very common. The organisms develop a persistent orlatent state and may become reactivated by appropri-ate stimuli. The association with increased coronaryheart disease and stroke risk is strongest for C pneu-moniae. A meta-analysis of 18 serologic studies hasnot supported that H pylori play an active role in coro-nary heart disease.3 The prevalence of periodontal dis-ease increases with age, and from one third to one halfof the elderly population are affected by moderate tosevere periodontal disease.4

Pathologic evidence of multipleinfections in atheromas

There have been many studies on the detection of Cpneumoniae directly in diseased and normal arteries bythe use of various pathologic techniques. The numberof positive specimens ranges from 28% to 100% inatheromatous vessels.5 C pneumoniae is rarely detectedin normal arteries.6 On the other hand, cytomegaloviruswas detected in significantly high proportions of bothatheromatous and nonatheromatous blood vessels.5,7

However, H pylori DNA was not detected in any of the50 patients with abdominal aortic aneurysm atheromas.8

By using an immunocytochemical staining method

An infection hypothesis of atherosclerosis was formulated about a century ago, but it was not until1978 that Fabricant et al1 drew renewed interest toinfection and atherosclerosis when they showed thatgerm-free chickens infected with avian herpesvirusdeveloped arterial lesions that resembled humanatherosclerosis. Associations have since been reportedbetween human atherosclerosis and chronic infectionswith Chlamydia pneumoniae, Helicobacter pylori,herpesviruses (cytomegalovirus and herpes simplexvirus [HSV]), and chronic dental infection.2 Asreviewed by Danesh et al,3 H pylori, C pneumoniae,cytomegalovirus, and HSV share some common charac-teristics. The proportion of adults in developed coun-tries who have antibodies is about one half. The proba-ble mode of spreading is through the aerodigestivetract. Infections may be acquired at an early age withmost of the infections asymptomatic and reinfection

From the Department of Laboratory Medicine and Pathobiology, St Michael’s Hos-pital and University of Toronto.Request reprints: Dr Brian Chiu, Department of Laboratory Medicine–Pathology, StMichael’s Hospital, 30 Bond St, Toronto, Ontario, Canada, M5B 1W8.Copyright © 1999 by Mosby, Inc.0002-8703/99/$8.00 + 0 4/0/101755

Multiple infections in carotid atherosclerotic plaquesBrian Chiu, MD Toronto, Canada

Background Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and recently, periodontal disease, havebeen associated with human atherosclerosis. Porphyromonas gingivalis and Streptococcus sanguis are major pathogens associ-ated with periodontitis, a common chronic inflammatory condition in adults. Investigators have found that these infectious agentsmay influence vascular cell functions by inducing thrombus formation, vascular cell proliferation, apoptosis, and cell death.

Methods and Results The main purpose of our study was to investigate the relation between the presence ofmultiple infectious agents in human carotid endarterectomy specimens and pathoanatomic features of the correspondingcarotid plaques. Histologically, plaque rupture of the fibrous cap and communication of the luminal thrombus with the centralnecrotic lipid core was seen in or at proximity to the macrophage-rich shoulder (unstable plaque region). Thrombus withinthe lipid core without plaque rupture was occasionally found near the internal elastic lamina, associated with increased vascu-larity and lymphocytic infiltrate. Apoptosis, as detected by both the immunohistochemical staining of apoptosis-related proteinsand in situ labeling of internucleosomally degraded DNA, was common in atherosclerotic plaques. Immunostainings forC pneumoniae, cytomegalovirus, herpes simplex virus-1, P gingivalis, and S sanguis were positive in the carotid plaques.From 1 to 4 organisms were found in the same specimen. The micro-organisms were immunolocalized in plaque shouldersand lymphohistiocytic infiltrate, associated with ulcer and thrombus formation, and adjacent to areas of strong labeling forapoptotic bodies.

Conclusions Our data provide evidence that multiple infectious agents may be found in atherosclerotic plaques,and sometimes in the same specimen. The current study is the first to report the detection of 2 major odontopathogens,P gingivalis and S sanguis, in atherosclerotic plaques. The immunolocalization of these micro-organisms within unstableplaque regions and their association with plaque ulceration, thrombosis, and apoptosis in vascular cells are intriguing.Multiple infectious agents may alter vascular cell function and provide a “trigger” for acute ischemic stroke events. Furtherevidence from human studies and animal models will be needed. (Am Heart J 1999;138:S534-S536.)

American Heart JournalVolume 138, Number 5, Part 2 Chiu S535

with specific monoclonal antibodies in a study of 76carotid atherectomy specimens, we detected the pres-ence of C pneumoniae, cytomegalovirus, and HSV-1. Atleast 1 of the micro-organisms was detected in 59(77.6%) of the diseased arteries, a single micro-organ-ism in 35 (46%), 2 micro-organisms in 18 (23.7%), andall 3 micro-organisms in 6 (7.9%) of the diseased arter-ies. The presence of C pneumoniae or cytomegaloviruswithin the plaque was associated with plaque thrombo-sis, which suggests that the presence of these micro-organisms may influence plaque morphology.9 Therehas not been any published report on the detection ofperiodontal pathogens in diseased arteries.

Unstable atherosclerotic plaque andinfections

Recent attention has focused on the mechanisms ofprogression of atherosclerotic plaques with plaque insta-bility and disruption and the subsequent thrombotic com-plication leading to acute coronary syndrome or strokeevents.10,11 Multiple mechanisms are likely to be involvedin plaque thrombosis. The determinants of plaque vulner-ability to rupture and thrombosis are thin fibrous cap,low smooth muscle cell count, high macrophage content,and large lipid core.12 Intraplaque neovascularization isoften seen associated with plaque macrophages.13 Capil-lary rupture within the plaque may be a causative factorin coronary thrombosis.14 Apoptosis, or programmed celldeath of vascular cells within plaques, is thought to play akey role in contributing to plaque instability.15,16 Matrix-degrading metalloproteinases (MMP) from plaque

macrophages have been implicated in plaque rupture.17

The presence of C pneumoniae in vascular cells withchlamydial and human heat shock protein 60 inducestumor necrosis factor-α and MMP production and mayprecipitate acute ischemic events.18 During periodontitis,periodontal pathogens may disseminate through theblood to infect vascular cells. The lipopolysaccharidecomponents of these micro-organisms evoke a systemicantibody response.19 Porphyromonas gingivalis, a majorodontopathogen, can actively invade human endothelialcells and activate latent host MMP, and P gingivalis pro-teases are essential for both processes.20,21 In an in vivomodel of thrombosis, certain dental plaque bacteriainduce platelets to aggregate by bacterial surface plateletaggregation–associated protein, triggering an acutemyocardial ischemic event.22

Methods and results

To investigate the relation between the presence ofmultiple infectious agents in human carotid atheroscle-rotic plaques and pathoanatomic features of the corre-sponding carotid plaques, 33 human carotid atherectomyspecimens were studied histologically. Plaque rupture of the fibrous cap and communication of the luminalthrombus with the central necrotic lipid core was seen in or at proximity to the macrophage-rich shoulder(unstable plaque region). Thrombus within the lipid corewithout plaque rupture was occasionally found near the internal elastic lamina, associated with increasedneovascularity and lymphohistiocytic infiltrate. Immuno-stainings with specific monoclonal antibodies against

Multiple infections in atherosclerosis (proportion in percentagesof micro-organisms in 33 human carotid atherosclerotic plaques).C.p, C pneumoniae; CMV, cytomegalovirus; HSV, herpesvirus-1;P.g, P gingivalis; S.s, S sanguis; org, organism(s).

Interaction of multiple infections in atherosclerosis in the initiation,development, progression, and acute coronary/stroke syndromestages of atherosclerosis, with emphasis on plaque disruption trig-gering an acute ischemic event: angiogenesis, apoptosis, andMMP.

Figure 1 Figure 2

American Heart JournalNovember 1999ChiuS536

C pneumoniae, cytomegalovirus, and HSV-1 and poly-clonal antibodies against P gingivalis and Streptococcussanguis (from R. Genco and R.T. Evans’ laboratory atNYSU at Buffalo) were positive for C pneumoniae(63.6%), cytomegalovirus (42%), HSV-1 (9%), P gingi-valis (42%), and S sanguis (12%) in the carotid plaques.From 1 to 4 organisms were found in the same specimen(30%, 24%, 21%, and 6%, respectively) (Figure 1). Themicro-organisms were immunolocalized in plaque shoul-ders and lymphohistiocytic infiltrate, associated withulcer and thrombus formation. None of the 15 controlpatients showed these changes. Apoptosis, as detectedby both the immunohistochemical staining of apoptosis-related proteins and in situ labeling of internucleosoma-lly degraded DNA (TUNEL method), was common in atherosclerotic plaques. The micro-organisms were im-munolocalized mostly in macrophages and adjacent toareas of strong labeling for apoptotic bodies.

Discussions and future directions

In the current study, we can detect and localizemultiple infectious agents, including for the first time 2odontopathogens, P gingivalis and S sanguis, in theatherosclerotic plaque. It is conceivable that multiplemicro-organisms may interact with each other23-26 aswell as with host vascular cells in a synergistic and/orinhibitory manner and may play a role in the initiation,development, and progression stages of atherosclerosis.Our data further suggest that the presence of thesemicro-organisms may influence atherosclerotic plaquemorphology, predisposing to plaque disruption, trigger-ing an acute coronary syndrome or ischemic stroke.Thus 3 major factors have to be taken into considerationin the determinants of an unstable plaque, namely: mul-tiple micro-organisms localized in the plaque, increasedapoptotic bodies of vascular cells, and increased neovas-cularity. There are potential implications of multipleinfections in the plaque in both animal studies and clini-cal trials of primary and secondary prevention to takeinto account the potential interactions of multiple infec-tions and with the vascular cells (Figure 2).

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