Background: The role of infections in pathogenesis of atherosclerosis has been a point of extensive discussion and research. Chronic infection has been proposed to account for the formation and progression of atherosclerotic plaques. Gastric mucosal damage caused by Helicobacter pylori (H. pylori) involves various bacterial and host-dependent toxic substances that have been recently associated with increased risk of coronary artery disease (CAD).

Aims: This study was designed to: 1) to determine the seroprevalence of H. pylori and its cytotoxin associated gene A (CagA) in patients with and without CAD (group A), 2) to evaluate the influence of infection with H. pylori expressing CagA (Cytotoxin associated gene A--as a determinant of high virulence) on coronary arterial lumen reduction in patients after percutaneous transluminal coronary angioplasty (PTCA) with stent (group B), 3) to assess the effect of H. pylori eradication on coronary artery lumen reduction after PTCA (group C), 4) to determine the influence of the H. pylori eradication on plasma levels of cytokines, lipids and coagulation factors in the same patients before and after PTCA (group C) and 5) to analyse coronary specimens in patients with severe CAD for the presence of H. pylori originated specific DNA (Group D).

Patients and methods: Group A included 96 patients with CAD (subgroup I) and 96 patients without CAD (subgroup II). For H. pylori seroprevalence, plasma anti-H. pylori and anti-CagA IgG were examined by ELISA and Western blot. Group B included 135 patients (86 male, 49 female, mean age 67 +/- 12 years) who had underwent PTCA with stent implantation initially and recoronaro-angiography about 6 months afterwards. All patients were tested for H. pylori--specific antibodies (IgG and CagA). Patients were divided into three subgroup "a": 34 patients with H. pylori IgG and CagA seropositivity, subgroup "b": 37 patients infected with H. pylori positive and CagA negative germs and subgroup "c": 64 patients with H. pylori IgG sero-negativity serving as control group. For all patients coronary lumen loss (percentage) in the dilated segment was measured at the end of PTCA and during re-coronaro-angiography and obtained values were considered taking into account the major risk factors of CAD (hypertension, hyperlipidemia, diabetes, obesity and smoking). Group C included 40 patients with significant single-vessel CAD and H. pylori infection confirmed by 13C-urea breath test (UBT) and serologically using anti-H. pylori and anti-CagA IgG. In addition, plasma interleukin (IL)-1beta and IL-8 and tumor necrosis factor alpha (TNFalpha) levels were measured by ELISA. Plasma total triglycerides, cholesterol, low (LDL) and high density lipoproteins (HDL), homocysteine levels, as well as some clotting factors such as thromboplastin and fibrinogen levels, thrombin time and platelet count were determined. All patients of group B undergoing PTCA were divided into two matched subgroups I and II used in exploratory study; subgroup I (20 patients) received H. pylori eradication triple-therapy for one week (Clarithromycin, Amoxicillin and Omeprazole), while subgroup II (20 patients) received similarly prepared placebo for the same time period starting immediately after PTCA. Six months after PTCA, the H. pylori status was re-assessed by UBT and found to be negative in all but two patients of subgroup I subjected to H. pylori therapy. Coronary angiography and laboratory tests were repeated in both subgroups of group B included into the trial and the reduction in coronary artery lumen in these subgroups was compared to baseline after PTCA considered as 100%. Large atherosclerotic plaques from coronary endatherectomy were obtained in 46 consecutive patients (9 female, 37 male, mean age 63 +/- 9 years) during coronary bypass procedures (group D). Serum was analysed for positive IgG antibodies specific for H. pylori by enzyme-linked-immunosorbent assay (ELISA). Antibodies specific for the CagA were detected by immunoblot analysis. Polymerase chain reaction (PCR) was used to identify bacterial DNA with primers encoding for the 16 S ribosomal RNA of H. pylori. Sequence analysis of PCR-products confirmed the specificity of the gene products for H. pylori. Coronary artery biopsies from 19 autopsies from a Forensic Medicine Department without coronary atherosclerosis were examined as a control group.

Results: The H. pylori seropositivity reached 69.79% (67 pts) of CAD (subgroup I of group A) and it was significantly higher than that in controls without CAD (subgroup II)--40.62% (39 pts), the odds ratio (OR) being 3.38 95% CI: 1.8598-6.1306 for H. pylori in CAD. CagA IgG detection was also significantly higher (58.20%) in CAD group than in controls (35.89%) giving the OR about 2.49 (95% CI: 1.1012-5.6175). Mean lumen loss in group B in H. pylori-positive subjects was 37.0% +/- 17.3%, whereas for H. pylori negative patients 29.9 +/- 13.8% were measured compared to initial values following PTCA (p = 0.0196). Even subgroup analysis and analysis regarding risk factors show significantly higher lumen loss for H. pylori-positive patients (especially CagA positive) compared to H. pylori negative patients. Mean coronary artery lumen reduction in patients undergoing PTCA + H. pylori eradication therapy (subgroup I of group C) was found to be significantly (P < 0.05) smaller compared to PTCA + placebo-treated subgroup II (22% vs 41%). The plasma cytokines such as TNFalpha, IL-1beta and IL-8 were significantly lower after the H. pylori-eradication in PTCA patients, while changes in plasma lipids, homocysteine and clotting factors were not significantly affected by H. pylori eradication. In group D thirty two patients (69.5%) were H. pylori-seropositive, 14 patients out of 32 H. pylori-positive were CagA positive. Eighteen of these patients showed positive results for H. pylori DNA, whereas 4 patients of the seronegative group also showed positive DNA results, but all 4 had undergone eradication therapy within the past two years. A total of 22 patients (47.8%) of the CAD group and none of the 19 controls revealed positive DNA assessed by PCR. Out of 14 anti-Cag A positive patients 11 showed positive detection of H. pylori DNA (p = 0.015)

Conclusions: 1) There is a significant link between CAD and infection with H. pylori, especially expressing CagA proteins; 2) Patients infected with CagA-positive H. pylori show significantly greater coronary artery lumen loss and arterial re-stenosis after PTCA with stent implantation; 3) H. pylori eradication significantly attenuates the reduction in coronary artery lumen in CAD patients after PTCA possibly due to the elimination of chronic inflammation and the decline in proinflammatory cytokine release and 4) The identification of DNA in atherosclerotic plaques of patients with severe CAD supports the hypothesis that infection with H. pylori (especially CagA positive) may influence the development of atherosclerosis.