I. Introduction
Only half of coronary artery
disease, and half of carotid plaque measured by ultrasound, can be
explained by the usual risk factors: age, sex, hypertension,
hyperlipidemia, smoking, and diabetes. It is likely that much of unexplained
atherosclerosis is genetic: a Swedish twin study showed that
myocardial infarction heritable this suggests that few environmental factors
remain to be discolved that would make a major contribution to
atherosclerosis.Recently, the notion that infection may be important in
atherosclerosis has been of interest.(Spence and Norris, 2003).
Recently, a potential link between
infectious agents and atherosclerosis
has been suggested.
Data obtained from several seroepidemological studies has given rise
to the hypothesis that an infection can initiate or maintain the
atherosclerotic process (Farsak et al, 2000). Chlamydia pneumoniae is a common cause of a usually
mild, community acquired pneumonia. This organism, however, can
spread from the respiratory tract into other parts of the body and
has been detected in up to 70% of atheromatous lesions in blood
vessels. Although the exact mechanism of the contribution of C. pneumoniae to the pathogenesis of
atherosclerosis remains unknown (Cagli et al, 2003).
Similarly, there is increasing
evidence that Chlamydia pneumoniae, a common respiratory tract pathogen, may play a role in
atherosclerosis. C. pneumoniae has been associated with coronary and carotid artery
disease in seroprevalence epidemiological studies, and in one prospective
cohort study C. pneumoniae elementary bodies have been detected in the
atherosclerotic plaques and fatty streaks of the aorta and coronary
arteries of autopsy cases. From atherectomy specimens of coronary
arteries, and from endarterectomy specimens of carotid arteries (Fong,
2000).
In the present study, the
presence of C. pneumoniae was
investigated by PCR in arterial plaque, as was the correlation between the
clinical status and DNA positivity of these bacteria.
II.
Materials and methods
A. Study design
The research in this study has been
done by descriptive methods. Samples were obtained in 2002 from 102 dead cases
with infarction due to Atherosclerosis. Basic demographic data and clinical
information such sex, smoking, familiar heart problem history coronary artery
diseases, diabetes, blood pressures and level of blood cholesterol (LDL &
HDL) in sera were extracted either from the files
B. DNA extraction for chlamydia
DNA from 50 ml of homogenized tissues was isolated
by proteinase K digestion (100 mg/ml for 1 to 2 h at 65¡C) followed by phenol-chloroform
extraction and ethanol precipitation.The DNA was then resuspended in 50 ml of Tris-EDTA buffer. For
PCR, 10 ml of DNA solution, was added per 50 ml of reaction mixture (Skowronski et
al, 1993).
C. PCR amplification
PCR targeting the 16S rRNA gene and a nested
PCR targeting the ompA gene were performed to detect C pneumoniae DNA. All amplification reactions were done in a volume of 50 ml containing 200 mM of four deoxynucleoside triphosphates. PCR
primers tested were CPN90 5' GGT CTC AAC CCC ATC CGT GTC GG 3',
CPN91 5' TGC GGA AAG CTG TAT TTC TAC AGT T 3', CP1 5' TTA CAA GCC
TTG CCT GTA GG 3', CP2 5' GCG ATC CCA AAT GTT TAA GGC 3' (Cagli et
al, 2003).
Briefly PCR was performed using
CPN90-CPN91 primer pair with a 0.25 mM concentration of each primer, 2.5
mM MgCl2 and 20 ml of the extracted DNA. Cycling
protocol was 75 seconds at 95¡C, followed by 60 cycles of
denaturation at 94¡C for 45 seconds, annealing beginning
at 64¡C and
ending at 52¡C for 45 seconds, and extension at 72¡C for one minute. The
annealing temperature was lowered 10¡C every four cycles until
52¡C and
this temperature was kept until the end of the cycling process.
CP1-CP2 primers with nested pair CPC-CPD were used for the ompA nested PCR. The first round of amplification used 1.5 mM MgCl2, 0.4 mM of each primer and 20 ml of the extracted DNA. Cycling consisted of nine minutes at 95¡C for Taq polymerase activation, 20 cycles of one minute at 94¡C, one minutes at 65¡C (temperature was decreased 0.5¡C for each cycle) and one minute at 72¡C plus an additional 20 cycles of one minute at 94¡C, one minute at 55¡C and one minute at 72¡C. The PCR products amplified by the outer primer pair were diluted 1:5 and 5 ml was added to a new PCR mixture containing 1 mM of each primer and 3 mM of MgCls. Cycling protocol entailed nine minutes at 95¡C for Taq DNA polymerase activation, 30 cycles of one minute at 94¡C, one minute at 50¡C and one minute at 72¡C.
III. Results
In Total, 102 patients (20 to 79 years old) Figure
2 from 75 males and 27 females had
been identified and died from atherosclerosis. Of these patients Chlamydia DNA
was detected in 23(22%) (Figure 1).
100(98%) of 102
atherosclerosis patients had the primary obstruction and the rest (2%), the
secondary obstruction. on the other hand majority of obstruction (91%) were
been displayed in left anterior decending artery (LAD).
Among of the Chlamydia DNA
positive patients (Picture 1 and 2),
some risk factors (sex, hyperlipidemia, blood pressure, diabetes, smoking and
family history of premature cardiovascular disease) were established. Out of
102 patients with atherosclerosis, 20 (36.30%) increasing of LDL, 18 (47.30%)
decreasing of HDL, 6 (24%) increasing of blood pressure, 7 (38.80%) diabetes,
12 (24%) smoking and 8 (29.90%) family history of premature cardiovascular
disease identified with DNA C.pneumoniae (Figure 1).
IV. Discussion
Human
atherogenesis appears to be of multifactorial etiology, and no single entity
can fully explain the pathogenesis. There is little doubt that risk
factors such as genetic predisposition, hypercholesterolemia, hypertension,
smoking, and diabetes mellitus are major predisposing conditions for
atherosclerosis. There is substantial evidence, albeit circumstantial, those
infectious agents are associated with atherosclerosis, but their
exact role in the pathogenesis of atherosclerosis is unknown. The
most compelling evidence to date is the presence of infectious
agents in the arterial wall, particularly in diseased vessels or within atherosclerotic plaques
(Chiu et al, 1997).
Figure 1. The frequency of
risk factors in Atherosclerosis patients with Chlamydia Pneumoniae (1-DNA
Chlamydia Positive and 2 –DNA Chlamydia negative).
Figure 2. The frequency of Chlamydia positive in Atherosclerosis patients.
Figure 3. The frequency of age in Atherosclerosis patients.
C. pneumoniae, an obligate intracellular gram-negative bacterium, has been
associated with atherosclerotic cardiovascular disease both by
seroepidemiological studies, indicating a significantly higher
prevalence of circulating C. pneumoniae
antibody or immune complexes among persons with clinical or
radiographic evidence of atherosclerotic disease. C pneumoniae has now been detected in atherosclerotic plaques
in several different arterial sites (coronary arteries, aorta, and
carotid arteries) and in early lesions (fatty streaks) and through
the use of various independent techniques. The organism has been detected
by electron microscopy, immunocytochemisty, direct immunofluorescence, and the
PCR in coronary artery and carotid artery plaque specimens (Shor et
al, 1992; Kuo et al, 1993a, b; Chiu et al, 1997; Farsak et al, 2000).
Some study was about comparison trial of DNA
extraction methods and PCR assay for detection of Chlamydia pneumoniae in endarterectomy specimens. There was no
consistent pattern of positive results among the various laboratories, and
there was no correlation between the detection rates and the sensitivity of the
assay used (Apfalter et al 2001).
Bartels et al, even found that occluded aorta-coronary
venous grafts harbour C pneumoniae (Bartels et al, 2000).
Using PCR and immunohistochemistry, C pneumoniae was
detected in arterial biopsies from femoral, popliteal, and coronary arteries,
as well as in the aorta, indicating that the organism is widespread
in atherosclerosis of the vascular system (Kuo et al, 1993a; Davidson
et al, 1998). Some studies found Between individuals, the percentage of
arteries with immunoreactivity to C pneumoniae was associated with the average area stenosis
throughout the arterial system. Their conclusion displayed C pneumoniae was mostly observed at locations that are related to
clinically relevant features. Within the individual, the distribution of C pneumoniae is associated with the distribution of
atherosclerosis (Vinik et al,2001).
On the other hand, Andreasen et al, could not detect C
pneumoniae in calcific or degenerative atherosclerotic aortic heart
valve disease and Nystromrosander et al did detect C pneumoniae in aortic valves
using electron microscopy (Nystrom-Rosander et al, 1997; Andreasen et al,
1998).
Furthermore, it is unclear whether C
pneumoniae initiates the process of atherosclerosis, facilitates progression
of existing plaques, or merely colonises the lesions. Some study Shown that the
adventitia of atherosclerotic coronary arteries frequently contains C. pneumoniae that seems to be located within macrophages. These
results might indicate a possible route for infected circulating macrophages to
home into atherosclerotic lesions in the artery via vasa vasorum (Vink et al,
2001a).
Another
study was to determine the presence of C. pneumoniae in coronary artery plaques, carotid artery plaques
and old vein grafts that were harvested at the time of surgery. But it failed
to find C. pneumoniae in any of
the vascular tissue. So was concluded that a large cooperative study involving
surgical specimen analysis is needed to assess the role of C. pneumoniae in the etiology of atherosclerosis (Johnson et
al,2001).
In our study C. pnemoniae was detected in 22 (23%) out of 102 tissue plaques
from dead atherosclerosis patients. Also, there were so many risk factors in
that patients. Therefore, In a condition with so many risk factors and genetic
influences it seems unlikely that infection will be the only or main cause
of atherosclerosis and events. The role of these newly emerging risk
factors and their relationship with traditional risk factors such as
hypertension or lipids, remains unexplored. The uncertainty of their
role and the types of infection or types of patients that should
be treated must be explored in properly conducted, prospective studies.
However, the findings to date are intriguing, and the hope that
anti-infective therapy may reduce the burden of stroke is worth
pursuing.
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Fatemeh
Fallah