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Association Between Chlamydia pneumoniae Antibodies and Intimal Calcification in Femoral Arteries of Nondiabetic Patients
Seppo Lehto, MD;
Leo Niskanen, MD;
Matti Suhonen, MD;
Tapani Rönnemaa, MD;
Pekka Saikku, MD;
Markku Laakso, MD
Arch Intern Med. 2002;162:594-599.
ABSTRACT
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Background Chlamydia pneumoniae, a gram-negative bacterium,
has been suggested to be a risk factor for atherosclerosis. Calcium is a well-known
component of atherosclerotic plaques, but it is uncertain whether infectious
agents play a role in the calcification process of the arteries.
Patients To address this issue we investigated the association of Chlamydia antibodies with intimal arterial calcification as assessed
by soft tissue radiograms from the thigh region of 1373 nondiabetic Finnish
individuals aged 45 to 64 years.
Results At baseline, radiologically detectable intimal calcification in femoral
arteries was found in 172 (27%) of 638 men and 43 (6%) of 735 women (P<.001). The presence of intimal artery calcifications
was strongly related to conventional atherosclerotic risk factors and to Chlamydia antibodies. In Cox regression analysis, association
of Chlamydia antibodies with intimal artery calcification
persisted after extensive adjustment for other cardiovascular risk factors
(P = .04). A dose-response relationship was observed
between Chlamydia antibodies and intimal femoral
artery calcification (P = .006). The presence of
intimal artery calcification was strongly associated with an increased risk
of future coronary heart disease mortality (P<.001).
Conclusion Chlamydia antibodies are strongly associated
with intimal calcification of the femoral arteries.
INTRODUCTION
CHLAMYDIA PNEUMONIAE, a gram-negative obligate
respiratory pathogen, has been suggested to be a possible trigger or even
causative agent in the pathogenesis of atherosclerosis. This hypothesis was
first presented by Finnish investigators,1-2
who showed that patients with coronary heart disease (CHD) had elevated IgG
and IgA titers of antibodies and specific circulating immune complexes to Chlamydia. This result has been replicated in other populations,3-5 and, moreover, the presence
of Chlamydia particles in atherosclerotic lesions
in coronary and carotid arteries, the aorta, and abdominal aortic aneurysms
has been directly demonstrated using polymerase chain reaction and immunohistochemical
methods.6-9
These findings have led to eradication trials with antibiotics. In the rabbit
model, Chlamydia infection accelerates the development
of atherosclerosis, and treatment with azithromycin prevents it.10
Also, in human studies, azithromycin treatment may reduce the risk of cardiac
events in patients with unstable angina or non–Q wave myocardial infarction
(MI)11 or in male survivors of MI.12
Calcium has been a largely neglected, although well-known, component
of atherosclerotic plaques. The presence of calcification in the coronary
arteries as evaluated by electron-beam computed tomography has been suggested
to be a sensitive, although not specific, marker of CHD that may yield information
beyond traditional risk assessment.13 In the
early phase, atherosclerotic lesions are composed mainly of lipids; from the
third decade of life onward, the lesions are formed by progressive accumulation
of intracellular and extracellular lipids and foam cells.14
From the fourth decade of life, atheromatous lesions may evolve primarily
from fibrotic or calcific lesions. These lesions may further progress by surface
defects, hemorrhage, or thrombus formation into complicated lesions and clinical
manifestations. The early phases and progression of arterial calcification
are poorly understood. In atherosclerotic plaques, calcium is found as hydroxyapatite,
the form found also in the bone.15-16
Further similarities with calcium and bone metabolism are that atherosclerotic
calcification is characterized by local expression of osteopontin, osteonectin,
osteocalcin, and bone morphogenetic protein type 2 and the presence of "calcifying
vascular cells."16-21
However, it is not known whether infectious agents play any role in the calcification
process of the arteries. On the basis of the aforementioned results, we reasoned
that Chlamydia pneumonia antibodies might have a
connection to artery calcification. Therefore, we investigated in a population-based
sample of middle-aged individuals the association of arterial calcification
as assessed by soft tissue radiograms from the thigh region with Chlamydia antibodies.
PATIENTS AND METHODS
STUDY POPULATION AT BASELINE
A random sample of nondiabetic individuals born and currently living
in the Kuopio University Hospital district (eastern Finland) or in the Turku
University Central Hospital district (western Finland) was taken from the
population register containing all individuals aged 45 to 64 years. Of the
827 individuals in eastern Finland and 863 in western Finland originally eligible
for the study, 651 in eastern Finland and 730 in western Finland participated
in the study, giving participation rates of 79% and 85%, respectively. Two
participants in eastern Finland and 6 in western Finland were excluded from
the final analyses because diabetes mellitus was diagnosed at baseline. The
final nondiabetic study population consisted of 649 individuals in eastern
Finland and 724 in western Finland.
Comparison regarding some background variables was made between participants
and nonparticipants by using the central register of the Social Insurance
Institution. Participating and nonparticipating groups were similar with respect
to clinical characteristics.
STUDY PROGRAM AND METHODS AT BASELINE EXAMINATION: 1982-1984
The study program was carried out during one outpatient visit to the
Clinical Research Unit of the University of Kuopio or to the Rehabilitation
Research Center of the Social Insurance Institution. The methods have been
described in detail elsewhere.22 The visit
included an interview on the history of chest pain symptoms suggestive of
CHD, smoking, alcohol intake, physical activity, and the use of drugs. All
medical records of participants who reported during the interview that they
had been admitted to the hospital because of chest pain or symptoms suggestive
of stroke were reviewed. Review of the medical records was performed by 2
of us (M.L. in Kuopio and T.R. in Turku) after careful standardization of
the methods between the reviewers. The World Health Organization criteria
for verified definite or possible MI based on chest pain symptoms, electrocardiographic
changes, and enzyme determinations were used in the ascertainment of the diagnosis
of previous MI.23
Blood pressure was measured with the patient in the sitting position
after a 5-minute rest using a mercury sphygmomanometer and was read to the
nearest 2 mm Hg. Patients were classified as having hypertension if they were
receiving drug treatment for hypertension or if their systolic blood pressure
was at least 160 mm Hg or their diastolic blood pressure was at least 95 mm
Hg.
BIOCHEMICAL METHODS
All laboratory specimens were obtained at 8 AM, after a 12-hour fast.
Fasting plasma glucose concentration was determined using the glucose oxidase
method (Boehringer Mannheim, Mannheim, Germany). Serum lipid and lipoprotein
levels were determined from fresh serum samples drawn after a 12-hour overnight
fast. Serum total cholesterol and triglyceride levels were assayed using automated
enzymatic methods (Boehringer). Serum high-density lipoprotein (HDL) cholesterol
levels were determined enzymatically after precipitation of low-density lipoprotein
(LDL) and very low-density lipoprotein cholesterols with dextran sulfate and
magnesium chloride.24 The LDL cholesterol was
calculated using the Friedewald formula as follows:
.
In patients with a triglyceride value greater than 354 mg/dL (>4.0 mmol/L),
the LDL cholesterol concentration was not calculated. Chlamydia IgG and IgA antibodies were determined from blood samples drawn during
baseline examination. A simplified microimmunofluorescence modification with
one spot was used.25 Antigen of the Kajaani
6 strain of Chlamydia pneumoniae26
was obtained from Labsystems Oy (Helsinki, Finland). Incubation for 4-fold
serum dilutions was 1 hour. For IgG titrations, dilutions starting at 1:32
were used. For IgA titrations, the serum samples were absorbed by Gullsorb
treatment (Gull Laboratories, Salt Lake City, Utah),27
and dilutions were started at 1:10. All titrations were read by the same person
(P.S.) using dry magnification (x400) in a Leitz fluorescence microscope
with a 100-W mercury vapor lamp. Participants were classified as having elevated
titers for Chlamydia if the IgA titer for Chlamydia was 1/40 or greater and the IgG titer was 1/128 or greater,
as has been shown previously.1
RADIOLOGICAL METHODS
Native soft tissue radiograms of the thigh were taken with the patient
in a recumbent position. Radiological findings were analyzed by a radiologist
(M.S.) in random order without knowledge of the Chlamydia antibody titers of the patient. The lower limb artery calcifications
were divided according to the method of Lindbom28
into discrete plaque (intimal type) and uniform linear railroad track (medial
type) calcifications. Grading of arterial calcifications was carried out separately
on both sides by assessing the involvement of arterial trunks or branches.
The extent of intimal and medial calcifications was originally graded as follows:
1 indicates none; 2, slight (calcifications just visible involving the arterial
trunks or their branches of  5 cm long); 3, moderate (intermediate grade,
neither grade 2 nor grade 4); or 4, marked (considerable calcification of  50%
of the length of the arterial trunk with or without involvement of the arterial
branches). For statistical purposes, intimal calcifications were divided into
absent (grade 1) or present (grades 2-4), except in Figure 1, where they were graded as none (grade 1), moderate (grade
2 and grade 3), or severe (grade 4). The  coefficients for intraobserved
variation were 0.87 for intimal calcification and 0.88 for medial calcification.
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Figure 1. The presence of Chlamydia pneumoniae antibodies by the degree of intimal femoral artery
calcification.
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COLLECTION OF FOLLOW-UP DATA
In 1990, a postal questionnaire containing questions about hospitalization
because of acute chest pain and symptoms suggesting stroke or lower limb amputation
was sent to every surviving participant of the original study cohort. All
medical records of participants who died between the baseline examination
and December 31, 1989, or who reported in the questionnaire that they had
been admitted to the hospital because of the aforementioned symptoms were
reviewed. The World Health Organization criteria for definite or possible
stroke based on a clinical syndrome consisting of neurological signs or symptoms
persisting for longer than 24 hours were used to ascertain the diagnosis of
stroke, as in the baseline study. The criteria for MI and stroke were identical
to those in the baseline study. Copies of death certificates of patients who
had died were obtained from the files of the Central Statistical Office of
Finland. In the final classification of the causes of death, hospital and
autopsy records were used, if available. The end point evaluated in this study
was mortality from CHD (International Classification of
Diseases, Ninth Revision, codes 410-414).29 The study was approved by the ethics committees of
the University of Kuopio and the University of Turku.
STATISTICAL METHODS
Data analyses were performed using a statistical software program (SPSS/PC;
SPSS Inc, Chicago, Ill). The results of continuous variables are given as
mean ± SE or percentage. Differences between the groups were assessed
using the  2 test or the 2-tailed t
test for independent samples, when appropriate. The multivariate Cox regression
model and Kaplan-Meier survival curves were used to investigate the association
of cardiovascular risk factors with the incidence of fatal CHD events.
RESULTS
Patient characteristics and cardiovascular risk factors in relation
to the occurrence of intimal femoral artery calcification are given in Table 1. Patients with intimal artery calcification
were more often men, were older, were less obese, had a higher frequency of
previously verified MI, and more often had a history of smoking than did those
without artery calcification; the prevalence of hypertension was similar between
the groups. Furthermore, higher insulin levels and adverse lipid profiles
(elevated LDL cholesterol and total triglyceride levels and low HDL cholesterol
levels) were also associated with intimal artery calcification. The frequency
of medial artery calcification was 22.1% (278/1259) in nondiabetic participants,
but, as in patients with type 2 diabetes mellitus,30
no evident association with respect to conventional cardiovascular risk factors
was found (data not shown). Chlamydia pneumoniae
antibodies were also significantly associated with the occurrence of intimal
artery calcification (Table 1).
In the following statistical analyses, the IgA and IgG antibodies were combined,
but the conclusions were identical whether we used either of these variables.
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Table 1. Patient Characteristics and Cardiovascular Risk Factors in
Relation to the Presence of Intimal Femoral Artery Calcification at Baseline*
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Although the presence of intimal artery calcifications was, as expected,
strongly related to conventional atherosclerotic risk factors (Table 1), the association with Chlamydia
antibodies remained statistically significant even after extensive adjustment
for confounding factors (age, sex, area of residence, total cholesterol level,
smoking status, and hypertension) (Table
2). Even further adjustment for HDL cholesterol level, total triglyceride
level, and body mass index did not change the magnitude of this association
(Table 2).
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Table 2. Adjusted Hazard Ratios for the Association of Chlamydia pneumoniae Antibodies With the Presence of Intimal Femoral
Artery Calcification (Cox Regression Model)*
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When the presence of intimal artery calcification was based on 3 categories
(none, moderate, and severe), a dose-response relationship was observed in
the occurrence of Chlamydia antibodies (Figure 1). In other words, the more severe
the arterial calcification, the more common the Chlamydia antibodies. No statistically significant association of medial artery
calcification with Chlamydia antibodies was observed
(none, 9.5%; moderate, 11.1%; severe, 12.4%; P =
.25).
Smoking was markedly associated with intimal artery calcification and Chlamydia antibodies, blunting this relationship in smokers,
but in nonsmokers, the presence of Chlamydia antibodies
was markedly higher in individuals with intimal artery calcification (Figure 2).
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Figure 2. Association of Chlamydia pneumoniae antibodies with intimal artery calcification
in relation to smoking status. Chlam+ indicates Chlamydia antibodies present; Chlam-, Chlamydia
antibodies absent.
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We also investigated the relationship between radiological intimal artery
calcification and CHD mortality during 7-year follow-up using the Cox regression
model. As shown in Figure 3, the
presence of intimal artery calcification was strongly associated with an increased
risk of CHD mortality. This relationship remained statistically significant
even after adjustment for other cardiovascular risk factors or risk modifiers,
including age, sex, body mass index, total cholesterol level, hypertension,
and smoking status. However, the risk of CHD mortality in patients with and
without intimal femoral artery calcification was not modified by the presence
of Chlamydia antibodies (Figure 3).
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Figure 3. Kaplan-Meier estimates of the
probability of death caused by coronary heart disease in participants with
(Calc+) and without (Calc-) intimal femoral artery calcification according
to the presence (Chlam+) or absence (Chlam-) of Chlamydia
pneumoniae antibodies (P<.001 between the
Calc-, Chlam- and Calc-, Chlam+ groups vs other groups).
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COMMENT
The major findings of this large, population-based study were that the
presence of chlamydia antibodies was associated in a dose-response fashion
with the degree of intimal artery calcification and that this association
was not explained by other cardiovascular risk factors. These findings suggest,
but do not prove, that Chlamydia may play a fundamental
role in the pathogenesis of calcification of atherosclerotic lesions.
In this study, native soft tissue radiographs were used to visualize
artery calcifications. Separation of the intimal and medial forms of calcification
is in most instances easily done.31 Chlamydia antibodies were associated with intimal artery calcifications
and not with medial artery calcifications. The former type of calcification
represents obstructive atherosclerosis, whereas the latter is a nonobstructive
calcification of the medial layer commonly associated with aging and diabetes
mellitus.32-33 However, as an
index of atherosclerosis, the radiological intimal artery calcifications are
rather crude, and radiological examination is likely to underestimate the
degree of atherosclerosis.34 However, this
method is sensitive, cheap, readily available, and, as shown in this study,
a strong predictor of CHD mortality.
Chlamydia antibody titers may decrease substantially
within a few years after seroconversion and may increase with the occurrence
of reinfection. This temporal variation implies that any association of vascular
disease with antibody titers for Chlamydia measured
only once is substantially weaker than associations of vascular disease with
long-term antibody concentration or more direct evidence of persistent infection
at the relevant anatomical site.35 Therefore,
it is remarkable that in our study the severity of intimal artery calcification
increased linearly with the occurrence of Chlamydia
antibodies. This association is suggestive of a close association of these
processes, although suggestions of causality must be viewed with great caution.
The mechanisms via which Chlamydia can increase
the risk of atherosclerosis remain unclear. Potentially it may precipitate
acute cardiovascular events (plaque rupture) or may increase the size of the
atherosclerotic plaque. This association could be mediated, at least in part,
by an indirect effect of an adverse pattern of known or potential cardiovascular
risk factors. Indeed, Chlamydia is associated with
an adverse profile of serum lipids and lipoproteins,36
coagulation factors, and oxidative metabolites.37
Furthermore, smoking is strongly associated with arterial calcification, and
as smokers more frequently have Chlamydia antibodies,38 smoking could account for a significant proportion
of this effect. The relationship between Chlamydia
antibodies and arterial calcification was not particularly strong among smokers
in our study but was most evident in nonsmokers. Therefore, in our study and
some previous studies,3-4 the
association of Chlamydia with atherosclerosis is
not explained statistically by conventional risk factors. Other indirect mechanisms
behind this link could be mediated by chronic inflammation and/or cross-reactive
antibodies.35, 39 However, there
is also in vitro evidence for a more direct association because Chlamydia may infect and multiply in smooth muscle cells, macrophages,
and endothelial cells,40 all which may contribute
to the formation of atherosclerotic plaques. Our findings that Chlamydia antibodies were associated with intimal calcification but
not medial calcification are in accordance with the hypothesis that Chlamydia could be directly involved in the process of
calcification.
Although the accumulation of smooth muscle cells is a hallmark of atherosclerosis,
the frequency of replication of smooth muscle cells in atherosclerotic plaques
is in fact rather low.20 Therefore, other processes,
for example, cell migration, extracellular matrix formation, and calcification,
may be of even more pathophysiological significance, leading to the expression
of genes specific to atherosclerotic plaque not found in uninvolved arteries.
Potential candidates could be the genes that regulate the calcification process
in bones and arteries. Recently, osteopontin, a potent regulator of bone mineralization,
has been shown to be synthesized by macrophages, smooth muscle cells, and
endothelial cells,20 and osteopontin seems
to be an important mediator of arterial neointima formation and dystrophic
calcification.17 Another possible candidate
of arterial calcification is bone morphogenetic protein, which is expressed
in human atherosclerotic lesions.16 Therefore,
one may postulate that chronic Chlamydia infection
could induce the expression of these proteins, which promote an increase in
extracellular matrix and arterial calcification. Degenerative aortic stenosis
is characterized by many similarities with atherosclerotic process, such as
an active inflammatory process with lipid deposition and protein and calcium
accumulation,41 and by the immunohistochemical
presence of Chlamydia.42
In our study, Chlamydia antibodies were strongly
associated with intimal calcification of the femoral arteries, and the effects
of cardiovascular risk factors did not explain this association. Therefore,
it is possible that Chlamydia may also play a role
in the calcification process of atherosclerosis, a hitherto largely neglected
component of this disorder.
AUTHOR INFORMATION
Accepted for publication July 16, 2001.
This study was supported by grants from the Academy of Finland (Helsinki),
the Finnish Heart Research Foundation (Helsinki), the Sigrid Juselius Foundation
(Helsinki), and the Aarne and Aili Turunen Foundation (Kuopio).
Corresponding author and reprints: Markku Laakso, MD, Department
of Medicine, University of Kuopio, PO Box 1777, SF-70210 Kuopio, Finland (e-mail: markku.laakso{at}kuh.fi).
From the Departments of Medicine (Drs Lehto, Niskanen, and Laakso)
and Radiology (Dr Suhonen), University of Kuopio and Kuopio University Hospital,
Kuopio, Finland; the Department of Medicine, Turku University Central Hospital,
Turku, Finland (Dr Rönnemaa); the Research and Development Centre, Social
Insurance Institution, Turku (Dr Rönnemaa); and the National Public Health
Institute, Oulu, Finland (Dr Saikku).
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