Association Between Chlamydia pneumoniae Antibodies and
Intimal Calcification in Femoral Arteries of Nondiabetic Patients
Arch Intern Med. 2002;162:594-599
Seppo Lehto, MD; Leo Niskanen, MD; Matti Suhonen, MD; Tapani Rönnemaa,
MD; Pekka Saikku, MD; Markku Laakso, MD
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.
Arch Intern Med.
2002;162:594-599
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.
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:
LDL Cholesterol = Total Cholesterol - HDL
Cholesterol - (0.45 Total Triglycerides).
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 (400) 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.
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.
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.
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).
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).
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).
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/Article Information
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).
Corresponding author and reprints: Markku Laakso, MD, Department of Medicine,
University of Kuopio, PO Box 1777, SF-70210 Kuopio, Finland (e-mail: [log in to unmask]).
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).
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Edward E.
Rylander, M.D.
Diplomat American
Board of Family Practice.
Diplomat American
Board of Palliative Medicine.