249

The role of the infectious agents in the pathogenesis and evolution of atherosclerosis

Claudio Blasi

molecules (“reactive oxygen species” or “superoxides”).

Such ions have a strong tendency to react with proteins caus-

ing serious damage: the so-called “oxidative stress”4-6.

Endothelial dysfunction, constituting a reaction to the

damage4,6,7, is the result. The transfer of information con-

tained in specific genes that control this reaction on mes-

senger RNA molecules is activated by means of a nuclear

transcription factor (NF-κB). This leads to the synthesis

of molecules (cytokines, factors of attraction and adhesion

to the endothelial surface of lymphocytes, monocytes,

etc.) that initiate the inflammatory process and reduce

the antithrombotic properties of the surface4,6. Also acti-

vated is the production of a protein called “heat shock pro-

tein” (HSP) that has a very important part in the role

played by infectious agents. Its exposure to the surface of

the endothelial cells is one of the antigenic stimuli acti-

vating the immune component of atherosclerosis (the

other are low-density lipoproteins [LDL] that have pen-

etrated into the subendothelial space due to the increased

permeability and have been oxidized)8.

Aside from describing the microorganisms involved, we

will also illustrate the mechanisms by which they are

supposed to act and the possible therapeutic and preven-

tive interventions.

U.O. Centro Prevenzione e Cura del Diabete (Responsabile: Dr. ClaudioBlasi), ASL RMB, 1° Distretto di Roma

The Italian version of this article is available addressing to the author.© 2004 CEPI Srl

Atherosclerosis is a chronic inflammatory process due to the endothelial reaction to stress riskfactors, only some of which are known. Clinical and experimental observations have suggestedthat several infectious agents are involved in this process. These agents, particularly the germChlamydia pneumoniae, and their relationship to the atheromata are described. Two hypothesesconcerning how these infectious agents act are suggested. Both hypotheses are based on thecapacity of these agents to induce the production, by endothelial cells, of the so-called heat shockprotein (HSP), one of whose characteristics is to provoke an immune system reaction: 1) induc-tion of a cross immune reaction, due to “molecular mimicry”, between the HSP of infectious ori-gin and the one that is produced by the endothelium as a consequence of stress due to the risk fac-tors; 2) infection of the endothelial cells, followed by the synthesis and exposure on their surfaceof the HSP and activation of innate immune surveillance. Numerous experimental studies havebeen performed and are still being performed with the aim of verifying the efficacy of antibiot-ic treatment in preventing or reducing the rate of acute cardiovascular events. The results are stillinconclusive. Probably, to be effective, treatment should be started at an earlier age. Preventionthrough vaccination against the involved microorganisms and the consequent induction ofimmune tolerance toward the HSP is also being investigated. As the mechanisms of action of infec-tious agents are further clarified, effective therapeutic and preventive measures could be takenwith important clinical spin-offs.(Ann Ital Med Int 2004; 19: 249-261)

Key words: Atherosclerosis; Chlamydia pneumoniae; Cytomegalovirus; Heat shock protein;Infectious agents.

Introduction

Atherosclerosis is a chronic pathology of the internal wallof the artery where inflammatory processes are present1.It is a complex process with diverse etiologies (risk fac-tors) acting simultaneously on the endothelium through twomain mechanisms: bio-mechanical stress and immunereaction. The natural chemical factors (such as smoking,dyslipidemia, hyperglycemia, etc.) as well as mechanicalfactors (e.g., high blood pressure) are well known. Inabout one third of the cases, conventional risk factors areabsent. This leads us to suppose that other yet unknownfactors may play an important role2. Among them, inrecent years, a number of infectious agents – both viral andbacterial – have been considered. They could act by meansof a not yet completely understood complex mechanismlinked to damage to and reaction of the endothelium3.According to the prevalent interpretation of the origin ofatherosclerosis, in fact, the various risk factors cause anincrease, within the endothelial cells, of ionized oxygen

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

250

Origins of the infective hypothesis for atherosclerosis

The idea that atherosclerosis could be related to an infec-tion is over a hundred years old. This hypothesis hadalready been made at the end of 1800 in Europe, and wasagain put forth at the turn of the century by Osler and otherAmerican physicians9,10. In the 1970s, the discovery thatcells present in the plaque were of monoclonal origin ledto the supposition of the action of cell transformationfactors such as viruses11. Moreover, researches were ableto induce atherosclerotic lesions in chickens infected withherpes virus12. In the 1980s, a group of Finnish researchersnoted a significant increase in antibodies against a bac-terium, Chlamydia pneumoniae, in subjects at greaterrisk of acute coronary events13. Numerous studies fol-lowing these first observations confirmed this relationshipbetween infectious agents and atherosclerosis14.

It is interesting to note that the association is withmicroorganisms that share two characteristics: they areobligate intracellular prokaryotic pathogens and are ableto provoke chronic infections with periodic reacutiza-tions that are accompanied by systemic immune reac-tions15. Diabetes mellitus, of which atherosclerosis is thechief complication, could constitute a favorable condition,because it also alters the defense capacity of the organismto infections and increases the number of infectiousprocesses which it undergoes (“increased infectious bur-den”)3,16.

The infectious agents involved

Before making a detailed examination of the agentsimplicated, the concept of “infectious burden” should beclarified. It has been observed that the greater the numberof microorganisms to which an individual has beenexposed, the greater the impact of the infectious factor onatherogenesis3,17. This suggests either a possible synergismbetween the agents on the initial and subsequent devel-opment of lesions, or an accumulative effect of the infec-tions that cause endothelial dysfunction and inflammationto precipitate3,18.

Viruses

Genus: herpes. The role of these viruses in man has notyet been well-defined, even though there is conclusive epi-demiological evidence of their involvement19. Other evi-dence includes: the presence, in the atherosclerotic lesions,of their antigens or of amino acid sequences of their DNA(although the same viral tracks have been found in non-involved aortic tissue)20; the demonstration of their capac-ity to provoke atherosclerosis in animal models, and,finally, in vitro experiments that have induced inflam-

matory and prothrombotic responses in infected endothe-lial cells19. Their infection amplifies the effect of hyper-cholesterolemia in experimental models21.

Cytomegalovirus. This virus is implicated in both thepathogenesis of systemic atherosclerosis and in athero-sclerosis subsequent to some types of heart surgery.

Many studies have demonstrated a significant associ-ation between the presence of antibodies against the virus,the incidence of atherosclerosis22,23 and the severity ofcoronaropathy24. In experimental models with elevatedcholesterolemia, infection with this virus induces aorticlesions similar to those observed during the first phases ofthe atherosclerotic process22,25. Such an effect could bebrought about either by its capacity to infect the endothe-lial cells and the smooth muscle cells (where it couldpersist for a long time in a latent form26) or could bemediated by a mechanism of immune origin27. The sec-ond possibility seems more probable22. In fact, human anti-bodies against viral peptides provoke a cross-reactionwith HSP-60 molecules present on the surface of stressedendothelial cells and contribute to the process of endothe-lial dysfunction28.

With regard to pathology following surgery, the viruscould be responsible for the proliferation of the smoothmuscle cells leading to restenosis after angioplasty. In fact,it has been observed that 43% of the patients who areseropositive for the virus have this complication as opposedto 8% of those who are seronegative22,29. Moreover, a cor-relation between antibody levels and the appearance of arapidly-progressive particular form of atherosclerosis inthe coronary arteries of transplanted hearts (“transplantationcardiac vasculopathy”)30 has been observed; it is charac-terized by diffuse and concentric intimal hyperplasia asso-ciated with proliferation of the smooth muscle cells andinflammation of the arterial wall31. This is the leading causeof heart transplant failure32. Moreover, transplanted patientspreviously infected by the virus have a significantly greaterlumen stenosis than those who have not had the infection31.

Two hypotheses have been proposed: either a directaction of the virus on the vessel wall or an action medi-ated by the formation of antigens on the endothelial sur-face that could further stimulate the rejection reaction31,32.In any case, prophylactic action with a drug that inhibitsviral replication for 28 days following transplant had low-ered the incidence (compared to treatment with a place-bo) of this form of atherosclerosis33.

Herpes simplex type 1 and type 2. A strong associationhas been noted between the presence of antibodies againstthis virus and coronaropathy (particularly among smok-ers)21.

Claudio Blasi

251

Epstein-Barr virus. The presence of antibodies againstthis virus is significantly associated with cardiovascularevents34. It is an obligate intracellular pathogen that per-sists in B lymphocytes for the patient’s entire lifetime,although it is not present in the plaques.

Hepatitis C and A virus. Seropositivity for hepatitis Cand hepatitis A virus is associated with the carotid plaqueand with myointimal carotid thickening independentlyof other risk factors35.

Enterovirus. The presence of antibodies against theseviruses is associated with an increased risk of myocardialinfarction36. The mechanism of action could consist in animmune response provoked by the HSP36 the productionof which is induced by the virus.

Bacteria

Helicobacter pylori. This bacterium, object of one of themost important pathogenetic discoveries in recent years(and emblematic of the difficulties that new ideas arefaced with in being accepted by the medical environ-ment37) also seems to be involved in atherosclerosis. Adirect relationship between an increased concentrationof antibodies against it and cardiovascular diseases hasbeen noticed37,38. Its DNA has been found in carotidplaques39. In this case too, it is suspected that its link toatherosclerosis passes through the immune cross-reac-tion against the HSP produced by the endothelium15. Infact, this bacterium also produces its own HSP, and thepresence of anti-HSP antibodies in patients with thisinfection has been described40. For reasons that are not yetclear to us, the contribution to atherosclerosis seems to beprovided above all by the most cytotoxic Helicobacterpylori species, the one characterized by the presence ofgene A, associated with cytotoxin41. In particular, anassociation between this bacterial species and cerebralictus caused by emboli originating in particularly advancedand unstable carotid plaques has been observed39. Themechanism of this association is not clear. Here too, crossimmune processes are invoked39.

Anaerobic gram-negative bacteria of the oral cavity. Thepathology they provoke, “periodontal disease”, consists ofa chronic, destructive infection of the gums, the connec-tive tissue and the alveolar bone. It causes bone reab-sorption, the formation of gingival pouches, progressivedamage to the fixing apparatus of the teeth and finally theirloss42. The greater risk of coronaropathy in the presenceof untreated periodontitis, with respect to healthy subjects,is 25% (increasing to 70% in men < 50 years)42. The

DARIC study (Dental Atherosclerotic Risk in Com-munities) pointed out the direct relationship between peri-odontal disease and the middle-intimal thickness of thearteries42. These germs produce HSP, and in patients withgingivitis, the level of anti-HSP IgA antibodies in thesaliva is increased43. It is thus hypothesized that the sametype of immune reaction described for other microorgan-isms occurs27,42.

Chlamydia pneumoniae. This is by far the infectiousagent most involved in the pathogenesis of atherosclero-sis44. Consequently, it will be discussed in more detail. Itis an ubiquitous gram-negative bacterium that depends onthe host cell for its survival and development. It is forcedto multiply within the host cell because it is unable to gen-erate the adenosine triphosphate molecule autonomouslyand consequently depends on the cell it occupies for itsenergy requirements45. It is transmitted among humans bymeans of aerosolized droplets and may infect the upper andlower respiratory tracts (atypical pneumonia, sinusitis,pharyngitis, bronchitis); it causes middle ear inflamma-tion46,47 and is at the origin of many cases of adult asth-ma. The infection often causes very mild symptoms (lowgrade fever, cough and hoarseness) or no symptoms at all,and hence, generally goes untreated48. Once an acuteinfectious episode is resolved, it may persist in a latentform47. This depends on the particular vital cycle of thebacterium that consists of an extracellular phase charac-terized by the so-called “elementary bodies” which are sur-rounded by a protective coating; these are metabolically-inactive and may penetrate the host cell and remain alivethere for long periods (with inclusion aspects similar tospores) without replicating. The bacterium may also ini-tiate an intracellular phase of transformation into meta-bolically active but non-infectious “reticulate bodies” thatmodify the host cell physiology to their advantage: theyreproduce by binary fission and again change into “ele-mentary bodies”. After about 72 hours, following celllysis, they may return to the external environment and ini-tiate a new cycle47,49. Still, in the presence of inhibitoryfactors (such as, for example, the immune-mediatedcytokines interferon-gamma and tumor necrosis factor[TNF-α]), the bacterium may remain for a long time inboth a metabolically and infectiously inactive form (“per-sistent bodies”). This form, important because of its path-ogenic mechanism that will be described later, has par-ticular characteristics: a diverse morphology and, from theimmunological point of view, the absence of proteins thatplay the dominant antigenic role, except for the increasedexpression of the HSP, on its external coating49. Thisimmune tactic, which enables it to escape control and

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

252

not be eliminated by the host, explains how the principalcharacteristic of Chlamydia pneumoniae infection is to setoff a chronic infectious state49,50 and how it is able to gaina role in the pathogenesis of atherosclerosis, particularlythe precocious type51. Its natural history, consisting of aprogressive exposition over time, is very similar to that ofthe Helicobacter pylori.

Relationship between the presence of anti-Chlamydia pneumoniae antibodies

and atherosclerosis

The previously-cited observations by Saikku et al.13,which, during the course of the Helsinki Heart Study, high-lighted a significant association between serological posi-tivity for this germ and acute cardiovascular events (50%with acute myocardial infarction and 68% with angina pec-toris with respect to 17% of serologically negative patients),were the first clues to the possible involvement of thismicroorganism in atherosclerosis. As it is a widespreadmicroorganism, many people are repeatedly infected by itduring their lifetime52. The prevalence of seropositivity at20 years of age is 50% and increases with age, reaching, at65 years of age, 80% in men and 70% in women46. The high-er seropositivity in men, as yet unexplainable, correspondssingularly with the higher incidence of atherosclerosisamong males53. This association has been confirmed in 30other studies carried out in 8 different countries54. Themost useful antibodies as reliable indicators of infection (pri-mary, chronic and recurrent) are the IgA that appear after6-8 weeks, like the IgG but, unlike the latter, disappear rapid-ly due to their brief half-life (5-6 days)44. Moreover, in con-trast to the IgG, they permit us to distinguish between a pastand a current infection51,55.

Relationship between hypercholesterolemia and Chlamydia pneumoniae infection

While some experiments showed that it is also possi-ble to provoke atheromatous lesions by infecting animalswith normal cholesterol levels10,52, the pro-atherogenicaction of Chlamydia pneumoniae cannot occur in theabsence of an increase in blood lipoproteins56,57. In ani-mals fed a diet rich in cholesterol, repeated infections viathe nose (to simulate the port of entry of human infection)have induced the rapid development of aortic atheroscle-rosis52,58,59. Treatment with the antibiotic azithromycin, towhich the bacterium is particularly sensitive, initiatedwithin a week after infection60, has impeded this effect59.In man too, a correlation between increased choles-terolemia and the role of the microorganism in the athero-matous process has been observed56,57. The incidence of

atherosclerosis in the tropics, where the diet is poor in fats,is low despite the widespread diffusion of Chlamydiapneumoniae infections61. It is here interesting to note,that while the early phase of atherosclerosis, that is the for-mation of lipidic stria, is widespread throughout the world,its evolution to the sclero-calcific plaque is typical insocieties characterized by diets rich in saturated fats andhigh levels of cholesterolemia54.

Some interpretations have been made concerning thepossible mechanisms that relate hypercholesterolemia toinfection. It is believed that LDL, if present in increasedquantities in the circulation, cross the endothelial barrierwhere they are oxidized due to the action of the oxygen ions(“hypothesis of modified LDL”)14,43,47. HSP, one of the vir-ulence factors of the bacterium, is able to cause oxidationof the LDL by monocytes, even in the absence of super-oxides62. Monocytes, attracted into the subendothelialspace by the molecules produced by the stressed cells,transform into macrophages and incorporate the oxidizedLDL. In macrophages there is normally an accurate equi-librium between the LDL that enter and those that exit. Thisdepends on the genetic control of the activity of the recep-tors for these lipoproteins (the apo-B/E receptor for theLDL)47. On the other hand, the oxidized LDL are capturedby the so-called “scavenger” receptors that cannot be sat-urated, that is that cannot be counter-regulated by an excessof phagocytized oxidized LDL4,49. The macrophages accu-mulate fat and transform into the so-called “foam cells”47.Even the macrophages infected in vitro by Chlamydiapneumoniae lose this capacity to control49, and increase thecaptation and accumulation of LDL47. This effect is inducedby one of the constituents of the external cell membraneof the bacterium, the lipopolysaccharide (LPS)47, andcould be finalized to its survival strategies63.

The oxidized LDL themselves represent, moreover, animportant antigenic stimulus that takes part in the immunereaction at the endothelial level together with that inducedby bacterial HSP14 (to be discussed further on). Accordingto a convincing theory, at the beginning of the process, theimmune-mediated inflammatory reaction provoked bythe microbial antigen and the oxidized LDL dominates thehyperlipidic effect but, due to the auto-alimentation of thephenomenon, the coexisting situation of hypercholes-terolemia impedes its resolution which would otherwisetake place within 2-3 weeks64.

The presence of Chlamydia pneumoniae in atherosclerotic lesions

Chlamydia pneumoniae has often been seen with theelectron microscope inside the atheromatous plaques65.Many other investigations, carried out with various tech-

Claudio Blasi

253

niques (the search for immunocomplexes with immuno-cytochemistry, the search for antibodies by means ofimmunofluorescence, the finding and amplification ofDNA of a bacterium by means of the polymerase chainreaction and of in situ hybridization) have confirmed itspresence66. According to a recent literature review, the bac-terium is identified in 46% of the samples of arteries withatheromatous plaque compared to less than 1% of thesamples of healthy arteries47. It has been identified in theplaque of all types of arteries45,67, particularly the carotidsof heavy smokers68. It has also been isolated from athero-matous tissue and cultivated in vitro69, although withsome difficulty47.

Experiments on animal models that do not sponta-neously develop atherosclerosis have demonstrated that thisparticular strain of Chlamydia, once introduced throughthe nose, may cause initial atheromatous lesions that con-tinue to grow if the animals are fed an atherogenic diet.Thus, the germ can aim at the internal arterial wall, infectit and constitute the primum movens of the plaque that thengrows in the presence of dyslipidemia70.

In man too, there is unquestionable evidence of thewidespread presence of the bacterium in the arteries65; thereis also, by now, little doubt that it has a strong tendencyto settle in the plaques52 (as well as in aneurysms andstenotic aortic valves45). It appears that the germ is trans-ported there from the initial site of infection by monocytes(chiefly alveolar macrophages) that are attracted by theendothelial inflammation (a phenomenon known as “path-ogenic opportunism”)52,56. Once they arrive here, theyare able to maintain a low grade infection of the cells51.

Attempts have also been made to exculpate the germby attributing to it a role of “innocent bystander”. However,the fact that it has been found in atheromatous plaques butnot in infective or non-infective granulomas or in otherhealthy tissues in the same patient leads us to doubt this“presumption of innocence”18,49,71.

The role of Chlamydia pneumoniae in thrombus formation

It has been observed that Chlamydia pneumoniaestrongly adheres to platelets and activates their aggrega-tion. It could favor the formation of thrombi by means ofthis mechanism72.

The role of Chlamydia pneumoniae in modulatingthe inflammatory reaction of the artery

Local action

Chlamydia pneumoniae is able to infect most of the cellspresent in the atheromatous lesion and to influence their

activity47,52-54,73,74. It acts, above all, by means of two“virulence factors” (factors responsible for its capacity tosettle in and reproduce): the above-cited surface LPS andthe HSP.

Actions induced by lipopolysaccharide

Aside from the already-mentioned stimulus to the for-mation of foam cells, the surface LPS (by means of the κBgenic transcription factor) is one of the responsible factorsfor the cascade of the molecules at the origin of inflam-mation in the endothelial cells75. It is able to act inde-pendently of the presence of live bacteria76. Moreover, thesmooth muscle cells are stimulated by it to proliferate77,and, once transformed into fibroblasts, produce the fibrouscapsule of the plaque that may be considered a defensemechanism against further passage of lipids and inflam-matory cells56. On the other hand, the LPS also stimulatesthe leukocytes to produce “metalloproteinase” enzymesthat degrade the collagen fibers of the capsule, renderingthe plaque unstable and predisposed to rupture47,78-80.

Actions of heat shock proteins

The often-cited HSP is a protein produced by the cellsof the organism in response to stress, that is, by any stim-ulus, internal or external, that leads to a central effect: thedenaturation of the proteins within the cells and the con-sequent activation of the gene for HSP81. These stimulimay include, aside from increased heat, infection, presenceof free oxygen radicals, lack of nutrition, etc.82. It has adouble function: one inside and the other outside the cell.The former consists of the role of “chaperon” of the youngproteins, that is to stabilize and protect them from denat-uration, from erroneous folding and from aggregation(both induced by stress83-85). It facilitates the folding of ris-ing proteins, renders the already-formed protein aggregatessoluble, transports the proteins across the mitochondrialmembrane and assists in the new, correct folding of thedenatured proteins56,86. All of this has the purpose ofimpeding the cascade formation of altered protein aggre-gates that are lethal to the cell83,85. The second functionis carried out on the cell surface to which it may move inresponse to stress40, activating the innate immune moni-toring system87, as a danger signal to the system (the“danger” theory88). Once the cell is identified, the latterdetermines activation of NF-κB and consequent expres-sion of the pro-inflammatory cytokines85.

HSPs are present in nature in about 24 molecules,grouped into five families according to their molecularweight81. They have an amino acid sequence that is verysimilar even in distant species such as bacteria and man

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

254

(particularly the HSP-60 family)61,82. All organismsrespond to potentially dangerous situations by inducing thetranscription of genes responsible for HSP synthesis49,86.The molecule produced by the endothelial cells of man hasan amino acid sequence that is very similar (> 50%) to thatproduced by Chlamydia pneumoniae82. Thus, the risk ofimmune cross reactions between the two proteins is high82.

Heat shock protein and the immune response

Chlamydia pneumoniae (in the form of “persistent bod-ies” of its vital cycle) may elaborate great quantities ofHSP-60 (from its molecular weight)79,80,86, which is high-ly antigenic to man84,89. On the other hand, in order tomaintain homeostasis in the arterial wall, the endotheliumproduces a great deal of HSP-60 in response to the stress-causing factors represented by the various risk factorsfor atherosclerosis90,91, including oxidized LDL92 and theimmune reaction against the oxidized LDL themselves86.It has been hypothesized that the immune system, alreadysensitized by bacterial HSP-60, may set off an allergic reac-tion40 in the presence of the HSP-60 produced by theendothelium and translocated onto its surface. This mech-anism is part of the immune surveillance against cellsaltered by stress. It has the purpose of eliminating themand protecting, in this way, the rest of the cellular com-munity83. This so-called “cross” reaction, due to theresemblance between the two molecules, has cytotoxiceffects on the endothelial cells through complement acti-vation or antibody-linked toxicity40,49,93. This could con-tribute to endothelial dysfunction and to cell apoptosis aswell, which are a part of the atherosclerotic process26,91.Clinical and experimental data confirm that indeed thisauto-immune reaction against HSP-60 is produced in theearly stages of atherosclerosis40,43,49,74,86,94,95. In man, astrong increase in IgG against HSP has been observed inconcomitance with acute coronary events96. Moreover, per-cutaneous angioplasty may induce an increase in the anti-bodies against Chlamydia pneumoniae96,97, and a rela-tionship between elevated antibody levels and coronaryrestenosis after angioplasty with or without stent implan-tation55 has been ascertained. The anti-HSP antibodies, too,provoked by the cross immune reaction between bacter-ial HSP and human HSP could play a role in the resteno-sis that follows angioplasty interventions, inasmuch as theantibodies against HSP could provoke an acceleration ofthe neointimal abnormal growth that narrows the arteriallumen98.

The fact that there is an immune component in theinflammation at the origin of atherosclerosis is further sug-gested by the presence, in the plaque, of activated T-lym-phocytes94 and also by memory T “asleep” lymphocytes

that may be reactivated in the presence of antigens simi-lar to those to which they had previously been sensi-tized95. The T-cells found in atherosclerotic lesions respondin vitro to Chlamydia pneumoniae, human and bacterialHSP and oxidized LDL92.

Finally, a bacterium of the same species, Chlamydia tra-chomatis, causes trachoma blindness and the obstructionof the fallopian tubes with a fibrosis that follows the infil-tration of lymphocytes and macrophages, through a delayedsensitization reaction in which the key antigen is HSP-6099,a process that has interesting analogies with the develop-ment of atherosclerotic plaque99.

Other atherogenic reactions of heat shock proteins

HSP would also act in an atherogenic sense by activatingpro-inflammatory genes both in the endothelial cells andin the macrophages and smooth muscle cells84, thus induc-ing a classic atherogenic effect: 1) the introduction ofinflammatory cytokines; 2) the production of adhesion andrecruitment molecules of the inflammatory cells; 3) the pro-duction of metalloproteases, 4) the oxidation of the LDLby monocytes49.

Pathogenetic hypotheses

The first, also called “molecular mimicry” may besynthesized as follows40,43,54,86 (Fig. 1): a) the immunereaction to bacterial HSP is set off in the course of the firstinfection; b) stress risk factors provoke the dysfunction ofthe endothelium followed by the activation of the inflam-matory process; moreover, they provoke cell proteindenaturation which is followed by the production ofendothelial HSP; c) HSP, translocated to the cell surfaceand identified by immune surveillance, triggers the aller-gic reaction set off by the antibodies followed by their cyto-toxic effect; also intervening are the T-lymphocytes that,aimed against the HSP, reach the target, proliferate andcomplete the attack; d) the monocytes with the bacteria set-tled within the initial infection site, become their vehiclesof spread. Attracted to the atheromatous lesion, they crossthe endothelium and transport the bacteria there; the HSPand the LPS of Chlamydia pneumoniae contribute to theinflammation as well as to the oxidation and subsequentphagocytosis of the LDL, and thus to the evolution of theatheroma, the rupture of the fibrous lining through theaction of the metalloproteases and TNF-α (to the pro-duction of which the presence of bacteria contributes)and the formation of the thrombus may follow.

This process makes one think of a sophisticated strat-egy of survival and reproduction of Chlamydia pneumo-niae49 that utilizes the atheromatous process to this pur-

Claudio Blasi

255

pose and that might be co-evolved with the species sub-ject to atherosclerotic disease.

The second hypothesis, not necessarily alternative to thefirst, could be applied particularly for the initial phases ofatherosclerosis, those that occur at a young age8,28,40,64,70,83

(Fig. 2). It maintains that the infective agent reaches theendothelial wall of an artery and stabilizes a persistent low-grade chronic infection there50. The infection would pro-voke the formation of HSP and its expression on the sur-face, the activation of innate immune surveillance, ofNF-κB, the production of pro-inflammatory molecules70,85,and the setting off of the atheromatous process.

The subintimal infiltration of the immuno-competentcells, in young people, occurs before lipids are deposited8.This leads us to believe that the process is subsequentlysustained by the oxidized LDL. These, in turn, play the roleof antigen in an autoimmune reaction with a cytotoxiceffect that induces the production of HSP86. In fact, theautoimmune process and the inflammation deriving fromit are amplified by high serum levels of cholesterol100. Theimmune-mediated inflammatory response, in the absenceof hypercholesterolemia, is unable to make atherogenesislast, and it tends to regress. On the other hand, the low-ering of cholesterol levels is much less effective in reduc-ing the media-intimal thickness in individuals positivefor anti-Chlamydia pneumoniae antibodies with respect tothose who are negative2. Thus, especially in the early, juve-nile phases, atherosclerosis could be the effect of a com-

FIGURE 1. Hypothesis of autoimmune pathogenesis of atherosclerosis provoked by infec-tious agents, called “molecular mimicry” (see text).HSPb = bacterial heat shock protein; HSPe = endothelial heat shock protein; LDL = low-density lipoprotein; LDL-OX = oxidized LDL.

FIGURE 2. Hypothesis of autoimmune pathogenesis of athero-sclerosis provoked by direct action of infectious agents on theendothelial cells (see text). Abbreviations as in figure 1.

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

256

plex interaction between lipid imbalances and an immunereaction induced by infectious agents64.

These hypotheses could also be valid for the otherinfectious agents involved in the pathogenesis of athero-sclerosis. As illustrated, the HSP expressed by them (withregard to bacteria) or incorporated into their membranewhen they abandon the infected cell (with regard to virus-es) is suspected to cause the immune cross reaction withthe human HSP at the level of the endothelium16,40,43.Moreover, the various bacteria and viruses both act byinducing the production of HSP by the infected cells83. Inthis way, they also stimulate the response of the innateimmune system85. Finally, we cannot exclude the possi-bility that the risk factors themselves determine the acti-vation of the immune reaction. The mechanism would be:endothelial stress, consequent denaturation of the cellularproteins and activation of the genes that lead to the syn-thesis of HSP and its expression on the cellular surface8,81.

It is interesting to note that Chlamydia pneumoniae isassociated with an increasing number of chronic diseasesincluding Alzheimer’s disease101. Some of them such asmultiple sclerosis, Reiter’s syndrome, reactive arthritis,Guillan-Barré syndrome, some types of thyroiditis, sar-coidosis, erythema nodosum and rheumatoid arthritis areconsidered autoimmune diseases102. The relation betweenthese diseases, Chlamydia pneumoniae infection and ath-erosclerosis could derive from the fact that a particulargenetic characterization (the presence of the ε4 allele inthe lipoprotein apo-E locus) is linked to an increased riskof these diseases and also of Chlamydia pneumoniaeinfection and of atherosclerosis103,104. Thus, the pathogenicmechanism would have a common autoimmune basis105.

Treatment and prevention

This aspect will be examined mainly for Chlamydiapneumoniae on which the researchers’ studies were con-centrated.

Antibiotic treatment

Theoretically, antibiotic treatment effective for Chla-mydia pneumoniae could prevent or attenuate the cardio-vascular events brought about by atherosclerosis. Still, thisbacterium is difficult to fight due to the characteristics ofits life cycle. Both during the inactive metabolic form ofits “elementary bodies” and during the latent form of its“reticulate bodies” it is not sensitive to antibiotics (whichare able to act only during the transformation from “retic-ulate bodies” to “elementary bodies”)106. Moreover, whenwithin the monocytes, its sensitivity is particularlyreduced70. Treatment with some antibiotics favors the

formation of the “persistent” and chronic form which isdifficult to eradicate. The macrolides, particularlyazithromycin53 are the most effective. The chinolones,tetracyclines and some anti-tubercular drugs are alsoeffective47,107,108.

Studies carried out

Various pilot studies have been carried out to ascertainthe usefulness of antibiotic treatment in patients with ath-erosclerosis, in particular, those with coronaropathy67.The results have been encouraging for some of them.After an acute myocardial infarction, patients with high lev-els of antibodies against the bacterium have been treatedwith azithromycin or with placebo: those treated with theantibiotic had a significantly reduced incidence of subse-quent cardiovascular events109. Studies that consideredother parameters, such as the growth of abdominal aorticaneurysms and the thickening of the carotid wall, have alsoyielded positive results, even though the number of caseswas limited and thus their statistical value reduced110.Still, other studies of brief duration (5 days), such as theAZACS study (Azithromycin in Acute Coronary Syn-drome) that have involved about 1500 subjects, did notconfirm a significant preventive effect either on the recur-rences of infarct or on the appearance of acute events insubjects with high levels of antibodies107,111. Studies withmore prolonged treatment, involving several thousandpatients, were subsequently carried out. In one of these,the WIZARD study (Weekly Intervention with Zythromaxfor Atherosclerosis and its Related Disorders), complet-ed in 2002 and including over 7000 subjects with a per-sonal history of heart attack and positivity for anti-Chlamydia pneumoniae antibodies, azithromycin wasadministered for 3 months. This resulted in a significant-ly decreased mortality due to a recurrent acute myocardialinfarction and occurring among patients treated with theantibiotic during the first 6 months of the experimentwith respect to those treated with placebo67,112. In theACADEMIC study (Azithromycin in Coronary ArteryDisease: Elimination of Myocardial Infection withChlamydia), that utilized azithromycin versus placebo inabout 300 patients for a period of 3 months, a tendencytowards the reduction of cardiovascular events wasobserved after 1 year and after 1 year and a half; this ten-dency, however, did not reach statistical significance113.A retrospective study, carried out on a large number ofpatients, to show the effects of the primary prevention ofantibiotic treatment on the onset of myocardial infarctionhas also yielded encouraging results114. Other large exper-iments such as the ACES trial (Azithromycin and CoronaryEvents Study) that will involve thousands of coronaro-

Claudio Blasi

257

pathic patients, sponsored by the United States NationalInstitutes of Health are presently underway, indicating theimportance that is attributed to the subject70. Some stud-ies have shown, in antibody-positive patients, a significantreduction in the levels of inflammatory markers such asC-reactive protein, interleukin-6 and TNF-α, that consti-tute the so-called “acute-phase response” of the liver toinflammation115. Still, the reduction of these markerscould be due to the aspecific anti-inflammatory effects ofthe antibiotics107. Among other things, antibiotics stabi-lize plaque: this could explain the improvement observedin patients with coronary syndromes, independently of anyantibacterial effect107.

It is hoped that new studies on primary prevention willbe undertaken as the pathogenic mechanisms are further clar-ified. They should be conducted during the first stages ofdisease progression70, particularly on young subjects52. Infact, the natural history of atherosclerosis develops over thecourse of decades, and more than 50% of young subjectsbetween 10 and 12 years old and living in western countriesalready have coronary atherosclerotic lesions in progress44.Already before 20 years of age the typical succession of inti-mal thickening begins, followed by partial healing andnew relapses linked to recurrent episodes of inflammatorystimuli; this goes hand in hand with episodes of infectionand reinfection (or reactivation of a chronic infection) byChlamydia pneumoniae115. Traces of the presence of the bac-terium have been found in the lipidic striae already inyoung adults18. Moreover, it is probable that antibiotictherapy is not particularly effective in preventing cardio-vascular events in grown-ups when the atheroscleroticprocess has already reached its advanced stages45.

Methods to evaluate the efficacy of antibiotic treatment

One could hypothesize that prevention may be achievedby the periodic administration of an antibiotic on thebasis of the course of reference values, something like whatwas done to prevent the cardiac alterations of acuterheumatic fever on basis of the course of the antistrep-tolysin titers and erythrocyte sedimentation rate. Still,what is missing here is a standardization of the variousmethods to prove the presence and evolution of Chlamydiapneumoniae infection, and this reduces the possibility ofevaluating with certainty the possible efficacy of treat-ment44,47. It could be useful to utilize the course of the IgAantibodies the titers of which normalize rapidly aftereffective treatment44 or parameters of the immune responseto members of the HSP family of bacterial origin55,70.The patients to treat could be selected on the basis of thetiters of anti-HSP antibodies.

Prevention via vaccine

An anti-Chlamydia pneumoniae vaccine that would pre-vent infection from the very beginning is being researchedin various laboratories. To date, no molecules towardswhich vaccines with a guaranteed protective action couldbe developed have been identified44,116. At the moment,there is a single vaccine, synthesized using the recombi-nant DNA technique, against some protein components ofthe bacterium; it is still under experimentation44. Potentialantigenic targets are the proteins of the external membranethat provide the bacterium with its adaptation properties,facilitating the interactions between the bacterial cell andthe host cell117. For example, the study of the genome ofthe bacterium was able to show the so-called “type IIIsecretion system”118. It is a secretion apparatus on the bac-terial surface, the formation of which is induced by genesactivated by host-bacterium contact, enabling the latter tosend invasion-favoring enzymes into the cell118,119. Theseproteins, since they represent factors of virulence, constitutea group that is potentially very important for the devel-opment of a vaccine derived from bacterial DNA118. Thepossibility of developing a vaccine with protective prop-erties against Chlamydia trachomatis by using DNAderived from the gene that governs the synthesis of the prin-cipal protein of the external membrane of the bacteriumhas been demonstrated in experimental studies with ani-mals120. This allows us to hope for the development of ananalogous vaccine against Chlamydia pneumoniae thatshares a “common immunobiological paradigm” with theother bacterium120. The worst problem for all types of vac-cine consists of the possible autoimmune and immuno-pathologic responses118, with the risk of provoking aninflammatory reaction of immune origin that would aggra-vate the vascular damage121.

Also examined has been the possibility of acting direct-ly on the HSP, given its relevant immune pathogenetic role.The oral or nasal mucosal administration of HSP in exper-imental animals has brought about the attenuation of ath-erosclerotic lesions122,123, allowing us to hypothesize that,in the future, tolerance to this antigen could be induced andits pathogenetic impact decreased8,124. One preventivestrategy could, in fact, be to vaccinate against the HSP orHSP peptides with the aim of shifting the autoimmunereactivity of the Th1 to the Th2 lymphocytes, and, in thisway, modulate the immune reaction against the antigen andattenuate it125.

Conclusions

The hypothesis of the contribution of infectious agentsto the origin of atherosclerosis, after many years since itsfirst formulation, has not yet been confirmed becauseproof of a direct causal relationship is still lacking and is

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

258

extremely difficult to obtain44,70. The most frequently-raised objection to this hypothesis is that the treatment ofsubjects at risk for cardiovascular events with antibiotics(in particular macrolides for Chlamydia pneumoniae) hasnot yielded very encouraging results in terms of reducingsuch events. Still, as already stated, it is difficult to thinkof reaching this objective when the plaques are already inan advanced state of evolution. This is because the actionof the infective agents probably occurs, in most cases, veryprematurely, at the beginning of the natural history ofthe lesion when the patient is still young44,70. The hypoth-esis, then, could be proposed in terms of probability ratherthan of certainty73. Still, as it has been attempted to demon-strate, it is based on a notable amount of associative andcorrelative evidences70.

To date, two mechanisms through which the infec-tious agents could contribute to the development of theatheromatous process have been proposed: 1) by induc-ing an immune cross reaction due to “molecular mimic-ry” against the HSP produced by the endothelium as aresponse to the traditional risk factors; 2) by directlyinfecting the endothelial cells, inducing the expression ofthe HSP on their surface and activating the immune sys-tem. The two mechanisms could coexist, and the secondone could be responsible for the most precocious andjuvenile phases of atherosclerosis. Both of them would inte-grate with the fundamental role of hyperlipidemia and ofthe other risk factors.

From a therapeutic and prophylactic point of view,antibiotic treatment should probably be initiated at ayoung age. Moreover, vaccination before 20 years of ageagainst various microorganisms (above all against Chla-mydia pneumoniae) and the modulation of the immunereactivity against HSP are promising.

All of this is particularly important for diabetics, whoundergo a notable “infectious burden” and are character-ized by an altered immune response18. It has been demon-strated, for example, that Chlamydia pneumoniae increas-es the risk of coronaropathy induced by the metabolic syn-drome126.

Many steps must still be taken65. When the patho-genetic mechanisms of the infectious component of ath-erosclerosis are further clarified, a properly aimed thera-peutic or prophylactic strategy could lead, in clinicalterms, to extremely significant outcomes.

Riassunto

L’aterosclerosi costituisce un processo infiammatoriocronico legato alla reazione dell’endotelio allo stress pro-vocato da fattori di rischio, alcuni ancora sconosciuti.Osservazioni cliniche e sperimentali hanno condotto a

ritenere che anche alcuni agenti infettivi siano coinvoltiin questo processo. Vengono descritti i vari microrgani-smi e i loro rapporti con le lesioni ateromasiche, in parti-colare la Chlamydia pneumoniae. Si possono proporre dueipotesi su come agirebbero. Ambedue si basano sulla lo-ro proprietà di indurre la produzione da parte delle cellu-le endoteliali di una proteina chiamata “proteina da shockdi calore” (HSP) che ha la caratteristica di provocare l’at-tivazione del sistema immunitario: 1) induzione di una rea-zione immunitaria crociata, da “mimetismo molecolare”,tra la HSP di origine infettiva e quella prodotta dall’en-dotelio in risposta ai fattori di rischio; 2) infezione dellecellule endoteliali, conseguente sintesi ed esposizionesulla loro superficie della HSP con attivazione della sor-veglianza immunitaria innata. Studi sperimentali sonostati eseguiti e sono tuttora in corso allo scopo di accer-tare l’efficacia del trattamento antibiotico nel ridurre l’in-cidenza di eventi cardiovascolari acuti come l’ictus e l’in-farto del miocardio. I risultati sono ancora inconcluden-ti. Probabilmente, per essere efficace, il trattamento deveessere iniziato in età giovanile. È stata anche intrapresa lavia della prevenzione tramite la vaccinazione contro imicrorganismi coinvolti o l’induzione di tolleranza im-munitaria verso la HSP. Quando verranno ulteriormentechiariti i meccanismi attraverso i quali gli agenti infetti-vi contribuiscono o sono all’origine dell’aterosclerosi po-trebbero essere messe in atto delle misure terapeutiche opreventive efficaci con importanti ricadute sul piano cli-nico*.

Parole chiave: Agenti infettivi; Aterosclerosi; Chlamydiapneumoniae; Cytomegalovirus; Proteina da shock da ca-lore.

Acknowledgments

The author wishes to thank Dr. Alessio Tommasettifor his skill and kind help in creating the figures.

References001. Ross R. The pathogenesis of atherosclerosis: a perspective for

the 1990s. Nature 1993; 362: 801-9.

002. Sawayama Y, Tatsukawa M, Okada K, et al. Association ofChlamydia pneumoniae antibody with the cholesterol-lower-ing effect of statins. Atherosclerosis 2003; 171: 281-5.

003. Prasad A, Zhu J, Halcox JP, et al. Predisposition to athero-sclerosis by infections. Role of endothelial dysfunction. Cir-culation 2002; 106: 184-90.

004. Nedeljkovic ZS, Gokce N, Loscalzo J. Mechanisms of oxida-tive stress and vascular dysfunction. Postgrad Med J 2003; 79:195-200.

*La versione italiana dell’intero articolo è disponibile su richiesta all’au-tore.

Claudio Blasi

259

005. Poredos P. Endothelial dysfunction and cardiovascular dis-ease. Pathophysiol Haemost Thromb 2002; 32: 274-7.

006. Wheatcroft SB, Williams IL, Shah AM, Kearney MT. Patho-physiological implications of insulin resistance on vascularendothelial function. Diabet Med 2003; 20: 255-68.

007. Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction.A marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol2003; 23: 168-75.

008. Wick G, Knoflach M, Xu Q. Autoimmune and inflammatorymechanisms in atherosclerosis. Annu Rev Immunol 2004; 22:361-403.

009. Osler W. Diseases of the arteries. In: Osler W, ed. Modern med-icine: its theory and practice. Philadelphia, PA: Lea & Febiger,1908: 426-47.

010. Gibbon RV. Germs, Dr. Billings, and the theory of focal infec-tion. Clin Infect Dis 1998; 27: 627-33.

011. Benditt EP, Benditt JM. Evidence of a monoclonal origin ofhuman atherosclerotic plaques. Proc Natl Acad Sci USA 1973;70: 1753-66.

012. Fabricant CG, Fabricant J. Atherosclerosis induced by infec-tion with Marek’s disease herpesvirus in chickens. Am HeartJ 1999; 138: S465-S468.

013. Saikku P, Leinonen M, Mattila K, et al. Serological evidenceof an association of a novel Chlamydia, TWAR, with chroniccoronary heart disease and acute myocardial infarction. Lancet1988; 2: 983-6.

014. Nicoletti A, Caligiuri G, Paulsson G, Hansson GK. Functionalityof specific immunity in atherosclerosis. Am Heart J 1999;138: S438-S443.

015. Mayr M, Kiechl S, Willeit J, Wick G, Xu Q. Infections, immu-nity and atherosclerosis: associations of antibodies to Chlamydiapneumoniae, Helicobacter pylori, and Cytomegalovirus withimmune reactions to heat-shock protein 60 and carotid orfemoral atherosclerosis. Circulation 2000; 102: 833-9.

016. Biondi-Zoccai GGL, Abbate A, Liuzzo G, Biasucci LM.Atherothrombosis, inflammation, and diabetes. J Am CollCardiol 2003; 41: 1071-7.

017. Epstein SE, Zhu J, Burnett MS, Zhou YF, Vercellotti G, HajjarD. Infection and atherosclerosis: potential roles of pathogen bur-den and molecular mimicry. Arterioscler Thromb Vasc Biol2000; 20: 1417-20.

018. O’Connor S, Taylor C, Campbell LA, Epstein S, Libby P.Potential infectious etiologies of atherosclerosis: a multifactorialprospective. Emerg Infect Dis 2001; 7: 780-7.

019. Nicholson AC, Hajjar DP. Viral activation of the coagulationcascade. Am Heart J 1999; 138: S461-S464.

020. Melnick JL, Hu C, Burek J, Adam E, De Bakey ME. Cyto-megalovirus DNA in arterial walls of patients with athero-sclerosis. J Med Virol 1994; 42: 170-4.

021. Nieto J. Viruses and atherosclerosis: a critical review of the epi-demiologic evidence. Am Heart J 1999; 138: S443-S460.

022. Zhou YF, Shou M, Guetta E, et al. Cytomegalovirus infectionof rats increases the neointimal response to vascular injurywithout consistent evidence of direct infection of the vascularwall. Circulation 1999; 100: 1569-75.

023. Sorlie PD, Nieto J, Adam E, Folsom AR, Shahar E, MassingM. A prospective study of Cytomegalovirus, herpes simplexvirus 1, and coronary heart disease. Arch Intern Med 2000; 160:2027-32.

024. Stöllberger C, Finsterer J. Atherosclerosis: infection-inducedinvolvement of mitochondrial chaperonins. Lancet 2003; 362:1949-50.

025. Bruggeman C. Cytomegalovirus is involved in vascular pathol-ogy. Am Heart J 1999; 138: S473-S475.

026. Jarvis M, Nelson J. Human Cytomegalovirus persistence and

latency in endothelial cells and macrophages. Curr OpinMicrobiol 2002; 5: 403-7.

027. Epstein SE, Zhou YF, Zhu J. Potential role of Cytomegalovirusin the pathogenesis of restenosis and atherosclerosis. Am HeartJ 1999; 138: S476-S478.

028. Bason C, Corrocher R, Lunardi C, et al. Interaction of antibodiesagainst Cytomegalovirus with heat-shock protein 60 in patho-genesis of atherosclerosis. Lancet 2003; 362: 1971-7.

029. Zhou YF, Leon MB, Waclawiw MA, et al. Association betweenprior Cytomegalovirus infection and risk of restenosis after coro-nary atherectomy. N Engl J Med 1996; 335: 624-30.

030. Grattan MT, Moreno-Cabral CE, Starnes VA, Oyer PE, StinsonEB, Shumway NE. Cytomegalovirus infection is associated withcardiac allograft rejection and atherosclerosis. JAMA 1989; 261:3561-6.

031. Potena L, Grigioni F, Ortolani P, et al. Relevance of Cytomega-lovirus infection and coronary-artery remodeling in the first yearafter heart transplantation: a prospective three-dimensionalintravascular ultrasound study. Transplantation 2003; 75: 839-43.

032. Hosenpud JD. Coronary artery disease after heart transplanta-tion and its relation to Cytomegalovirus. Am Heart J 1999; 138:S469-S472.

033. Valantine HA. Cardiac allograft vasculopathy: central role ofendothelial injury leading to transplant “atheroma”. Transplan-tation 2003; 76: 891-9.

034. Rupprecht HJ, Blankenberg S, Bickel C, et al. Impact of viraland bacterial infectious burden on long-term prognosis inpatients with coronary artery disease. Circulation 2001; 104:25-31.

035. Ishizaka N, Ishizaka Y, Takahashi E, et al. Association betweenhepatitis C virus seropositivity, carotid-artery plaque, and inti-ma-media thickening. Lancet 2002; 359: 133-5.

036. Roivainen M. Enteroviruses and myocardial infarction. AmHeart J 1999; 138: S479-S483.

037. Kusters JG, Kuipers EJ. Helicobacter and atherosclerosis. AmHeart J 1999; 138: S523-S527.

038. Mendall MA, Goggin PM, Molineaux N, et al. Relation ofHelicobacter pylori infection and coronary heart disease. BrHeart J 1994; 71: 437-9.

039. Gabrielli M, Santoliquido A, Cremonini F, et al. CagA-posi-tive cytotoxic H. pylori strains as a link between plaque insta-bility and atherosclerotic stroke. Eur Heart J 2004; 25: 64-8.

040. Zhu J, Katz RJ, Quyyumi AA, et al. Association of serumantibodies to heat-shock protein 65 with coronary calcificationlevels. Suggestion of pathogen-triggered autoimmunity in earlyatherosclerosis. Circulation 2004; 109: 36-41.

041. Mayr M, Kiechl S, Mendall MA, et al. Increased risk of ath-erosclerosis is confined to CagA-positive Helicobacter pyloristrains: prospective results from the Bruneck study. Stroke2003; 34: 610-5.

042. Beck JD, Pankow J, Tyroler HA, Offenbacher S. Dental infec-tions and atherosclerosis. Am Heart J 1999; 138: S528-S533.

043. Wick G, Perschinka H, Xu Q. Autoimmunity and atheroscle-rosis. Am Heart J 1999; 138: S444-S449.

044. Higgins JP, Higgins JA, Higgins PM, Ahuja S, Higgins DL.Chlamydia pneumoniae and coronary artery disease: legit-imized linkages? Expert Rev Cardiovasc Ther 2003; 1: 367-84.

045. Boland JP. Chlamydia pneumoniae and cardiovascular dis-ease: current status. W V Med J 2000; 96: 458.

046. Kuo CC, Jackson LA, Campbell LA, Grayston JT. Chlamydiapneumoniae (TWAR). Clin Microbiol Rev 1995; 8: 451-61.

047. Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae asan emerging risk factor in cardiovascular disease. JAMA 2002;288: 2724-31.

048. Meier CR. Antibiotics in the prevention and treatment of coro-nary heart disease. J Infect Dis 2000; 81 (Suppl 3): S558-S562.

Ann Ital Med Int Vol 19, N 4 Ottobre-Dicembre 2004

260

049. Kalayoglu MV. Chlamydial heat shock protein 60 and lipo-polysaccharide: potential virulence determinants in atheroge-nesis. Curr Drugs Targets Inflamm Allergy 2002; 1: 249-55.

050. Stratton CW, Sriram S. Association of Chlamydia pneumoni-ae with central nervous system disease. Microbes Infect 2003;5: 1249-53.

051. Tasaki N, Nakajima M, Yamamoto H, et al. Influence ofChlamydia pneumoniae infection on aortic stiffness in healthyyoung men. Atherosclerosis 2003; 171: 117-22.

052. Campbell LA, Kuo CC. Chlamydia pneumoniae and athero-sclerosis. Semin Respir Infect 2003; 18: 48-54.

053. Lindholt JS, Stovring J, Andersen PL, Henneberg EW,Ostergaard L. A review of macrolide treatment of atheroscle-rosis and abdominal aortic aneurysms. Curr Drug TargetsInfect Disord 2003; 3: 55-63.

054. Siscovick DS, Schwartz S, Caps M, Wang SP, Grayston JT.Chlamydia pneumoniae and atherosclerotic risk in popula-tions: the role of seroepidemiology. J Infect Dis 2000; 81(Suppl 3): S417-S420.

055. Zairis MN, Papadaki OA, Paraskevi K. Serologic markers ofpersistent Chlamydia pneumoniae infection and long-termprognosis after successful coronary stenting. Am Heart J 2003;146: 1082-9.

056. Rosenfeld ME, Blessing E, Lin TM, Moazed TC, Campbell LA.Chlamydia, inflammation and atherogenesis. J Infect Dis 2000;81 (Suppl 3): S492-S496.

057. Hu H, Pierce GN, Zhong G. The atherogenic effects ofChlamydia are dependent on serum cholesterol and specific toChlamydia pneumoniae. J Clin Invest 1999; 103: 747-53.

058. Muhlestein JB. Chlamydia pneumoniae-induced atherosclerosisin a rabbit model. J Infect Dis 2000; 181 (Suppl 3): 505-7.

059. Muhlestein JB, Anderson JL, Hammond EH, et al. Infectionwith Chlamydia pneumoniae accelerates the development of ath-erosclerosis and treatment with azithromycin prevents it in arabbit model. Circulation 1998; 97: 633-6.

060. Fong IW, Chiu B, Viira E, Jang D, Mahony JB. Influence ofclarithromycin on early atherosclerotic lesions after Chlamydiapneumoniae infection in a rabbit model. Antimicrob AgentsChemother 2002; 46: 2321-6.

061. Stille W, Dittmann R. Atherosclerosis as a sequela of chronicChlamydia pneumonia infection. Herz 1998; 23: 185-92.

062. Kalayoglu MV, Hoerneman B, LaVerda D, Morrison SG,Morrison RP, Byrne GI. Cellular oxidation of low-densitylipoprotein by Chlamydia pneumoniae. J Infect Dis 1999; 180:780-90.

063. Kalayoglu MV, Morrison RP, Morrison SG, Yuan Y, Byrne GI.Chlamydial virulence determinants in atherogenesis: the roleof chlamydial lipopolysaccharide and heat shock protein 60 inmacrophage-lipoprotein interactions. J Infect Dis 2000; 81(Suppl 3): S483-S489.

064. Ludewig B, Zinkernagel RM, Hengartner H. Arterial inflam-mation and atherosclerosis. Trends Cardiovasc Med 2002; 12:154-9.

065. Taylor-Robinson D, Thomas BJ. Chlamydia pneumoniae inatherosclerotic tissue. J Infect Dis 2000; 81 (Suppl 3): S437-S440.

066. Kuo CC, Campbell LA. Detection of Chlamydia pneumoniaein arterial tissues. J Infect Dis 2000; 81 (Suppl 3): S432-S435.

067. Muhlestein JB. Antibiotic treatment of atherosclerosis. CurrOpin Lipidol 2003; 14: 605-14.

068. Ciervo A, Petrucca A, Cassone A. Identification and quantifi-cation of Chlamydia pneumoniae in human atheroscleroticplaques by LightCycler real-time-PCR. Mol Cell Probes 2003;17: 107-11.

069. Ramirez JA. Isolation of Chlamydia pneumoniae from thecoronary artery of a patient with coronary atherosclerosis. The

Chlamydia pneumoniae/Atherosclerosis Study Group. AnnIntern Med 1996; 125: 979-82.

070. Belland RJ, Ouellette SP, Gieffers J, Byrne GI. Chlamydia pneu-moniae and atherosclerosis. Cell Microbiol 2004; 6: 117-27.

071. Gaydos CA. Growth in vascular cells and cytokine productionby Chlamydia pneumoniae. J Infect Dis 2000; 81 (Suppl 3):S473-S478.

072. Kälvegren H, Majeed M, Bengtsson T. Chlamydia pneumoni-ae binds to platelets and triggers P-selectin expression andaggregation. A causal role in cardiovascular disease? Arterio-scler Thromb Vasc Biol 2003; 23: 1677-83.

073. Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis.Emerging mechanistic paradigms. Circulation 1999; 100: E20-E28.

074. Lopes-Virella MF, Virella G. The role of immune and inflam-matory processes in the development of macrovascular diseasein diabetes. Front Biosci 2003; 8: S750-S768.

075. Summersgill JT, Molestina RE, Miller RD, Ramirez JA.Interactions of Chlamydia pneumoniae with human endothe-lial cells. J Infect Dis 2000; 81 (Suppl 3): S479-S482.

076. Baer JT, du Laney TV, Wyrick PB, et al. Nuclear factor-κB acti-vation in endothelium by Chlamydia pneumoniae withoutactive infection. J Infect Dis 2003; 188: 1094-7.

077. Wong YK, Gallagher PJ, Ward ME. Chlamydia pneumoniaeand atherosclerosis. Heart 1999; 81: 232-8.

078. Hortovànyi E, Illyés G, Kàdàr A. Early atherosclerosis andChlamydia pneumoniae infection in the coronary arteries.Pathol Oncol Res 2003; 9: 42-6.

079. Choi EY, Dongsoo K, Hong BK, et al. Upregulation of extra-cellular matrix metalloproteinase inducer (EMMPRIN) andgelatinases in human atherosclerosis infected with Chlamydiapneumoniae: the potential role of Chlamydia pneumoniaeinfection in the progression of atherosclerosis. Exp Mol Med2002; 34: 391-400.

080. Kol A, Sukhova GK, Lichtman AH, Libby P. Chlamydial heatshock protein 60 localizes in human atheroma and regulatesmacrophage tumor necrosis factor-α and matrix metallopro-teinase expression. Circulation 1998; 98: 300-7.

081. Cornelussen RN, Vanagt WY, Prinzen FW, Snoeckx LH.Proteins involvement in salvage of the myocardium. Adv ExpMed Biol 2003; 543: 277-91.

082. Zügel U, Kaufman SH. Role of heat shock proteins in protec-tion from and pathogenesis of infectious diseases. Clin MicrobiolRev 1999; 12: 19-39.

083. Gleimer M, Parham P. Stress management: MHC class I andclass I-like molecules as reporters of cellular stress. Immunity2003; 19: 469-77.

084. Hirono S, Dibrov E, Hurtado C, Kostenuk A, Ducas R, PierceGN. Chlamydia pneumoniae stimulates proliferation of vascularsmooth muscle cells through induction of endogenous heatshock protein 60. Circ Res 2003; 93: 710-6.

085. Asea A. Chaperokine-induced signal transduction pathways.Exerc Immunol Rev 2003; 9: 25-33.

086. Lamb DJ, El-Sankary W, Ferns GA. Molecular mimicry in ath-erosclerosis: a role of heat shock proteins in immunization.Atherosclerosis 2003; 167: 177-85.

087. Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA.Cutting edge: heat shock protein (HSP) 60 activates the innateimmune response: CD14 is an essential receptor for HSP 60 acti-vation of mononuclear cells. J Immunol 2000; 164: 13-7.

088. Campisi J, Leem TH, Fleshner M. Stress-induced extracellu-lar Hsp 72 is a functional significant danger signal to theimmune system. Cell Stress Chaperones 2003; 8: 272-86.

089. Mayr M, Metzler B, Kiechl S, et al. Endothelial cytotoxicitymediated by serum antibodies to heat shock proteins of Esche-richia coli and Chlamydia pneumoniae. Circulation 1999; 99:1560-6.

Claudio Blasi

261

090. Pockley AG. Heat shock proteins, inflammation, and cardio-vascular disease. Circulation 2002; 105: 1012-7.

091. Knoflach M, Kiechl S, Kind M, et al. Cardiovascular risk fac-tors and atherosclerosis in young males. ARMY study (Athero-sclerosis Risk-Factors in Male Youngsters). Circulation 2003;108: 1064-9.

092. Zal B, Kaski JC, Arno G, et al. Heat shock protein 60-reactiveCD4+CD28 nullT cells in patients with acute coronary syn-dromes. Circulation 2004; 109: 1230-5.

093. Schett G, Xu Q, Amberger A, et al. Autoantibodies against heatshock protein 60 mediate endothelial cytotoxicity. J Clin Invest1995; 96: 2569-77.

094. Tousoulis D, Davies G, Stefanadis G, Toutouzas P, AmbroseJA. Inflammatory and thrombotic mechanisms in coronaryatherosclerosis. Heart 2003; 89: 993-7.

095. DeGraba TJ. The role of inflammation in atherosclerosis. AdvNeurol 2003; 92: 29-42.

096. Biasucci LM, Liuzzo G, Ciervo A, et al. Antibody response tochlamydial heat shock protein 60 is strongly associated withacute coronary syndromes. Circulation 2003; 107: 3015-7.

097. Tiran A, Tio RA, Ossewaarde JM. Coronary angioplastyinduces rise in Chlamydia pneumoniae-specific antibodies. JClin Microbiol 1999; 37: 1013-7.

098. Gorge J, Greenberg S, Barshack I, et al. Immunity to heatshock protein 65 - an additional determinant in intimal thick-ening. Atherosclerosis 2003; 168: 33-8.

099. Shi Y, Tokunaga O. Chlamydia pneumoniae and multipleinfections in the aorta contribute to atherosclerosis. Pathol Int2002; 52: 755-63.

100. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q. Is ath-erosclerosis an immunologically mediated disease? ImmunolToday 1995; 16: 27-39.

101. Balin BJ, Gerard HC, Arking EJ, et al. Identification and local-ization of Chlamydia pneumoniae in the Alzheimer’s brain. MedMicrobiol Immunol 1998; 187: 23-42.

102. Gilden DH. Chlamydia a role for multiple sclerosis or more con-fusion? Ann Neurol 1999; 46: 6-14.

103. Gérard HC, Wang GF, Balin BJ, Schumacher HR, Hudson AP.Frequency of apolipoprotein E (APOE) allele types in patientswith Chlamydia-associated arthritis and other arthritides.Microb Pathog 1999; 26: 35-43.

104. Ewald PW, Cochran GM. Chlamydia pneumoniae and car-diovascular disease: an evolutionary perspective on infectiouscausation and antibiotic treatment. J Infect Dis 2000; 81 (Suppl3): S394-S401.

105. Sriram S, Stratton CW, Yao SY, et al. Chlamydia pneumoni-ae infection of the central nervous system in multiple sclero-sis. Ann Neurol 1999; 46: 6-14.

106. Beatty WL, Morison RP, Byrne GI. Persistent Chlamydiae: fromcell culture to a paradigm for chlamydial pathogenesis. Micro-biol Rev 1994; 58: 686-99.

107. Higgins JP. Chlamydia pneumoniae and coronary artery dis-ease: the antibiotic trials. Mayo Clin Proc 2003; 78: 321-32.

108. Fong IW. Value of animal models for Chlamydia pneumoni-ae-related atherosclerosis. Am Heart J 1999; 138: S512-S513.

109. Gupta S, Leatham EW, Carrington D. Elevated Chlamydiapneumoniae antibodies, cardiovascular events and azithromycinin male survivors of myocardial infarction. Circulation 1997;96: 404-7.

110. Grayston JT. Antibiotic treatment of atherosclerotic cardio-vascular disease. Circulation 2003; 107: 1228-30.

111. Cercek B, Shah PK, Noc M, et al. Effect of short-term treat-ment with azithromycin on recurrent ischaemic events inpatients with coronary syndrome in the Azithromycin in AcuteCoronary Syndrome (AZACS) trial: a randomised controlledtrial. Lancet 2003; 361: 809-13.

112. Grayston JT. Background and current knowledge of Chlamydiapneumoniae and atherosclerosis. J Infect Dis 2000; 81 (Suppl3): S402-S410.

113. Anderson JL, Muhlestein JB, Carlquist J, et al. Randomized sec-ondary prevention trial of azithromycin in patients with coro-nary artery disease and serologic evidence for Chlamydiapneumoniae infection: the Azithromycin in Coronary ArteryDisease: Elimination of Myocardial Infection with Chlamydia(ACADEMIC) study. Circulation 1999; 99: 1540-7.

114. Meier CR, Derby LE, Jick SS, Vasilakis C, Jick H. Antibioticsand risk of subsequent first-time myocardial infarction. JAMA1999; 281: 427-31.

115. Liuba P, Persson J, Luoma J, Ylä-Herttuala S, Pesonen E.Acute infection in children are accompanied by oxidative mod-ification of LDL and decrease of HDL cholesterol, and are fol-lowed by thickening of carotid intima-media. Eur Heart J2003; 24: 515-21.

116. Stephens RS. Chlamydial genomics and vaccine antigen dis-covery. J Infect Dis 2000; 81 (Suppl 3): S521-S523.

117. Shirai M, Hirakawa H, Ouchi K, et al. Comparison of outermembrane protein genes omp and pmp in the whole genomasequences of Chlamydia pneumoniae isolates from Japan andthe United States. J Infect Dis 2000; 81 (Suppl 3): S524-S528.

118. Brunham RC, Zhang DJ, Yang X, McClarty GM. The poten-tial for vaccine development against Chlamydia infection anddisease. J Infect Dis 2000; 81 (Suppl 3): S538-S543.

119. Slepenkin A, Motin V, de la Maza LM, Peterson EM. Temporalexpression of type III secretion genes of Chlamydia pneumo-niae. Infect Immun 2003; 71: 2555-62.

120. Brunham RC, Zhang DJ. Transgene as vaccine for chlamydia.Am Heart J 1999; 138: S519-S522.

121. Fong IW. Antibiotics effects in a rabbit model of Chlamydiapneumoniae-induced atherosclerosis. J Infect Dis 2000; 181(Suppl 3): S514-S518.

122. Harats D, Yacov N, Gilburd B, Schoenfeld Y, George J. Oraltolerance with heat shock protein 65 attenuates Mycobacteriumtuberculosis-induced and high-fat-diet-driven atheroscleroticlesions. J Am Coll Cardiol 2002; 40: 1333-8.

123. Maron R, Sukhova G, Faria AM, et al. Mucosal administrationof heat shock protein-65 decreases atherosclerosis and inflam-mation in aortic arch of low-density lipoprotein receptor-defi-cient mice. Circulation 2002; 106: 1708-15.

124. Moling O, Mian P. Induction of oral tolerance as treatment orprevention of chronic diseases associated with Chlamydiapneumoniae infection - hypothesis. Med Sci Monit 2003; 9:HY15-HY18.

125. Birk OS, Gur SL, Elias D, et al. The 60-kDa heat shock pro-tein modulates allograft rejection. Proc Natl Acad Sci USA1999; 96: 5159-63.

126. Leinonen M. Chlamydia pneumoniae and other risk factors foratherosclerosis. J Infect Dis 2000; 81 (Suppl 3): S414-S416.

Manuscript received on 23.4.2004; accepted on 1.9.2004.

Address for correspondence:Dr. Claudio Blasi, Largo T. Solera 7, 00199 Roma. E-mail: clblasi@tin.it