Ischemic heart disease appears to be of multifactorial etiology . Results of large epidemiologic studies have established the concept of traditional risk factors, such as hypercholesterinemia, systemic hypertension, and smoking, with fundamental implications for current treatment. Most recently, however, it has been shown that inflammation too can play a pivotal role in the pathogenesis of coronary artery disease (CAD).
Results of animal studies , seroepidemiologic observations [3–5], pathology-based investigations [6–8], and data from small randomized clinical trials [9,10] have provided evidence implicating direct pathogenic involvement of infectious agents in the process of atherogenesis and especially in the development of CAD. The potential pathogenic effects by means of which these agents may act involve both direct effects on the arterial wall and indirect effects mediated via the circulation [11,12]. This theory is further supported by a large body of basic laboratory and experimental data complementing the clinical evidence relating inflammation and infection to vascular risk .
Infection with cytomegalovirus has consistently been implicated in atherogenesis, restenosis after coronary angioplasty, and transplant vasculopathy [14–16]. In addition, Chlamydia pneumoniae (C. pneumoniae) and Helicobacter pylori (H. pylori) have also been suggested to be independent risk factors for CAD [5,8,17]. The statistical significance of seroepidemiologic associations, however, could not be taken as evidence of causal pathogenic involvement. Most authors investigating the endovascular presence of infectious agents used directional coronary atherectomy and were therefore limited to small samples, possibly underestimating the true incidence of pathogens or were not able to identify coinfections within the coronary arteries.
This study was intended to determine the prevalence and distribution of cytomegalovirus, C. pneumoniae, and H. pylori in atherosclerotic lesions in a systematic, serial analysis of coronary specimens obtained during aortocoronary bypass surgery. In addition, the role of concurrent infections and possibility of morphologic or clinical implications of the pathogen presence were studied.
We studied 53 patients (aged 63 ± 9 years, 81% men) with symptomatic CAD, for whom coronary endatherectomy during aortocoronary bypass surgery had been successful. Their cardiovascular risk profile included 57% of patients with arterial hypertension (blood pressure ≥ 140/90 mmHg, or being administered antihypertensive medication), 64% hypercholesterolemic (≥160 mg/dl low-density lipoprotein cholesterol, or being administered lipid-level-lowering medication), 43% smokers (current cigarette smoking), and 30% diabetics (≥ 140 mg/dl fasting blood glucose, or being administered antidiabetic medication). Forty-three patients presented with stable angina, the remaining 10 patients had unstable angina (defined as Braunwald class IIB or greater). A history of previous myocardial infarction was reported by 33 patients (62%). In total 15 atheromas (28%) were taken from the infarct-related artery; in addition, seven coronary atheromas (13%) contained restenotic lesions after balloon angioplasty (secondary lesions) without implantation of a stent.
Preoperative angiography and coronary endatherectomy
Results from preoperative coronary angiograms were available for all patients with digital storage of the data. Offline quantitative coronary angiography (CAAS II system; Pie Medical, Maastricht, The Netherlands) was performed to obtain the minimal lumen diameter (MLD) and the percentage diameter stenosis (PDS) of the target lesion (MLD and PDS calculations included total occlusions). The presence of angiographically detectable coronary calcifications or total chronic occlusions was also documented.
Decisions for thrombendatherectomy were always made intraoperatively on the basis of coronary morphology at the proposed site of the distal anastomosis; coronary samples were never taken exclusively for the present study. Briefly, atherosclerotic cylinders were obtained by blunt dissection from the adventitial layer after longitudinal incision of the artery. Immediately after harvesting, samples were stored in sterile tubes and frozen at −20°C for further pathologic examination.
Control group without symptomatic coronary disease
In order to provide a control group without symptomatic coronary disease, 52 coronary samples were obtained within 24 h of death from 26 patients who had died for reasons other than CAD. In detail, the proximal part of the dedicated coronary artery was removed from the epicardium surgically. Consecutively, great care was taken to remove as much adventitial tissue as possible before extracting DNA. Precautions to avoid contaminations were taken throughout the procedure.
Pathologic examination and detection of pathogens
Extraction of DNA
DNA was extracted from the proximal and distal end of each atheroma and the control samples using the QIAamp extraction procedure (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, the samples were lyzed with proteinase K at a concentration of 1.8 mg/ml at 55°C for 12 h and afterwards loaded onto a spin column. DNA was then absorbed by brief centrifugation onto the QIAamp silica membrane, washed twice, and eluted with buffer.
Polymerase chain reaction
Amplifications of target DNA sequences of cytomegalovirus and H. pylori were performed as described previously [18,19]. Polymerase chain reaction (PCR) conditions for the detection of C. pneumoniae DNA were as follows. Amplification was performed as a semi-nested PCR in 50 μl of a reaction mixture containing DNA template, 10 mmol/l TRIS-HCl (pH 8.3), 50 mmol/l KCl, 3 mmol/l MgCl2, 50 μm of each dNTP (100 μmol/l for the second round of amplification), 0.5 μmol/l of each primer, and 1 U Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The nucleotide sequences of the primers were 5′-GTG TCA TTC GCC AAG GTT AA-3′ for HM-1, 5′-TGC ATA ACC TAC GGT GTG TT-3′ for HR-1, 5′-TCC GTG TCG TCC AGC CAT-3′ for CP-1. For the first round of amplification, primers HM-1 and HR-1 were used. Each PCR cycle consisted of a denaturation step at 94°C for 60 s, annealing of primer at 50°C for 60 s, and extension at 72°C for 60 s. Initial denaturation was performed at 94°C for 5 min and, in the final cycle, duration of extension was prolonged to 5 min at 72°C. Samples were amplified through 30 cycles in an Omnigene-Thermocycler (Hybaid, Ashford, UK).
We used 1.5 μl PCR product as template for the second amplification with primers HM-1 and CP-1. Each PCR cycle consisted of a denaturation step at 94°C for 30 s, annealing of primer at 56°C for 30 s, and extension at 72°C for 15 s. Initial denaturation and last extension step were the same as those described before. Samples were amplified through 20 cycles in an Omnigene-Thermocycler (Hybaid).
Negative control reactions were performed with samples containing distilled water. Sterile materials were used throughout, with strict precautions to avoid contaminations. Extraction of DNA, PCR, and analysis of PCR products were performed in separate laboratories.
Identification of amplicon sequences
Amplification products were first analyzed by electrophoresis through a 2% agarose gel by standard methods. The expected amplicon sizes were 110 bp for cytomegalovirus, 132 bp for H. pylori, and 229 and 81 bp for the first and the second PCR for C. pneumoniae, respectively.
Furthermore, the identities of digoxigenin-labeled amplified cytomegalovirus, C. pneumoniae, and H. pylori sequences were checked by liquid hybridization assays with biotin-labeled specific probes according to a protocol described previously . Due to a wide variability of prevalences of C. pneumoniae in atherosclerotic samples, PCR products were further analyzed by cycle sequencing using the ABI Prism BigDye Terminator Kit (PE Applied Biosystems). Cycle-sequencing reactions were performed according to the manufacturer's protocols on the Gene Amp PCR System 9600 (PE Applied Biosystems). Electrophoresis of samples was performed on the ABI Prism 310 capillary electrophoresis unit (PE Applied Biosystems, Perkin Elmer, Überlingen, Germany).
Statistical analysis was performed using SPSS for Windows version 7.5 (SPSS Inc., Birmingham, UK). Data are expressed as proportions or means ± SD for continuous variables. Univariate analysis was performed on all clinical and angiographic variables to determine correlations between characteristics of patients and presence of pathogens. Statistical comparisons were performed using χ2 statistics for categorical variables and Student's t test for continuous variables. P < 0.05 was considered statistically significant.
Detection of cytomegalovirus, C. pneumoniae, and H. pylori in coronary lesions
A total of 53 coronary atherectomy specimens (of lengths 9–104 mm) were investigated. Samples for extraction of DNA were taken from the proximal and distal ends of every lesion, resulting in a total of 106 probes.
PCR for cytomegalovirus yielded positive results for 11 samples (10%) from 10 patients (19%). Hybridization confirmed results for all positive samples and identified another six samples as positive for cytomegalovirus, resulting in a total of 17 of 106 (16%) cytomegalovirus-positive samples and 16 of 53 (30%) cytomegalovirus-positive patients. For one of 16 (6%) cytomegalovirus-positive patients both samples from a particular coronary lesion were identified as positive for cytomegalovirus, indicating a homogenous virus distribution. However, the remaining 15 of 16 (94%) positive samples were located next to a cytomegalovirus-negative specimen, indicating that the pathogens were not homogeneously distributed in most of these cases.
C. pneumoniae was detected by PCR in 22 of 106 samples (21%) from 17 of 53 (32%) patients. Twelve of 17 (71%) patients yielded one positive sample, whereas for five of 17 (29%) patients both coronary samples were identified as positive for C. pneumoniae, indicating that there was an inhomogeneous coronary distribution pattern for C. pneumoniae also. In contrast to the results obtained for cytomegalovirus, hybridization confirmed assignment of all positive samples without detection of further positive samples. The identity of all PCR-positive samples was confirmed by sequencing.
At least one of the pathogens was detected in 27 of 53 patients (51%). Concurrent infection with C. pneumoniae and cytomegalovirus was observed for 5 of 106 lesions (5%) and five of 53 patients (9%). All 106 coronary samples were negative for H. pylori according to the PCR protocol and the PCR enzyme-linked immunosorbent assay.
Control group without symptomatic coronary disease
All 52 control samples were negative for C. pneumoniae and H. pylori. Four samples (from three patients) were positive for cytomegalovirus; therefore, 48 of 52 samples (92%) and 23 of 26 (88%) control patients were negative for cytomegalovirus.
Clinical and angiographic correlations to endovascular presence of cytomegalovirus and C. pneumoniae
The demographic, clinical, and angiographic characteristics of patients positive for cytomegalovirus and C. pneumoniae compared with those of corresponding pathogen-negative subjects are provided in Table 1
. In summary, the presence of cytomegalovirus was significantly associated with secondary lesions and lesser severity of calcifications (assessed by angiography), whereas the endovascular presence of C. pneumoniae was associated with the severity of angina.
A comparison between patients with endovascular presence of pathogens (cytomegalovirus, C. pneumoniae, or both) and pathogen-free patients is provided in Table 2
. Pathogen-positive patients tended to be older and their coronary lesions were found to be significantly less calcified by angiography.
The main findings of this study indicate that DNA from cytomegalovirus and C. pneumoniae can be detected in coronary atherosclerotic lesions from about 30% of patients with severe CAD. Cytomegalovirus but not C. pneumoniae can also be detected in coronary arteries from patients without symptomatic coronary disease. The endovascular distributions of both pathogens, C. pneumoniae and cytomegalovirus, are not homogeneous in most cases. H. pylori DNA is present neither in coronary lesions nor in control samples. The concurrent presence of cytomegalovirus and C. pneumoniae occurs coincidentally.
Pathologic findings: prevalence and distribution
The prevalences of cytomegalovirus and C. pneumoniae within coronary atherosclerotic lesions found by the detection of genomic DNA were 30 and 32%, respectively, in this study. Cytomegalovirus and C. pneumoniae have been detected in advanced human atherosclerotic lesions of diverse origins (coronary arteries, carotid arteries, and aorta) [6–8,20–22] by the use of various techniques (immunocytochemistry, electron microscopy, and PCR techniques). The incidence of cytomegalovirus in coronary arteries has been reported to range from 0% in primary coronary lesions  to 67% or even 90% at peripheral arterial sites . In comparison, C. pneumoniae has been detected in coronary atherosclerotic lesions with a prevalence of up to 79%, compared with 4% in normal coronary arteries and in explanted hearts with chronic transplant rejection . Additionally, a significant proportion of atherosclerotic coronary arteries (17%) harbor viable C. pneumoniae, further supporting the hypothesis of a chlamydial contribution to CAD .
This is the first report to assess concurrent coronary infection with cytomegalovirus and C. pneumoniae in the same patient, for which we found a prevalence of 9%. These data clearly indicate that coinfection of the coronary vasculature by these two pathogens occurs independently and coincidentally. Our results are further supported by data in a recent report by Chiu et al. , assessing the presence of cytomegalovirus and C. pneumoniae in the same atherosclerotic tissue sample from carotid arteries. Clinical implications of vascular coinfection and the pathogenic impact on atherosclerosis remain unknown and need further investigation.
The distributions of cytomegalovirus and C. pneumoniae within diseased coronary arteries are not homogeneous, as we found by serial analysis of two different sites of the same coronary atheroma. The true prevalence of infection of coronary arteries could therefore even be higher than that found in this study. Furthermore, the wide variability of prevalences of pathogens reported in the literature (0–90%) might result from the apparently uneven patterns of distribution and a resulting sampling error. In addition, the observation of an uneven distribution of pathogens might simply reflect the different cellular compositions of the samples taken (macrophage-rich versus fibrous tissue).
It can not be ruled out that the true prevalence of a pathogen within the vasculature is equal to the seroprevalence of the pathogen investigated; however, the data available so far indicate that there is a rather poor correlation between serum levels of antibodies and the endovascular presence of C. pneumoniae [23,24]. Authors of subsequent studies investigating the presence of pathogens in atherosclerotic lesions should therefore also use serial analysis and perform systematic sampling of serum.
Angiographic and clinical findings
Latent infection with cytomegalovirus has recently been implicated in atherogenesis and restenosis after directional coronary atherectomy [15,16,19]. Two key findings of this study may further strengthen the hypothesis that there is a causal relationship between infection with cytomegalovirus and endothelial hyperplasia (and restenosis as the clinical correlate). First, the proportion of restenotic lesions in the cytomegalovirus-positive atheromas was significantly higher than that in cytomegalovirus-negative atheromas (31% versus 5%, P = 0.021). Furthermore, angiographically identifiable calcifications were less commonly found in the cytomegalovirus positive vessels (33% versus 94%, P < 0.0001). In obstructive CAD, the lack of heavy calcification, albeit indirectly, implies rather that accumulation/proliferation of cells is the main reason for obstruction of a vessel. Both observations fit well with the hypothesis that cytomegalovirus induces an excessive proliferative response of vascular endothelial cells via IE84-mediated inhibition of p53 (IE84 is a major immediate-early gene protein of cytomegalovirus) .
Indisputably the most important clinical finding associated with a latent coronary C. pneumoniae infection is the significantly greater proportion of patients with unstable angina among patients who are positive for C. pneumoniae (41% versus 8%, P = 0.008). Furthermore, C. pneumoniae -positive patients tend to have more severe angina than do C. pneumoniae -negative subjects (Canadian Cardiac Society functional class 2.9 ± 1.2 versus 2.3 ± 0.9, P = 0.048). A higher prevalence of coronary infection with C. pneumoniae among patients with unstable angina as compared to patients with stable angina has also been reported . The potential mechanisms by which C. pneumoniae may cause CAD remain unknown; however, a recent report by Bachmaier et al.  indicates that infection with C. pneumoniae can lead to a local immune response and activation of autoreactive T and B lymphocytes. The pathogenic background of the possible link to acute coronary syndromes indicated by our results remains to be investigated.
Of interest is that 51% of the diseased coronary arteries contain at least one of the pathogens. These patients tended to be older than the pathogen-free patients. In addition, coronary lesions of the pathogen positive patients were significantly less calcified.
H. pylori and CAD
No H. pylori DNA was found in coronary samples from patients with severe CAD in this study. Causative involvement of this pathogen in CAD, however, is not completely excluded by the lack of detection of antigen. The postulated pathophysiologic concept of the ‘inflammatory–infectious’ contribution to atherogenesis potentially includes both direct and indirect effects of pathogens on the arterial wall [11,12]. Possibly indirect effects of low-grade systemic infections may lead to inflammatory or infectious activation of vascular wall cells or lesional leukocytes, finally promoting evolution of lesions.
The seroepidemiologic association between infection with H. pylori and CAD is reported to range between strong positive and negative, mainly due to the association of infection with H. pylori with confounding factors, such as age and social class [4,5]. Data in a recently published paper by Pasceri et al.  however, support the hypothesis that H. pylori promotes atherogenesis via low-grade, persistent inflammatory stimulation by more virulent strains of H. pylori in CAD patients .
This study included 106 lesions from 53 patients. Despite a significant correlation between the presence of pathogens and certain clinical variables (angina and restenosis), the study population is still relatively small. In particular, the number of patients with concurrent infections with cytomegalovirus and C. pneumoniae (n = 5) was too small to allow us to draw definite conclusions with regard to clinical implications. The incidence of pathogen-positive patients was derived by the serial analysis of two samples per patient. It can not be ruled out that the analysis of more than two different lesion locations would have resulted in detection of a higher incidence of cytomegalovirus-positive or C. pneumoniae -positive patients. Samples taken from restenotic segments do not necessarily reflect the precise area of angioplasty. In addition, it is difficult to identify the unstable plaque in diffusely diseased coronary arteries with multivessel CAD, such as those seen in this population of patients. Furthermore, total occlusions (amounting to about 60% of lesions) are not likely to cause instability. Therefore, the conclusions derived from these observations (associations between presence of pathogens and restenosis and unstable angina) have to be interpreted with caution. The number of serum samples obtained preoperatively was too small to draw definite conclusions with regard to whether there is an association between antibody titers and the endovascular presence of pathogens.
In conclusion, inhomogeneous infections of coronary atherosclerotic lesions with cytomegalovirus and C. pneumoniae are common. Concurrent infection with both pathogens occurs coincidentally; however, possible clinical implications of this new observation and the pathogenic impact on atherosclerosis need further investigation. The association between H. pylori and CAD is still limited to results of seroepidemiologic studies, as this pathogen cannot be detected in the coronary vasculature. The use of antiviral substances or antibiotic treatment for the prevention or treatment of CAD and restenosis, however, seems justifiable only in well-designed and placebo-controlled trials on the basis of current data.
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