Association Between Chlamydia pneumoniae Antibodies and Intimal
Calcification in Femoral Arteries of Nondiabetic Patients

 Arch Intern Med. 2002;162:594-599

Author Information
<http://archinte.ama-assn.org/issues/v162n5/rfull/#aainfo>   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
IOI10094
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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r1> , 2
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r2>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r3>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r6>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r10>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r11>  or in male
survivors of MI. 12 <http://archinte.ama-assn.org/issues/v162n5/rfull/#r12>
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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r13>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r14>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r15> , 16
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r16>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r16>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r22>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r23>
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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r24>  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 <http://archinte.ama-assn.org/issues/v162n5/rfull/#r25>
Antigen of the Kajaani 6 strain of Chlamydia pneumoniae 26
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r26>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r27>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r1>
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 Lindbom 28
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r28>  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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_f1.html> ,
where they were graded as none (grade 1), moderate (grade 2 and grade 3), or
severe (grade 4). The kappacoefficients 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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r29>  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 plusmnSE or percentage. Differences between the groups were assessed
using the chi2 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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_t1.html> .
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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r30>  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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_t1.html> ). 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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_t1.html> ), 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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_t2.html> ).
Even further adjustment for HDL cholesterol level, total triglyceride level,
and body mass index did not change the magnitude of this association ( Table
2 <http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_t2.html> ).
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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_f1.html> ). 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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_f2.html> ).
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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_f3.html> , 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
<http://archinte.ama-assn.org/issues/v162n5/fig_tab/ioi10094_f3.html> ).



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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r31>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r32> , 33
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r33>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r34>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r35>  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 <http://archinte.ama-assn.org/issues/v162n5/rfull/#r36>
coagulation factors, and oxidative metabolites. 37
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r37>  Furthermore,
smoking is strongly associated with arterial calcification, and as smokers
more frequently have Chlamydia antibodies, 38
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r38>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r3> , 4
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r4>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r35> , 39
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r39>  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 <http://archinte.ama-assn.org/issues/v162n5/rfull/#r40>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r20>  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
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r20>  and osteopontin
seems to be an important mediator of arterial neointima formation and
dystrophic calcification. 17
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r17>  Another possible
candidate of arterial calcification is bone morphogenetic protein, which is
expressed in human atherosclerotic lesions. 16
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r16>  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 <http://archinte.ama-assn.org/issues/v162n5/rfull/#r41>
and by the immunohistochemical presence of Chlamydia. 42
<http://archinte.ama-assn.org/issues/v162n5/rfull/#r42>
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] <mailto:[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.