the need to keep an open mind and seek further information through continued research focused on the relationship between infectious agents and vascular diseases. Certainly, increased understanding of the basic vascular biology of infection in relation to arterial diseases and creation of relevant animal models would facilitate resolution of these issues.

	Summary and Assessment

Top Introduction Evidence Supporting Involvement... Chlamydia Chlamydia Herpesviridae, Including CMV... Direct Infection of Cultured... Effects of Infection and... The Weight of the... On Transfer to a... Unanswered Questions and Areas... Summary and Assessment References

 
Observational studies in humans provide intriguing hints relating infectious agents such as certain viruses and bacteria to atherosclerosis, coronary events, or restenosis after arterial intervention. A number of recent seroepidemiological and pathological studies support an association between C pneumoniae and coronary atherosclerosis. Also, emerging data have rekindled interest in the role of CMV in arterial diseases such as restenosis. A large body of in vitro work provides a pathophysiological foundation for a contribution of Herpesviridae, including CMV, in vascular pathology. Information regarding C pneumoniae at the basic science level remains scant. The Marek's disease in chickens model provides strong experimental support for in vivo potentiation of cholesterol-induced fibrofatty lesion formation in arteries. Similar data regarding the relationship between bacterial infection and atherosclerosis have not yet emerged.

The pressing question that remains unanswered is whether infections are a cause, a cofactor, or a commensal of no pathological import in the context of atheroma.⁸⁸ Only further basic science and clinical research can shed light on this issue and promote a better understanding of the roles of infection in arterial disease. Perhaps such efforts will aid in the identification of a population appropriate for particular surveillance or a specific intervention such as antibiotic therapy.

Note added in proof
Since acceptance of this manuscript, Danesh et al published a related review,⁸⁹ and two intriguing preliminary studies showing reduction in recurrent coronary events in patients treated with macrolide antibiotics have appeared.^90,91

	Acknowledgments

 
The authors acknowledge the members of the special emphasis panel, who each contributed to the concepts explored in this paper, including Drs James T. Grayston, James Melnick, David P. Hajjar, Steven E. Epstein, Hannah Valantine, George Byrne, Priscilla Schaffer, Gregory Vercellotti, Maurice Nachtigal, Lewis H. Kuller, Gail H. Cassell, and David S. Siskovick. We thank Drs Amir Kol and Paul Ridker for critical reading of the manuscript.

	Footnotes

 
¹ The authors convened a special emphasis panel on September 19, 1996, under the auspices of the National Heart, Lung, and Blood Institute to examine the role of infectious agents in atherosclerosis and restenosis. The members of the panel included James T. Grayston, James Melnick, David P. Hajjar, Steven E. Epstein, Hannah Valantine, George Byrne, Priscilla Schaffer, Gregory Vercellotti, Maurice Nachtigal, Peter Libby, and Lewis H. Kuller. Invited discussants included Dr Gail H. Cassell and Dr David S. Siskovick. The information and views expressed in this article are based on the deliberations of the panel, edited and commented on by the authors. The views expressed in this document do not constitute an official position of the panelists, the National Heart, Lung, and Blood Institute, the National Institutes of Health, or the US Government.

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