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Missbichler A, Hawa G, Woloszczuk W, Schmal N, Hartter E Enzyme immunoassays for proBNP fragments (8–29) and (32–57) J Lab Med 1999;23:241–244 54 http://www.roche.com/ diagnostics/news/2002/020128.html 55 Downie PF, Talwar S, Squire IB, Davies JE, Barnett DB, Ng LL Assessment of the stability of N-terminal pro-brain natriuretic peptide in vitro: implications for assessment of left ventricular dysfunction Clin Sci (Colch) 1999;97:255–258 56 Nakamura M, Endo H, Nasu M, Arakawa N, Segawa T, Hiramori K Value of plasma B type natriuretic peptide measurement for heart disease screening in a Japanese population Heart 2002;87:131–135 Infectious Diseases in Atherosclerosis and ACS 425 Part VI Role of Infectious Diseases and Genetics in Heart Disease 426 Möckel Infectious Diseases in Atherosclerosis and ACS 427 27 Infectious Diseases in the Etiology of Atherosclerosis and Acute Coronary Syndromese Focus on Chlamydia pneumoniae Martin Möckel INTRODUCTION Several infectious agents including Herpes simplex virus, Cytomegalovirus, Helicobacter pylori, and Chlamydia pneumoniae have been investigated with respect to their role in the genesis of atherosclerosis Although the increasing number of articles published on this issue suggests a causal role of infectious agents, the matter is far from settled and yet not proven The fascination of the “infection hypothesis” of atherosclerosis has been stimulated by the recognition of H pylori (HP) playing a causal role in the pathogenesis of peptic ulcers Owing to the possibility of eradication of HP by antibiotic treatment, the disease has been changed completely during the last years Earlier reviews by Libby et al (1) and Danesh et al (2) in 1997 have summarized the evidence at that time that infectious agents may initiate, propagate, and complicate atherosclerosis During the last yr several new works have been published on this topic The data on C pneumoniae as an infectious agent that potentially plays a causal role in the development and progression of atherosclerosis in general and especially coronary artery disease seem to be most compelling Another important cause for the focus on C pneumoniae is that a cheap and well-tolerated antibiotic therapy is available DIFFERENT INFECTIOUS AGENTS STUDIED WITH RESPECT TO ATHEROSCLEROSIS AND MYOCARDIAL INFARCTION The different agents studied mostly were Cytomegalovirus, H pylori, and C pneumoniae Cytomegalovirus Cytomegalovirus has been recognized as a possible cause of atherosclerosis Published studies have been summarized by Danesh et al (2) and Libby et al (1) in 1997 Recent studies led to the cumulation of evidence that Cytomegalovirus does not play a crucial role in atherosclerosis (3–5) Analysis of blood samples from the Physicians’ Health Study with respect to antibodies against H simplex virus and Cytomegalovirus showed no increase of atherothrombotic risk in individuals with positive titers (3) A clear argument against a significant role of From: Cardiac Markers, Second Edition Edited by: Alan H B Wu @ Humana Press Inc., Totowa, NJ 427 428 Möckel Cytomegalovirus in the development of myocardial infarction comes from the study of Hernandez et al (5) in patients after renal transplantation It is well known that patients under immunosuppression are prone to viral infections and therefore have a higher incidence of new infection and reactivation of Cytomegalovirus In this population, incident cases of myocardial infarction should be in some way related to Cytomegalovirus, if this agent would play a causative role for atherosclerosis In fact, that is not the case Hernandez and colleagues clearly show that despite of a high event rate (11.6%) and a high rate of Cytomegalovirus disease (around one third of the 1004 consecutive patients) this was no significant risk factor (5) Helicobacter pylori As early as 1997, the study overview of Danesh et al (2) showed only a weak association of H pylori with atherosclerosis and myocardial infarction This has been confirmed by data from the Physicians’ Health Study (6) Recently, two reports have re-emphasized the role of H pylori Hoffmeister et al found an altered, atherogenic lipid profile in current H pylori but not in C pneumoniae or Cytomegalovirus seropositivity (7) This study concludes that “proof of principle” has been shown but did not report any association between H pylori and incident ischemic events in patients H pylori “infection” has been determined by [13C]urea breath test in this study A positive breath test is not strictly associated with local or systemic infection but could reflect local colonization only Therefore, the reported associations of lipid profile changes and H pylori positivity are perhaps not free of chance The second study by Hara et al (8) was a case control study in patients with acute or old myocardial infarction, stable or vasospastic angina, and age-matched controls The main result is an odds ratio of 4.09 (95% CI 0.79–21.11) for 21 patients with elevated IgA levels and acute myocardial infarction versus the other patient groups As the confidence limits include 1.0, the result did not show a clear association between H pylori infection and acute myocardial infarction In the light of other negative studies with more patients, the lack of animal models and a clear concept of principle, H pylori cannot be considered to play definitely a causal role in atherosclerosis Chlamydia pneumoniae The studies with respect to C pneumoniae are numerous and have conflicting results Danesh et al have summarized evidence that C pneumoniae may play a role in the development of atherosclerosis and myocardial infarction (2) The same group presented data from 5661 British men aged 40–59 yr who provided blood samples during 1978–1980 (9) The study results show an odds ratio of 1.7 comparing highest with lowest IgG titer tertiles with respect to incident ischemic heart disease After adjustment for age, town, smoking, and social class, the odds ratio was still 1.6 The authors then additionally adjust for childhood social class, which reduced the odds ratio to 1.2 I believe with others (10) that Danesh et al (9) did an “overadjustment” in this case and in some way “threw the baby out with the bathwater.” As newer data suggest especially an association of C pneumoniae with premature myocardial infarction (11), early infection may play an causative role In contrast to this positive study, the data of the Physicians’ Health Study have negative results irrespective of the IgG titer (12) Infectious Diseases in Atherosclerosis and ACS 429 Fig Immunofluorescence stain reveals infection of Hep-2 host cells with replicating C pneumoniae isolated from the occluded coronary artery of a 62-yr-old man (passage 15 after primary isolation) Multiple inclusions in the host cells are characteristic of C pneumoniae This cardiovascular strain is morphologically identical to the common respiratory isolates (From Maass et al [34] with permission.) SEROEPIDEMIOLOGICAL STUDIES Several seroepidemiological studies have been published with respect to the risk of atherothrombotic complications and C pneumoniae seropositivity or infections with other infectious agents Saikku et al were the first to show an association between C pneumoniae IgG titer of ³32, chronic coronary heart disease (CCHD), and acute myocardial infarction (AMI) The authors reported on paired sera from 40 male patients with AMI, 30 male patients with CCHD, and 41 age- and sex-matched controls The IgG titers were increased in 65% AMI patients, 50% CCHD patients, and 17% of controls (13) Danesh et al summarized the studies up to 1997 (2) Table gives an overview of the important studies published including the more recent articles It has to be mentioned that most of the studies were cross-sectional in design, and, because of the limited number of patients, control for all potential confounders was not possible In addition, the different cutoff antibody titers make comparison of the studies difficult, and it seems unlikely that all of these cutoffs were prospectively defined The study by Ridker et al (12), which was longitudinal in design, could not show any association of CAD and previous C pneumoniae infection In summary, the studies show conflicting data on the association between CAD and past C pneumoniae infection More recent studies show a positive correlation with high antibody cutoffs (21) or premature AMI (11) as target variable Prospective studies with other pathogens such as cytomegalovirus and Herpes simplex virus + showed an increased risk of MI or death with increased pathogen burden in a dose–response fashion (22,23) Thus, the inconsistencies in the seroepidemiological data could be due to the broad spectrum of different disease intensities and missing differentiation between past and chronic persistent infection 430 Möckel Table Seroepidemiological Studies with Respect to Elevation of C pneumoniae IgG Antibody Titers and Risk of CAD or Complications Source No of cases/controls Titer cutoff Saikku et al 1988 (6) 70/41 Age/sex matched IgG ³ 32 Thom et al 1991 (7) 461/95 IgG ³ 64 Matched for age and sex; controls were angiography patients without CAD 103/103 IgG ³ 64 Patients from Helsinki Heart Study, matched for treatment, locality and time point 171/120 IgG ³ Adjustment for age, sex, and calender quarter of blood drawing 326/326 IgG ³ Matched by age, race, sex, examination period, field center (ARIC substudy) 60/60 IgG ³ 32 Sex matched Saikku et al 1992 (8) Thom et al 1992 (9) Melnick et al 1993 (10) Dahlén et al 1995 (11) Ridker et al 1999 (4) Nieto et al 1999 (12) multiSiscovick et al 2000 (13) Chandra et al 2001 (14) Gattone et al 2001 (15) 343/343 (All male) Age/smoking matched 246/550 3.3 yr ARIC follow up IgG ³ 32 IgG ³ 64 213/405 IgG ³ Controls matched for several variables including major risk factors 830/IgG ³ 1024 120/120 Age matched; post AMI patients £ 50 Jahre IgG ³ 16 Results Titer positive in 68% of AMI and 50% of CCHD patients; 17% positivity in controls Odds ratio for CAD (compared to subjects with low [less or equal than 1:8] antibody titer): 2.0, 95% CI: 1.0/4.0 Odds ratio for the development of CHD: 2.3, 95% CI: 0.9/6.2 Odds ratio for CAD: 2.6, 95% CI: 1.4/4.8 Odds ratio for asymptomatic atherosclerosis: 2.0, 95% CI: 1.19/3.35 Odds ratio for angiographic CAD was 3.56, 95% CI: 0.99/16.10 with smoking as covariable Relative risk 1.0 for future MI (12-yr follow-up) Odds ratio for CHD: 1.6 (p < 0.01); not significant in multivariate analysis (1.2) Odds ratio for risk of MI and CV death: 1.1, 95% CI: 0.7/1.8, adjusted for several matching factors Odds ratio for ACS vs non-ACS: 1,62; unselected patients admitted to chest pain center Odds ratios for premature AMI: 2.4, 95% CI: 1.3/4.6; additional smoking: 3.7; additional CMV infection: 12.5 AMI, (acute) (old) myocardial infarction; ACS, acute coronary syndrome; ARIC, Atherosclerosis Risk in Communities Study; CAD, coronary artery disease; (C)CHD, (chronic) coronary heart disease; CI, confidence interval; CV, cardiovascular Infectious Diseases in Atherosclerosis and ACS 431 Fig Direct effects of infectious agents on intrinsic vascular wall cells (Reproduced from Libby et al [1] with permission.) C PNEUMONIAE IN ATHEROSCLEROTIC LESIONS Although the matter of seroepidemiologic evidence has become more confusing due to several negative studies published in the last yr, in the mid-1990s several research groups had been able to demonstrate C pneumoniae antigen in atherosclerotic lesions and therefore added some evidence to the infectious hypothesis of atherosclerosis In 1993, Kuo et al were first to identify C pneumoniae in atheromas of autopsy cases by use of the polymerase chain reaction (PCR; 43% positive) and immunocytochemistry (42% positive) (24) Further studies confirmed these findings (25–31) Except for a few studies using PCR only (32), C pneumoniae has been found in >50% and up to 100% of the lesions studied (33) It must be emphasized that not only could the antigen be detected but also isolation of viable bacteria was possible (34) Maass and co-workers were able to recover viable C pneumoniae from 11 (16%) of 70 atheromas (from cardiovascular surgical procedures) investigated (from surgical procedures, see Fig 1) (34) Therefore, the association of C pneumoniae and atherosclerosis appears to be established beyond a reasonable doubt The significance of the association for the development of atherosclerosis, the disease progression, and complications remains uncertain POSSIBLE MECHANISMS OF ATHEROSCLEROSIS DEVELOPMENT DUE TO C PNEUMONIAE The mechanisms that are possibly involved in the development of atherosclerotic lesions due to infectious agents are summarized in Fig (1) Atherosclerosis is becoming increasingly recognized as an inflammatory disease (35) The process probably starts with endothelial dysfunction in distinct arteries (36) In the progression of the disease “fatty streaks” appear in children and young adults (37) In subsequent decades, the disease progresses depending on concomitant risk factors such as diabetes mellitus, arterial hypertension, smoking, and so on The inflammatory process of atherosclerosis includes transformed macrophages (“foam cells”) as important players (35) Prior to the onset of complications such as acute coronary syndrome (ACS) or stroke, the atheromas 432 Möckel fibrous cap undergoes thinning The rupture of the fibrous cap can occur spontaneously or it is triggered by exhaustive exercise, extensive rise of blood pressure, or other factors Infectious agents may lead to a chronic inflammatory response with increased concentrations of proinflammatory cytokines and C-reactive protein (CRP) (35), which themselves contribute to a progression of the disease (38) Adhesion molecules may have an additional impact on ACS depending on the mode of therapy (39) It has been demonstrated in recent studies that CRP is an independent risk factor for complications of CAD (40–43) and that antiinflammatory properties of cholesterol synthetic enzyme (CSE) inhibitors may be beneficial in these patients (44–47) Taking into consideration that CRP itself appears to contribute to atherosclerotic lesion formation (38), it could be hypothesized that chronic inflammation, for example, by C pneumoniae or other infectious agents, therefore propagates the disease This concept was supported further by a study that showed that an increased pathogen burden increases the risk of adverse events in CAD patients (23) Several cofactors are potentially involved in the disease progression by chronic C pneumoniae infection including interleukin-1 gene polymorphism (48) and NF-kB-activation, induction of tissue factor, and plasminogen activator inhibitor (PAI)- expression (49) Future studies will need to address further important cofactors and the exact molecular mechanisms of the atherosclerosis development and progression by infectious agents such as C pneumoniae ANIMAL MODELS OF ATHEROSCLEROSIS DUE TO INFECTION To determine an etiological role for C pneumoniae for the development of atherosclerosis, some animal studies have been performed (50–53) In a rabbit model (New Zealand White rabbits), 11 animals were infected via the nasopharynx with C pneumoniae (TWAR strain VR 1310) Animals were killed after 7, 14, 21, and 28 d Atherosclerotic lesions were detected in two animals with fatty streaks at d and an intermediate lesion at d 14 (51) In a second study with New Zealand White rabbits, animals were infected and reinfected after wk Of nine reinfected rabbits, six (67%) showed inflammatory changes of the aorta consisting of intimal thickening or fibroid plaques resembling atherosclerosis 2–4 wk after reinfection (50) The third study with these rabbits included treatment with azithromycin, a macrolide antibiotic known to be effective against C pneumoniae and a modest cholesterol-enhanced diet In this study, 20 animals were infected by three separate intranasal inoculations of C pneumoniae Ten animals served as controls The infected animals were then divided into treatment and no treatment groups The main result was an increased maximal intimal thickness (MIT) in infected and nontreated (0.55 mm) vs control animals (0.16 mm, p = 0.009) Infected rabbits receiving antibiotics had a significantly lower increase of MIT (0.20 mm, p < 0.025 vs both other groups) Chlamydial antigen was detected in two untreated, three treated, and no control animals (52) Finally, Moazed et al investigated the influence of C pneumoniae infection on the aortic atherosclerotic areas in apolipoprotein E-deficient mice They found at 16 wk of age, a 1.6-fold larger atherosclerotic area compared to uninfected controls (53) In conclusion, the animal models consistently suggest a pathogenetic role of C pneumoniae in the development and progression of atherosclerosis It is not clear which cofactors are necessary, which molecular mechanisms are involved, and if the results can be attributed to humans because no studies in primate models have been undertaken yet 454 Wu showed an inverse relationship between the number of D alleles present and the minimum lumen diameter at mo after successful stent implantation (56) The follow-up to these observations was a therapeutic drug trial where these investigators examined the success of ACE inhibitors on the restenosis rate in a randomized double-blind placebocontrolled trial (57) To their surprise, they showed that the restenosis rate was not reduced by ACE inhibitor treatment, but in fact was exaggerated Others have shown no association with ACE DD genotype and stent placement with regard to 1-yr outcomes (58) The precise role of ACE polymorphism for ACS remains unclear at this time Because the RAA system is important in the pathophysiology of congestive heart failure (CHF), the effect of ACE polymorphisms has also been studied to determine if there is a correlation to CHF Like results for CAD, the studies conducted to date are conflicting Candy et al correlated ACE genotypes with left ventricular systolic performance as measured by ejection fraction using both echocardiography and radionuclide ventriculography (59) McNamara reported higher survival rates for CHF patients who were not treated with b-blockers (60) However, Montgomery was unable to find an association of ACE polymorphism in 99 patients with idiopathic dilated cariomyopathy (61) One explanation for the lack of an association between the DD polymorphism was suggested by Spruth et al (62), who found no difference in mRNA expression between the DD, ID, and II genotypes Finding genetic markers for patients with CHF may be more difficult than for patients with ACS because there may be more heterogeneity in the pathophysiology of CHF A polymorphism also exists in the gene for angiotensin receptor type 1, where there is a change from an adenine in nucleotide 1166 to a cytosine This mutation may be linked to increased prevalence of hypertension and coronary vasoconstriction A early study suggested that this polymorphism was linked to the ACE D/I polymorphism, that is, the combination of the DD and 1166CC phenotype produced a significant association for AMI (odds ratio: 3.95 95% CI: 1.26–12.4) (63) This association has not been confirmed in more recent studies (64) In a study in which angiotensin II was given to volunteers, investigators concluded that the A1166C polymorphism does not have an effect on the actions of angiotensin II (65) b -Blockers Cardiac inotropy and chronotropy are modulated in part by b1 and b2-adrenergic receptors expressed in the human heart Patients with heart failure have an overstimulation of neurohormones such as the catecholamines, resulting in a down-regulation of the b-receptors The use of b-blocker therapy improves clinical symptoms and cardiac output by slowing the heart rate and improving the rhythm Under a situation of increased RAA axis stimulation, polymorphisms in the b-receptors may predispose an individual to the development of heart failure There are many polymorphisms in the b1 receptor gene that encodes for a 478-aminoacid protein Many of these polymorphisms, however, are silent and thus their significance in development of CHF can be questioned (66) Two of these polymorphisms have been studied with regard to idiopathic dilated cardiomyopathy One involves nucleotide 145, in which there is a change from an adenine to a guanosine resulting in the substitution of serine for a glycine The other is at nucleotide 1165, where there is a change Polymorphisms Related to CAD 455 from a guanine to a cytosine resulting in a arginine to glycine substitution The allele frequencies for the 145G and 1165C are 15% and 25%, respectively These polymorphisms were studied in a cohort of patients with CHF In a study of 821 cases in controls, the 1165G polymorphism was not associated with idiopathic dilated cardiomyopathy (67) In another study, Borjesson et al found the 49G allele frequency was higher than for the 49S allele (68) These investigators also reported significant differences in survival rates with the mutation (odds ratio: 2.34, 95% CI: 1.30–4.20) The amino- (N)terminal sequence where the 49S mutation resides may be important to fold the receptor within the membrane, while not being the target for receptor activation These observations may help explain why some individuals respond to b-blockade while others not Prospective pharmacogenomic trials for different b1 and b2 blockers will be the next logical step in this area Three common polymorphism in the b2 receptor are in amino acid position 16 substituting an arginine for a glucine, position 27 substituting a glutamine for a glutamic acid, and 164 substituting an arginine for an isoleucine The allele frequencies for the respective polymorphisms are 61%, 43%, and 2% The polymorphism in positions 16 and 27 have different susceptibilities to agonist-induced down-regulation The 164 polymorphism exhibits a decreased affinity for b2-adrenogeric angonists, and an uncoupling of receptors from the Gs protein In volunteer subjects, the increase in heart rate and systolic blood pressure among those with the Thr164Ile polymorphism was less than for the wild-type (69) In a study of 471 case and controls, Liggett et al found no difference in the allele frequencies for CHF patients and controls (70) However, when the 1-yr outcomes were compared, patients with the 164 ile polymorphism had a higher death risk rate and need for cardiac transplantation than CHF patients with the wild-type mutation SUMMARY Although many of these genes demonstrate a significant correlation with polymorphisms and the incidence of CAD or heart failure, the degree of risk is not particularly high in any case (odds ratio typically between and 2) Most investigators agree that the pathogenesis of these diseases is too complex and diverse to be explained on the basis of a single gene mutation Discrepancies in results of individual studies may be the result of inadequate numbers of subjects enrolled or heterogeneity in the sex and/or racial make of the population studied However, several studies have shown that the presence of some mutations is linked to others and their effect on disease progression may be additive Unfortunately, all of the studies published to date involve only a handful of gene mutations on a limited number of subjects enrolled The costs for performing the DNA analysis prohibits the analysis for more than just of few of these genes at any given time This chapter discussed only a minority of the polymorphisms that are known to exist among the various proteins and factors known to participate in the disease However, the technology for detecting gene mutations is changing with the development of reusable microarrays or “gene chips.” In the very near future, it will become practical to examine 50 or 100 genes following a single amplification of DNA from the same individual Polymorphisms that are not correlated to CAD alone may be important when used in combination with other factors 456 Wu Genes that have been initially targeted are those proteins known to participate in the pathophysiology of cardiac diseases Given that none of these factors studied to date have produced particularly high odds ratios (i.e., they are typically