Serum chlamydial lipopolysaccharide as a prognostic factor for a new cardiovascular event

ISSUES IN CARDIOVASCULAR NURSING

Serum chlamydial lipopolysaccharide as a prognostic factor for a new cardiovascular event Erkki Pesonen, MD, PhD,a Terttu Tiirola, PhD,d Eeva Andsberg, BM,a Matti Jauhiainen, PhD,d Matti Paldanius, MD, PhD,d Kenneth Persson, MD, PhD,c Pekka Saikku, MD, PhD,e Seppo Sarna, D Soc Sc,f Hans Öhlin, MD, PhD,b and Maija Leinonen, PhDd

BACKGROUND: Infections caused by Chlamydia pneumoniae are considered to participate in inflammatory processes leading to coronary artery disease. After a primary infection, the bacteria remain dormant intracellularly causing a chronic inflammatory stimulus. MATERIALS AND METHODS: Blood samples were obtained from 235 patients with acute myocardial infarction (AMI) and 108 patients with unstable angina pectoris (UA). We evaluated the prognostic significance of bacterial and viral antibody titers, serum troponin T, C-reactive protein, and chlamydial lipopolysaccharide (cLPS) concentrations during acute coronary syndrome of patients with AMI and UA for cardiovascular death and new UA and AMI that required hospital care during a 6-year follow-up. RESULTS: Serum cLPS levels correlated with C-reactive protein and serum troponin T concentrations during acute coronary events. Patients with AMI had significantly higher serum concentration of cLPS compared with patients with UA. Enterovirus antibody titers and cholesterol-lowering therapy at admission of the index event were negatively correlated with cLPS concentration (r ⫽ ⫺.198, P ⫽ .0003 and r ⫽ ⫺.26, P ⫽ .019, respectively). The presence of circulating cLPS was associated with a hazard ratio of 2.04 for a new cardiovascular event during the follow-up period (P ⫽ .006). The area under the curve in the receiver operating graph was .572. CONCLUSION: cLPS is evidently liberated from the infected atherosclerotic tissue during an acute coronary event. Our study supports the view that inflammation caused by C. pneumoniae infection is an important but as yet poorly understood factor in the development of atherosclerosis and may play a role in acute vascular events. (Heart Lung® 2009;38:176 –181.)

A

therosclerosis is currently considered an inflammatory disease. Inflammatory cells and cytokines indicative of an active local inflammatory response are present in atherosclerotic lesions.1 During an acute respiratory infecFrom aPediatric Cardiology, bCardiology, University Hospital of Lund, Sweden; cMedical Microbiology, University Hospital of Malmö, Sweden; dNational Public Health Institute, Oulu and Helsinki; eDepartment of Medical Microbiology, University of Oulu, Finland; and fDepartment of Public Health, University of Helsinki, Finland. The study was supported by grants from Lund University, Lund University Hospital, Academy of Finland, and Finnish Funding Agency for Technology and Innovation. Corresponding author: Erkki Pesonen, MD, PhD, Department of Pediatrics, Lund University Hospital, Getingevägen 4, SE-221 85 Lund, Sweden. 0147-9563/$ – see front matter Copyright © 2009 by Mosby, Inc. doi:10.1016/j.hrtlng.2008.06.001

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tion, Chlamydia pneumoniae bacteria may invade the lungs and enter the circulatory system in monocytes. They subsequently invade the subendothelial space of arteries and establish a persistent infection in the vasculature.2-4 Recent studies indicate that systemic and local inflammatory processes are associated with atherosclerotic disease.5 It has been suggested that infections, such as those caused by C. pneumoniae, induce inflammatory processes that lead to progression of atherogenesis. C-reactive protein (CRP) is a component of the acute phase response, and its production is induced by infection and tissue damage, such as that caused by acute myocardial infarction (AMI). Slightly elevated CRP, measured with highly sensitive assays, is a marker of low-grade systemic inflammation and seems to be a risk factor for cardiovascular disease,

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myocardial infarction (MI), and chronic coronary heart disease.5 Chlamydial lipopolysaccharide (cLPS) is a genusspecific antigen that is surface exposed and present in elementary and reticular bodies.6 LPS is considered a major antigen in gram-negative bacteria, and it can elicit a variety of pathophysiologic responses, including fever, coagulant activity, and septic shock7-9 by activating an acute inflammatory response that includes the production of various inflammatory cytokines.7,10,11 We recently developed a quantitative enzyme immunoassay (EIA) for measurement of serum cLPS.12 Serum cLPS is first solubilized with detergent and then captured by LPS-binding protein (LBP). LBPLPS complexes are bound to the solid phase with anti-cLPS monoclonal antibodies, and the bound complexes are detected with anti-LBP antibodies. By using this method, we have shown that the patients with acute coronary syndrome have higher positivity rate and higher levels of serum cLPS than their age- and sex-matched controls12 and that the cLPS levels decrease after an acute event concomitantly with CRP levels.13 In the present study, we compared the presence of several viral (eg, enterovirus) and bacterial antibodies, measured as described in our earlier study with the same patients,14 as well as cLPS, CRP, and troponin T (TNT) concentrations in acute-phase serum obtained from patients with AMI or unstable angina pectoris (UA). We analyzed whether they predict future cardiac events. We hypothesized that elevated viral and bacterial titers or the presence of serum cLPS reflecting chronic C. pneumoniae infection are risk factors for future coronary events.

MATERIALS AND METHODS We followed 355 patients (276 male and 75 female) for 6 years (mean 6.02 years, range 4.06-7.13 years) who were admitted for AMI or UA to the coronary care unit, Lund University Hospital, between March of 1999 and April of 2002. The original inclusion criteria were age less than 80 years, no signs of cognitive intellectual disability, and competence to apply to the research protocol. Of these patients, 21 died before they could be interviewed. Of the invited patients, 48 chose not to participate. After admission, 12 patients were excluded from the study with the following diagnoses: unspecified precordial pain in 7 patients, atrial fibrillation in 1 patient, pericarditis in 1 patient, myocarditis in 1 patient, pulmonary embolism in 1 patient, and aortic aneurysm in 1 patient. AMI was diagnosed in 235

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patients, and UA was diagnosed in 108 patients. An earlier MI had been diagnosed in 37 patients. Informed consent was obtained from each patient for the participation and follow-up in the study. The Lund University Hospital ethical committee approved the study. The follow-up data were obtained from answers to the letters sent to the patients, hospital records, or mortality data from the population register. AMI was diagnosed if 2 of the following criteria were fulfilled: ST-segment elevation followed by Twave inversion or new Q-waves in the electrocardiogram, chest pain that lasted more than 20 minutes, and increase of CK-MB to more than twice the upper limit of the normal value, which at that time was more than 5 ␮g/L. UA was diagnosed in patients with continuous ischemic chest pain and transient or persistent ST-segment depression (⬎1 mm) or T-wave inversion (⬎1 mm), and/or elevation of CK-MB (10 ␮g/L ⬍ CKMB ⬎ 5 ␮g/L), or troponin T (0.10 ␮g/L ⬍ TNT ⬎ 0.05 ␮g/L). Blood and serum samples were collected within 24 hours of diagnosis during the first hospital admission for analyses of TNT, CRP, cLPS levels, and viral and bacterial antibodies. Bacterial serology included measurement of immunoglobulin (Ig)G and IgA antibodies against C. pneumoniae and C. pneumoniae heat shock protein 60 (CpnHSP60) and Helicobacter pylori. Viral serology included determination of antibodies against cytomegalovirus, enterovirus, and herpes simplex virus (HSV) titers as recently described.14 Serum cLPS was quantified by our recently developed EIA in the acute phase sera of patients with AMI and UA. Quality control samples were included in each analysis series. The intra-assay and interassay coefficients of variation were 6% and 12.8%, respectively. New patient events, such as hospitalization, MI, and death, were analyzed for correlations with the variables listed above. The authors had full access to the data to take responsibility for its integrity. All authors have read and agreed to the article written. The Lund University Hospital ethical committee approved the study. A written informed consent for the participation in the study was obtained from all subjects. There are no conflicts of interest among the authors.

Statistical methods Spearman rank correlations were used to evaluate correlations between variables. The chi-square test was used to compare the prevalence of smoking, diabetes, and lipid medication in the patient

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Table I Demographic data of patients with acute myocardial infarction or unstable angina pectoris No. of patients Percentage (%) with Smoking Diabetes Lipid medication Total cholesterol mmol/L LDL cholesterol mmol/L TNT ␮g/L CRP mg/L

AMI

UA

235

113

19 14 16 5.31 (1.37) 3.36 (1.19) .70 (2.22) 25.7 (44.5)

9 13 42 5.02 (.85) 3.08 (.83) .05 (.00) 3.36 (11.8)

P value

NS NS ⬍.001 NS NS ⬍.001 ⬍.001

AMI, Acute myocardial infarction; UA, unstable angina pectoris; LDL, low-density lipoprotein; TNT, troponin T; CRP, C-reactive protein; NS, not significant. Prevalence of diabetes, smoking, and lipid medication is presented as percentages (%) and concentrations as means; standard deviations are in parentheses.

groups. The Mann–Whitney test was used to compare cLPS concentrations in patients with AMI and UA. A linear-by-linear association test was used to compare cLPS concentrations in the patients with UA and patients with Q-wave and non–Q-wave MI. The Cox proportional hazards model was used to calculate hazard ratios (HRs) and 95% confidence intervals for hospitalization or death during the follow-up. The receiver operating curve was generated and area under the curve was calculated to estimate the predictive value of the model. Data were analyzed using the Statistical Package for the Social Sciences for Windows software, version 15.0 (SPSS Inc, Chicago, Ill).

RESULTS The mean age of the patients was 63 years (32-82 years, median 64 years). Twenty percent of the patients were aged 55 years or less, and 30% of the patients were aged 70 years or more. At the first hospital admission, AMI was diagnosed in 235 patients and UA was diagnosed in 108 patients with LPS measurements. Thirty-seven of the patients had experienced a previous AMI. cLPS was present in the sera of 140 patients (60%) with AMI and 44 patients (41%) with UA. Demographic data, including smoking history, diabetes, and lipid medication, are shown in Table I. In addition, 84% of the patients were positive for CpnHSP60 IgG, 80% of the patients were positive for C. pneumoniae IgG, and 64% of the patients were positive for C. pneumoniae IgA antibodies. cLPS was

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present in the serum of 66% of the patients with AMI and 64% of the patients with UA who died during follow-up. All patients with cLPS present also had serum CpnHSP60 IgA antibodies. Serum total cholesterol levels were inversely correlated with the presence of cLPS in the serum (odds ratio ⫽ .771; 95% confidence interval, .621.958; P ⫽ .019). No association was found between cLPS concentration and diabetes, smoking, or lipid medication at admission of the index event. cLPS concentrations correlated significantly with CRP (r ⫽ .433, P ⬍ .001) and TNT (r ⫽ .251, P ⬍ .001) concentration. They were higher in patients with AMI compared with patients with UA (26.2 ng/mL vs 13.2 ng/mL) (Table II). A linear-by-linear association test showed a significant trend for higher cLPS concentrations in patients with AMI compared with patients with UA (P ⫽ .004). cLPS did not correlate with C. pneumoniae antibody titers or with other bacterial and viral antibody titers (data not shown), except those of enterovirus, which displayed an inverse correlation with cLPS concentration (r ⫽ ⫺.198, P ⫽ .0003) (Table III). During the 6-year follow-up, 62 patients died, and the cause of death had a cardiovascular origin in 36 of those patients. A new MI occurred in 37 patients, and 95 patients received hospital care for severe UA. Thus, death or a new MI or severe UA that demanded hospitalization occurred in 194 of the 343 patients. In a Cox proportional hazards model, the presence of serum cLPS had an HR of 1.89 (P ⫽ .13) for

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Table II Chlamydial lipopolysaccharide concentration in acute myocardial infarction and unstable angina pectoris

No.

Median

Mean (range)

All Q Non Q Males Females

235 113 122 180 55

9 9 9 10 2

26.2* (0–181) 26.3 (0–181) 26.0 (0–177) 28.2 (0–181) 19.5 (0–122)

All Males Females

95% CI for HR

Serum cLPS ng/mL

Acute myocardial infarction

Unstable angina pectoris

Table IV Hazard ratios for a new cardiovascular event during 6-year follow-up

Serum cLPS ng/mL No.

Median

Mean (range)

113 95 18

9 10 2

13.3* (0–121) 14.2 (0–121) 8.2 (0–54)

cLPS, Chlamydial lipopolysaccharide; Q, Q-wave infarction; Non Q, non–Q-wave infarction. *P value between patients with acute myocardial infarction and unstable angina pectoris ⫽ .0007 (Mann– Whitney test).

HR

P Lower Upper value

Troponin T .859 .617 CRP .996 .989 Enterovirus 1.938 .830 antibody Anti-lipid 1.813 1.067 medication cLPS1 2.042 1.233

1.198 1.003 4.522

.371 .280 .126

3.081

.028

3.380

.006

CI, Confidence interval: HR, hazard ratio; CRP, C-reactive protein; cLPS, chlamydial lipopolysaccharide. Dichotomous cLPS 0 or ⬎ 0.

addition to death during the 6-year follow-up, HR for cLPS was significant (HR 2.04, P ⫽ .006). HR was adjusted for TNT, enterovirus antibody titer, lipid medication, and cLPS (Table IV). The area under the curve in the receiver operating graph was .572, indicating a poor predictive value of the model.

DISCUSSION Table III Spearman rank correlation between chlamydial lipopolysaccharide and C-reactive protein concentrations, troponin T concentration, and enterovirus titers CRP

cLPS r .433 P value ⬍.001

Troponin T Enterovirus

.251 ⬍.001

⫺.198 .003

CRP, C-reactive protein; cLPS, chlamydial lipopolysaccharide.

death associated with cardiovascular disease, but TNT concentrations during the first cardiovascular event reflecting the size of myocardial damage estimated was not a risk factor for future events (HR for TNT .80). HR for cardiovascular-related death or an additional cardiac event was significant for lipid medication at admission of the index event (HR 1.81, P ⫽ .028). When a new MI or UA event that required hospital care was taken into account in

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Perhaps the most interesting clinical observation of the present study was that the presence of serum cLPS during the first acute cardiac event indicated an increased risk for a new cardiovascular event (HR ⫽ 2.04) even if its predictive value was poor, possibly because of frequent false-positive samples. Elevated viral or bacterial titers at the coronary event did not predict later morbidity to coronary disease, which supports the idea that they were more closely related to an acute infection process precipitating a coronary event. cLPS seems to be a marker of a chronic inflammatory process. cLPS was present in approximately half of the patients, which is similar to the reported incidence of previous C. pneumoniae infection in the adult population,15 whereas 66% and 64% of those patients with AMI and UA who died were cLPS positive. We previously reported that cLPS concentration decreased within 3 months after a coronary event in another patient population13 but remained at a slightly elevated level, evidently as a marker of ongoing persistent C. pneumoniae infection. We reported earlier that the patients in the present series have higher enterovirus, HSV, and CpnHSP60 anti-

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body titers than their paired controls.14 New coronary events may subsequently be precipitated by any microbial infection, stress, or immunosuppressant that reactivates the latent C. pneumoniae infection. In mouse models, it is demonstrated that immunosuppression can reactivate chronic pulmonary C. pneumoniae infection.16 An interesting finding was a significant negative correlation between cLPS and enterovirus antibodies. Antagonism and synergy of different microbial species are well-known phenomena. Enteroviruses and chlamydiae are intracellular microbes, and it is possible that cells infected by one organism may produce cytokines and chemokines that suppress the other. Antibodies to CpnHSP60 have been shown to cross-react with enterovirus structures,17 providing the possibility of cross-protection. Enterovirus antibodies measured years before an acute coronary event have been shown to be a risk factor for coronary disease.18,19 In those studies, the presence of enterovirus antibodies in patients and controls were compared, and time from sampling to coronary event was not taken into account. In the present series, enterovirus and HSV antibody titers and antibody levels against CpnHSP60 were elevated in association with an acute cardiac event,14 but they did not predict a future cardiac event. This suggests that an acute infection (new, reinfection, or reactivation of a chronic infection) is needed to trigger an acute coronary event. Lipid-lowering therapy at the admission of the index event was associated with an increased risk of coronary events, suggesting that patients with high cholesterol values are treated and that they have a more severe disease. On the other hand, patients with UA were treated significantly more often with lipid-lowering therapy than patients with AMI, which shows the protective effect of lipid therapy. We do not know which type of lipid medication the patients in this series received. Statins reportedly reduce coronary heart disease mortality, nonfatal MI, the need for revascularization, and stroke.20 The presence of cLPS in the serum indicates active, productive C. pneumoniae infection, whereas the presence of C. pneumoniae antibodies shows that a person has been exposed to chlamydia in the past. Thus, high cLPS levels during acute coronary syndrome suggest the liberation of cLPS from damaged tissue with active infection. This is also supported by our recent finding that cLPS is found in more than 20% of atherosclerotic carotid plaques obtained from patients with symptomatic carotid stenosis by the commercial EIA method.21

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The EIA used for the measurement of cLPS in serum is a new method; the reliability, validity, and specificity of this method have been widely discussed in our previous articles.12,13 The EIA-method for measurement of cLPS used in the present study detects cLPS from all chlamydial species. In genital infections, the amount of cLPS in the lesions is evidently too low to be detectable in serum. However, in ascending complications of C. trachomatis infections, such as in pelvic inflammatory disease and perihepatitis, cLPS could be present in serum, leading to the positive finding in cLPS test. When there is a chronic C. pneumoniae infection in the lungs or in the vasculature, cLPS has easy access into circulation, and the present cLPS-EIA may be most useful in the diagnosis of chronic infection associated with chronic lung and cardiovascular diseases, which are most common in elderly populations, in whom C. trachomatis infections are rare. TNT concentration is a sensitive marker of the size of tissue damage, and CRP concentration reflects the severity of tissue damage. The significant correlation of TNT and CRP to cLPS concentrations suggests that cLPS may be liberated from infected, rupture-prone tissue during acute coronary events. Similarly, coronary angioplasty has been shown to induce an increase in C. pneumoniae-specific antibodies, suggesting that chlamydial components are liberated from plaques during the angioplasty procedure.22 Studies linking C. pneumoniae to inflammation and accelerated atherosclerosis have accumulated during the past years, but the precise mechanisms whereby this and perhaps many other pathogens could exert noxious effects on the vessel wall remain elusive. Most of the evidence suggests that C. pneumoniae reaches the intima via circulating monocytes, which get “entrapped” in the arterial wall, particularly in areas affected by local inflammation. This may explain why, in some but not all studies, the prevalence of MI was found to be increased in patients with simultaneous signs of inflammatory activity before the event. Our study shows the organism persistence, not only of serologic markers but also of earlier infections. This is consistent with the view that persistent C. pneumoniae infection induces a chronic inflammatory response seen as a slightly elevated CRP level. In our earlier study,13 both cLPS and CRP levels decreased during a 1-year follow-up, but the correlation between them was even higher 1 year after than during an event. In agreement with the present findings, our previous findings also showed that immune complexes containing cLPS are present in the sera of patients with

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AMI and in chronic coronary heart disease.23,24 In addition, circulating monocytes with C. peumoniae DNA has been found to be a predictor of coronary artery disease in men and is considered to be a direct indicator of current infection.25 These findings support the idea that C. pneumoniae infection plays a role in the genesis of acute coronary events. The present study was limited because there was a small number of deaths. Therefore, the statistical power for risk of death was low. However, the presence of circulating cLPS seemed to be associated with an increased morbidity in patients with coronary disease. The finding is pathophysiologically important even if the risk for a new coronary event cannot be predicted from the presence of LPS in the serum because of the large number of false-positive cases.

CONCLUSIONS The study supports the hypothesis that C. pneumonia bacteria plays a role as a risk factor for coronary artery disease. The circulating cLPS is probably released from the infected atherosclerotic tissue after an acute coronary event. Chronic C. pneumonia infections seem to be a risk factor for a secondary coronary event. New studies in different study populations are needed to elucidate whether the presence of cLPS is a risk factor for the first cardiac event. The authors thank research nurses Laura Darcy and Annica Maxedius for their excellent job in interviewing the healthy control subjects, blood sampling, data collection, and its registration.

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