Detection of Helicobacter pylori DNA in Aortic and Left Internal Mammary Artery Biopsies

Abstract

We investigated the relationship between acute coronary ischemia and the presence of Helicobacter pylori DNA in aortic regions that were absent macroscopic atheromatous plaques.

The study group (Group 1) consisted of 42 patients who underwent coronary artery bypass grafting. Biopsy samples were obtained from 2 different locations: from regions of the aorta that were free (macroscopically) of atheromatous plaque (Group 1A), and from the internal mammary artery (Group 1B). The control group (Group 2) of 10 patients who had no atherosclerotic vascular disease provided aortic tissue samples for comparison. The real-time polymerase chain reaction method was used to detect H. pylori DNA in all biopsy samples.

Eleven of 42 aortic tissue samples (26%) in Group 1A were positive for H. pylori DNA. Neither biopsies from the left internal mammary arteries of those patients nor biopsies from the aortas of the control group (Group 2) were positive for H. pylori DNA. There was a statistically significant difference between 1A and 1B in terms of H. pylori positivity (P=0.001). In Group 1 as a whole, acute coronary ischemia was more prevalent in the H. pylori-positive patients than in the H. pylori-negative patients (P=0.001).

To our knowledge, this is the 1st study to investigate the detection of H. pylori DNA in aortic biopsy samples that are macroscopically free of atheromatous plaque. Such detection in patients who have atherosclerotic coronary artery disease could be an important indication of the role of microorganisms in the pathogenesis of atherosclerosis.

Currently, widely accepted atherogenic factors such as hypertension, smoking, hypercholesterolemia, diabetes mellitus, and sex account for only 50% of the causes of atherosclerosis.¹ This causal uncertainty has led investigators to focus on infectious agents and on consequent inflammatory processes as important contributors to the development of atherosclerosis.^1–7

Heterogeneous inflammatory infiltrates of T-lymphocytes and activated macrophages—which could possibly be attracted by infectious agents—are commonly encountered within atherosclerotic lesions and intimal fibrocellular proliferations. These histopathologic findings suggest the role of infectious agents and inflammatory processes in the pathogenesis of atherosclerosis.⁸

The gram-negative bacillus Helicobacter pylori, which colonizes the gastrointestinal tract of more than 50% of the adult population, has been associated with chronic gastritis, peptic ulcer, and gastric cancer.^9,10 Recently, several studies have described the association of H. pylori with extragastric diseases.¹¹ The putative role of H. pylori in cardiovascular system diseases has garnered much attention. In the carotid arteries, the serologic evidence of an H. pylori–atherosclerosis association has preceded the confirmation of H. pylori DNA in the plaques themselves.¹²

For many years, the internal mammary artery (IMA) has been used as a conduit graft for coronary artery bypass and has been believed to be more resistant to atherosclerosis than are other conduits.^13–15 Elevated endothelial nitric oxide synthetase (eNOS), angiotensin-converting enzyme (ACE), and endothelin-A receptor expression at the tissue level of the IMA have been proposed to account for this resistance.¹³

Our clinical study was carried out from January 2004 through January 2006 on patients who were undergoing coronary artery bypass grafting (CABG) due to atherosclerotic coronary artery disease. Our aim was to investigate the presence of H. pylori DNA in biopsy samples taken from aortic tissues that did not contain macroscopic atheromatous plaques and from IMA biopsy samples. In addition, we investigated other atherosclerotic risk factors and preoperative infections in patients with and without H. pylori in order to determine whether a correlation existed between H. pylori and acute myocardial infarction (AMI) or acute coronary ischemia in these patients.

Patients and Methods

Obtaining and Preserving Tissue Biopsy Samples

The required approvals to conduct this study were obtained from the ethics review board of our institution. Informed-consent forms were signed by all participants. The study group (Group 1) consisted of 42 patients who were undergoing coronary artery bypass surgery due to coronary artery disease. We collected aortic punch samples from patients who were undergoing proximal anastomosis of the saphenous vein (Group 1A, n=42) and IMA tissue samples from patients who were undergoing IMA anastomosis (Group 1B, n=42). Ten patients who were undergoing either aortic surgery or aortic homograft preparation—but who had no atherosclerotic vascular disease—were included as a control group (Group 2), from whom aortic biopsy samples were collected during operation. All biopsy materials were transported in saline and stored at −86 °C until they were processed.

The mean ages of the patients in Groups 1 and 2 were 57.3 ± 11.4 and 56.4 ± 8.6 years, respectively (Table I). Group 1 consisted of 33 (78.6%) male and 9 (21.4%) female patients. Similarly, Group 2 consisted of 8 (80.0%) male and 2 (20.0%) female patients.

TABLE I. Broad Descriptive Data and Preoperative Blood Values of Study (Group 1) and Control (Group 2) Patients

Detection of H. pylori DNA with Real-Time Polymerase Chain Reaction

The aortic and IMA tissue samples were minced by means of sterile razor blades. Approximately 10 mg of tissue samples were digested by incubation for 3 hours at 52 °C in 500 μL of digestion buffer (10 mM Tris pH, 8.5), 1 mM ethylenediaminetetraacetic acid, and 0.5% sodium dodecyl sulfate (SDS) containing 300 μg/mL of proteinase K. Deoxyribonucleic-acid purification from digested tissues was performed by use of a spin-column DNA purification kit (Invitek GmbH; Berlin, Germany).

Helicobacter pylori DNA in the samples was detected by using real-time fluorometric polymerase chain reaction (PCR), directed to amplify the 23S rRNA gene. Each reaction tube contained TaqMan® Universal Master Mix (Applied Biosystems; Foster City, Calif) at 1× concentration, 0.9 μM of forward primer (5′-GCTCTTATGGAGYCATCCTTGA-3′), 0.9 μM of reverse primer (5′-TCAAACTACCCACCAAGCATTG-3′), 0.25 μM of TaqMan® probe (5′FAM-CCACCCTTGATGTTTCTGTTAGCTAACT-TAMRA 3), and approximately 1 μg of template DNA in a total reaction volume of 50 μL. Deoxyribonucleic acid extracted from cultured H. pylori J99 cells was used as positive amplification control. Polymerase chain reaction was performed in an ABI Prism® 7000 Sequence Detection System (Applied Biosystems). The amplification protocol included 10 minutes of incubation at 95 °C for denaturation and enzyme activation and 48 cycles of denaturation at 95 °C for 15 sec and extension at 60 °C for 90 sec. The results were analyzed with the software of the device by using log fluorescence versus cycles of amplification plot (ABI Prism® 7000 SDS Software Version 1.0).

Statistical Analysis

All of the data from both groups were compared by using the Student t, Kruskall-Wallis, and χ² tests. McNemar's test was used to compare H. pylori DNA positivity between Group 1A and 1B. A P value ≤0.05 was considered statistically significant.

Results

Preoperative atherosclerotic risk factors were surveyed. In Group 1, 30 of the 42 patients (71.4%) were heavy smokers, 23 (54.8%) had hypertension, 21 (50.0%) had hypercholesterolemia, and 8 (19.0%) had diabetes mellitus. In specific regard to smoking, hypertension, and hypercholesterolemia, Group 1 patients manifested these risk factors to a significantly higher extent than did Group 2 patients (P=0.001).

Table I displays the mean preoperative serum total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, plasma fibrinogen levels, and leukocyte counts for Groups 1 and 2. In Group 1, the mean serum total cholesterol, triglyceride, and fibrinogen levels of patients were significantly higher than in Group 2.

Helicobacter pylori DNA was detected in 11 (26.19%) of the aortic biopsies of the patients in Group 1. In contrast, none of the aortic samples in Group 2 and none of the corresponding IMA samples in Group 1 was found to be positive (McNemar, P=0.001). Furthermore, 9 of the 11 patients who were positive for H. pylori DNA had emergency operations for AMI or acute coronary ischemia, whereas only 1 of 31 H. pylori-negative patients underwent surgery for AMI or acute coronary ischemia (Table II). These results reveal a statistically significant association between H. pylori DNA positivity in the aorta and AMI or acute coronary ischemia (P=0.001).

TABLE II. Heliobacter pylori PCR Positivity Values in Emergency and Elective Revascularization Patients (P=0.001)

We also studied the association, in aortic biopsy samples, between the presence of H. pylori DNA and plasma acute-phase reactants. Elevated serum C-reactive protein levels were detected in 9 of the 11 (81.82 %) patients who had H. pylori DNA (χ², P=0.035) (Table III). In contrast, plasma fibrinogen levels were not so sharply different between H. pylori-positive and -negative patients (P=0.074). In regard to factors that might influence or facilitate the presence of this organism in the aortic wall, elevated blood leukocyte counts (P=0.030), serum LDL cholesterol levels (P=0.001), and total cholesterol levels (P=0.001) were found in association with H. pylori DNA (Fig. 1). Interestingly, serum HDL cholesterol levels were also elevated in H. pylori-DNA-positive patients, compared with the rest of the Group I patients (P=0.040).

TABLE III. Group 1: Heliobacter pylori PCR Positivity Was Associated with Elevated C-Reactive Protein Levels (P=0.035)

Fig. 1 In H. pylori PCR-positive (hp+) patients, the blood leukocyte (P=0.03), plasma low-density lipoprotein (LDL) cholesterol (P=0.001), high-density lipoprotein (HDL) cholesterol (P=0.03), and total cholesterol (P=0.001) values were all significantly higher.

PCR = polymerase chain reaction

Discussion

Although a strong association has been shown between H. pylori and atherosclerosis² and between H. pylori and atherosclerotic stroke,¹² the relationship between H. pylori and ischemic coronary artery disease or AMI is still debatable. Even though H. pylori's existence in carotid atheromatous plaques has been shown by DNA extraction,³ it was not known until recently whether H. pylori DNA was present in aortic tissue, coronary arteries, or atheromatous plaques of the aorta and the coronary arteries. Hence, there was no effort to go a step farther to seek an association between H. pylori's existence in these tissues and acute coronary ischemia or the need for post-AMI CABG.

Our study is a preliminary investigation of the association between H. pylori-related atherosclerosis and the development of atheromatous plaques in the aorta, the coronary arteries, and the IMA.

The association between infectious agents and atherosclerosis has been known since 1856.¹⁶ At early stages of atherosclerosis, monocytes and T-lymphocytes migrate to the subendothelial space.¹⁷ These cells have been seen in human atheromatous plaques.^18,19 Three theories have been proposed to explain the atherosclerotic process induced by infectious agents. In the 1st such theory, which might be called the theory of plaque instability,³ factors such as the cytotoxin association gene (CagA) of H. pylori play a role in the acute infection of patients who have one or more vascular risk factors, and in the subsequent development of atherosclerotic plaques. In the 2nd theory, chronic infection facilitates the development of atherosclerotic plaque: a protein molecule called heat-shock protein 60 stimulates a cross-reaction²⁰ and a protein secreted by Chlamydia pneumoniae and H. pylori stimulates plaque development.²¹ The 3rd theory holds that the presence of microorganisms in the vascular wall induces the atherosclerotic process. The existence of this microorganism might account for the progression and complication of atherosclerotic plaques.^1,8,22 Indeed, in support of the 3rd theory, in vitro studies have shown that H. pylori resides intracellularly in large cytoplasmic vacuoles; and the appearance of H. pylori in extracellular media parallel to its disappearance from intravacuolar compartments suggests that H. pylori is secreted from an intravacuolar source.²³

Recent studies have shown that the inflammatory process—when initiated by an injury to the vascular wall or by a cross-reaction against certain proteins (heat-shock proteins and oxidized LDL cholesterol) as a result of the autoimmune process—can play a significant role in the pathogenesis of atherosclerosis.^1,4–7 In our study, the finding of H. pylori DNA in a region of aorta devoid of macroscopic atheromatous plaques rendered the autoimmune process less likely. We believe that, upon reaching the vascular wall, H. pylori is directly involved in the development of atheromatous plaque in that region. These H. pylori and C. pneumoniae microorganisms, which are capable of intracellular penetration,²³ can reach the aortic wall from distant sites, hidden inside macrophages,¹ and can initiate an inflammatory process, causing irreversible changes in the vascular wall. Studies involving cytomegalovirus have shown the association between microorganisms and severe changes in the arterial wall.¹ Cytomegalovirus causes smooth-muscle-cell proliferation in the arterial wall,²⁴ inhibition of apoptosis,²⁵ endothelial dysfunction,²⁶ and increased expression of cytokines, chemokines, and cellular adhesion molecules.^27,28 We believe that similar pathologic processes might be induced by H. pylori. This helicobacter-mediated activation of an inflammatory process in the vascular wall is possibly the initial step in the development of atheromatous plaque.

In our study, 11 of the 42 patients tested positive for H. pylori DNA in the aortic wall biopsies. While 9 of these 11 patients required emergency operation due to AMI or acute coronary ischemia, only 1 of the remaining 31 patients (all of whom were seronegative for H. pylori) required such an operation. This could be due either to H. pylori-caused development of plaques or to H. pylori-induced instability in preexisting plaques. The finding that most of our H. pylori-positive patients (9 of 11) required early revascularization supports the cause-and-effect association between H. pylori and acute coronary syndrome or AMI.

Recent serologic studies have shown associations between H. pylori and the instability of atherosclerotic carotid plaque or the proliferation of aortic atheromatous plaque.^2,12 In a study conducted by Shmuely and associates,² in which the presence of aortic atheromatous plaques was evaluated by transesophageal echocardiography, it was found that the incidence of aortic atherosclerosis was higher in patients who were seropositive for H. pylori and CagA than in patients who were seronegative for H. pylori and CagA.

In a clinical study by Majka and colleagues,²⁹ it was found that patients with a stroke history and H. pylori seropositivity had significantly higher levels of serum total cholesterol, LDL cholesterol, and interleukin-8, compared with members of a H. pylori-seronegative group. Besides, in the same study, it was shown that patients with a stroke history had statistically significant reductions in the serum values of total cholesterol, LDL cholesterol, and interleukin-8, after 6 months of anti-H. pylori therapy. In accord with these findings, we detected significantly higher levels of total cholesterol, LDL cholesterol, leukocytes, and HDL cholesterol in H. pylori-positive patients than in other patients in our study. In H. pylori-mediated atherosclerosis, such alterations in blood values might reflect irregularity in lipid metabolism induced by this microorganism and might indicate its role as a causative and facilitative factor in atheromatous plaque development.

Another notable finding in our study was that none of the 11 patients who tested positive for H. pylori DNA in the aortic wall tested positive in IMA biopsies. The IMA is known to be resistant to atherosclerosis before and after its use as a bypass conduit.^13,14 This resistance to atherosclerosis is currently attributed to the IMA's superior endothelial function, compared with that of the saphenous vein and the aorta.¹⁵ In a clinical study initiated upon this premise,¹³ elevated eNOS levels with higher expression of ACE and endothelin-A were detected in the IMA than in the saphenous vein. The resistance of this vessel to atherosclerosis might be attributed to its dynamic endothelial functions and its high cellular turnover.

In our study, the presence of H. pylori DNA in aortic tissue that (macroscopically, at least) was atheroma-free, but not in IMA biopsy samples, could indicate yet another reason for the resistance of the IMA to atherosclerosis. Indeed, the superior endothelial function of the IMA could well be linked to the absence of H. pylori DNA in the IMA, in comparison with the aortic wall: resistance to endothelial dysfunction could aid the IMA's resistance to H. pylori-mediated atherosclerosis. The absence of H. pylori DNA in the IMA and its simultaneous presence in the aorta (of the same patients) is a good indication of why the IMA is resistant to infection-caused atherosclerosis.

We conclude that H. pylori, upon reaching the aortic or other arterial wall, becomes directly involved in the development of atheromatous plaque in that region. It is possible that H. pylori's presence in the aorta may indicate the simultaneous presence of H. pylori-associated atherosclerosis in the coronary arteries. Further, H. pylori's presence in the coronary arteries may contribute to plaque instability and to the development of acute coronary syndrome. Our findings support the hypothesis of a pathogenetic link between H. pylori infection and the formation of atherosclerotic plaque in the aorta and coronary arteries. However, further prospective studies are needed; our small sample size may render our results inapplicable to the general population.

We believe that the existence of H. pylori DNA in the aorta but not in the IMA of the same patients might indicate the resistance of the IMA to atherosclerosis. Moreover, we believe that a strong correlation exists between H. pylori positivity in aortic punch biopsy samples and the presence of AMI or acute coronary ischemia in the patients from whom the tissue samples were drawn. Our group is performing further studies on the possible role of H. pylori in the atherosclerotic process.