Pathohistological changes in diffuse coronary atherosclerosis and chronic infection caused by Chlamydia pneumonia.

BACKGROUND AND PURPOSE: To investigate the histopathologic characteristics of atherosclerotic lessions in diffuse coronary artery disease and to evaluate the possible inflammatory role of chronic infection with Chlamydia pneumoniae (CP). MATERIALS AND METHODS: For 10 patients (males, mean age 61 years) who were surgically treated for grave diffuse coronary artery disease, histomorphological analyses of endarterectomized segments of the coronary arteries were performed. Serological analyses for the detection of CP antibodies in peripheral blood were done, preoperatively. RESULTS AND CONCLUSIONS: Diffuse and concentric atherosclerotic changes from VI to VIII stage according to the Stary classification were found. Immunohistochemical methods revealed infiltrates of T-lymphocytes (80% of cases), B-lymphocytes (40% of cases) and macrophages (80%). Using the nuclear marker for proliferation activity MIB-1, single MIB-1 positive cells were found in 40% of cases. Features of arteriologenesis and vasculitis of newly formed arterioles (as well as thickening of the wall of newly formed arterioles) were found in the vessel wall of 8 patients, 7 of them had chronic infection with CP (preoperative micro-immunofluorescent test results: 1:32<IgG ≤1:512 and IgA≥32), one had passed CP infection (1:32 ≤IgG<1:512, IgA negative). These features were absent in 2 patients, both recovered from CP infection and had not the chronic CP infection at the time of surgery. DNA of Chlamydia pneumoniae was detected using the polymerase chain reaction (PCR) method in the vessel wall of 3 patients who were chosen randomly for this method. This study suggests an inflammatory and proatherogenic role of CP in a high grade atherosclerotic coronary artery wall in diffuse coronary artery disease. INTRODUCTION The modern view on the aethiopathogenesis of atherosclerosis includes the inflammatory process on the vessel wall. This inflammation might at least partly be caused by certain infectious agents, among them Chlamydia pneumoniae is a visible candidate (1, 2). Pathogenetic mechanisms of possible Chlamydial involvement in the progression of atherosclerosis have been already discused (3). Seroepidemiological, laboratoratory and pathological studies (4-7) revealed CP as »being there« in atherosclerosis, however, whether CP is an initiator, promoter or an innocent bystander in the atherosclerotic process remains unproven (8). Diffuse coronary atherosclerosis is a special entity of coronary atherosclerosis where long segments of coronary arteries are diffusely atherosclerotically damaged (9). During the surgical treatment with the classic by-pass techinque, many times it is necessary to make an endarterectomy of the diffusely involved artery segment. This is the procedure where the surgeon with a special knife has to cut off the damaged intima of the coronary artery wall (10). Endarterectomized sequesters offers the unique opportunity for studying the atherosclerotic vessel wall. The aim of our research was to study pathohistological changes in the endarterectomized segments of the coronary artery wall in patients with serologically proven chronic CP infection where we were expecting to find distinct pathomorphological features regarding the patients without chronic CP infection. MATERIALS AND METHODS We histologically analysed the endarterectomized segments of the coronary arteries for 10 patients who were surgically treated because of diffuse coronary artery disease. All our patients were males, their mean age was 61+/-3 years. Eight of them suffered myocardial infarction before the operation, and angina pectoris was present for 3–24 months before the operation. All patients had arterial hypertension, their mean blood pressure was 145/90 mm Hg (receiving therapy!). Also, all were obese, 19 Bosnian Journal of Basic Medical Sciences IV (1) 2004. PATHOHISTOLOGICAL CHANGES IN DIFFUSE CORONARY ATHEROSCLEROSIS AND CHRONIC INFECTION CAUSED BY CHLAMYDIA PNEUMONIAE Mateja Legan1, Olga Vraspir-Porenta1, Darja Ke{e2, Ruda Zorc-Pleskovi}1, Marjeta Zorc1 1. Institute of Histology & Embryology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia 2. Institute of Microbiology & Immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia their mean BMI was 30.0+/-4.4 kg/m2. Nine patients were hyperlipidemic and had been receiving therapy with statin, 4 patients had diabetes. 90% of our patients were smokers, preoperatively. Immediately after the operation, the tissue samples of the endarterectomy were fixed in 10% buffered formalin for 24 hours and embedded in paraffin. Four mm sections were cut, deparaffinized, and stained with hematoxylineosin (HE), Masson-trichrome and Weigert. For the immunohistochemical analyses the sections were heated in a microwave oven (15 minutes for CD3, CD79α, CD68, and 25 minutes for the proliferation marker Ki67). For the detection of the proliferation marker Ki-67, the MIB-1 monoclonal antibody was used. The sections were washed with a phosphate-buffered saline solution (PBS). Then, the primary antibodies were applied: CD3 monoclonal antibody, incubation overnight in a wet chamber at 4oC (DAKO, Glostrup, Denmark; dilution 1:40); CD79a monoclonal antibody, incubation for 30 minutes at room temperature (DAKO, Glostrup, Denmark; dilution 1:80); CD68 monoclonal antibody, incubation for 30 minutes at room temperature (DAKO, Glostrup, Denmark; dilution 1:40); MIB-1 monoclonal antibody, incubation overnight in a wet chamber at 4oC (DIANOVA, Hamburg, Germany; dilution 1:100). After washing in PBS, strepatavidin-biotin complex/horseradish peroxidase was applied for 30 minutes at room temperature. Positive controls for CD3, CD79a, CD68, and MIB-1 tonsils were used. In the step serial sections of endarterectomized segments, the severity of atherosclerosis was graded according to the Stary classification (11, 9). Cell infiltration, proliferation activity of cells and tissue, capillarogenesis and arteriologenesis in the endarterectomized segments were investigated. Preoperatively, a venous blood sample was taken for the determination of the antibody levels IgG, IgM and IgA to CP, at least 3 times in each patient over a period of 6 months to prove a stable titer. Serological studies were performed in the Institute of Microbiology of the Medical Faculty, University of Ljubljana by the microimmunofluorescence method (MIF), utilising Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis elementary bodies (MRL Diagnostics, USA) as antigens to detect specific IgG, IgM and IgA antibodies. Serological evidence of CPn infection was based on the criteria published by Grayston et al. (12). A fourfold rise in IgG/IgA titer in paired sera or an IgM titer of ≥ 1:20 in any serum were considered as presumptive evidence of acute or recent infection with CP. Titers of IgG ≥ 1:32 and < 1:512 were presumed to be due to past infection with Cp. Titers with stable IgG and IgA titers ≥ 1:32 were presumed to be chronic infection with CP. A negative result was defined as an IgG/IgA titer < 1:32 and IgM titer < 1:20. The sera tested for IgM or IgA antibodies were pre-treated to remove possible free and complexed IgG antibodies, following the manufacturer’s instructions (MRL Diagnostics, USA). The study was approved by the National Ethics Committee and an informed consent was obtained by