Endothelial Dysfunction Is Reversible in Helicobacter Pylori-Positive Subjects

CLINICAL RESEARCH STUDY

Endothelial Dysfunction Is Reversible in Helicobacter Pylori-Positive Subjects Arnon Blum, MD,a,b,c Snait Tamir, PhD,d Keren Mualem, BSc,c Rotem Shelly Ben-Shushan, MSc,d Lital Keinan-Boker, MD, PhD,e Maya Paritsky, MDb a

Interdisciplinary Stem Cell Research Institute, University of Miami, Miami, Fla; bDepartment of Medicine and the Gastroenterology Unit, Baruch Padeh Poria Medical Center, Lower Galilee, Israel; cRuth and Baruch Rappaport Faculty of Medicine, Technion Institute of Technology, Haifa, Israel; dLaboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Technology Center, Kiryat Shmona, Israel; eSchool of Public Health, Haifa University, Haifa, Israel.

ABSTRACT BACKGROUND: Studies have shown an association between Helicobacter pylori (HP) infection and atherosclerosis. Although epidemiological studies have suggested an association between HP infection and atherosclerosis, the issue is still controversial. It is not clear whether HP eradication will reverse endothelial damage and prevent cardiovascular events. METHODS: Thirty-one dyspeptic subjects (16 men, 15 women; 50.8 ⫾ 16.7 years) were diagnosed as HP positive using histopathological evaluation. Eleven dyspeptic subjects (5 men, 6 women; 55.4 ⫾ 9.3 years) were negative to HP (controls). Interleukin-6 level and vascular measurements (ankle brachial index and flow-mediated diameter percent change) were done twice: on entry and 3 months afterwards. HP-positive subjects were treated with the triple therapy. RESULTS: Thirty-one HP-positive subjects (50.8 ⫾ 16.7 years, 16 men, weight 79.6 ⫾ 14.8 kg, height 1.70 ⫾ 0.1 m, body mass index [BMI] 27.5 ⫾ 4.4, waist circumference 97.6 ⫾ 16.7 cm) were treated accordingly. There were 11 HP-negative subjects (controls) (55.4 ⫾ 9.3 years, 5 men, weight 83.4 ⫾ 16.8 kg, height 1.68 ⫾ 0.1 m, BMI 29.6 ⫾ 6.1, waist circumference 104.4 ⫾ 13.7 cm). No difference in age (P ⫽ .27), weight (P ⫽ .51), height (P ⫽ .50), BMI (P ⫽ .30), or waist circumference (P ⫽ .20) was observed. HP-positive subjects had severe endothelial dysfunction (⫺1.26 ⫾ 8.4%) that improved after treatment (8.4 ⫾ 9.0%) (P ⫽ .001). HP-negative subjects had endothelial dysfunction (1.9 ⫾ 9.7%) that was not improved (5.6 ⫾ 8.3%) (P ⫽ .41). Interleukin-6 levels in serum were not elevated in HP-positive subjects before or after HP eradication (8.4 ⫾ 17.5 vs 13.5 ⫾ 30.7 pg/mL; P ⫽ .45). CONCLUSIONS: The novel finding of our study was that HP eradication can improve endothelial dysfunction. © 2011 Elsevier Inc. All rights reserved. • The American Journal of Medicine (2011) 124, 1171-1174 KEYWORDS: Cardiovascular prevention; Endothelial function; Helicobacter pylori

Many observational studies have indicated that elevated levels of C-reactive protein are associated with future risk of coronary heart disease and stroke,1 supporting the hypothesis that inflammation plays an important role in the patho-

Funding: None. Conflict of Interest: None. Authorship: All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Requests for reprints should be addressed to Arnon Blum, MD, Interdisciplinary Stem Cell Research Institute, University of Miami, Miami, FL 33136. E-mail address: [email protected]

0002-9343/$ -see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjmed.2011.08.015

genesis of atherosclerosis.2 Many epidemiological studies have shown association between previous infections with Helicobacter pylori (HP),3,4 Chlamydia pneumoniae,5 and cytomegalovirus6 and the presence of atherosclerosis in coronary, carotid, or peripheral vessels. According to the response-to-injury hypothesis of atherosclerosis, endothelial dysfunction is the first step in atherosclerosis.2,7 The pathogenesis of atherosclerosis includes abnormal lipid metabolism, endothelial dysfunction, inflammatory and immunological factors, plaque rupture, and smoking.8 Infectious processes can contribute to the pathogenesis of atherosclerosis by producing chronic infection of blood vessels, vascular inflammation, and endothelial dysfunction.9 It has

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been suggested that exposure to sustained high levels of Soluble interleukin-6 (IL-6) levels were measured by an endotoxin constitute a risk factor for atherosclerosis in anenzyme-linked immunosorbent assay method according to imal models and in humans.9,10 Although epidemiological the manufacturer instructions (R&D Systems Inc., Minnestudies have suggested a relationship between HP infection apolis, Minn). and atherosclerosis, the issue is still controversial. It is not An ABI was measured while the patient was supine, and clear whether HP eradication will measurement of the systolic blood improve vascular inflammation and pressure in all 4 extremities was endothelial damage and thus, may done. To measure an ankle sysCLINICAL SIGNIFICANCE prevent atherosclerosis and future tolic pressure, a standard adult cardiovascular events. blood pressure cuff was placed ● Helicobacter pylori infection, usually subOur purpose was to study the around the ankle just above the clinical, may cause endothelial dysfunceffect of HP eradication on endothemalleoli. While using the Doppler tion and atherosclerosis with future carlial function vascular inflammation. flow-meter to monitor the signal diovascular events. from the posterior of the anterior ● H. pylori eradication by conservative tibial artery, distal to the cuff, the METHODS cuff was inflated to a pressure apmanagement (antibiotics) can change Thirty-one patients (16 men, 15 proximately 30 mm Hg above the the clinical outcome, improve endothewomen; 50.8 ⫾ 16.7 years old) systolic pressure to occlude flow lial dysfunction, and may prevent fuwith dyspepsia were diagnosed as temporarily. As the cuff was ture atherosclerosis and cardiovascular having a positive HP using antral slowly deflated (2-5 mm Hg/s), events. histopathologic evaluation. Eleven the pressure at which the Doppler subjects with dyspepsia (5 men, 6 flow signal was first heard and rewomen; 55.4 ⫾ 9.3 years old) had corded was the ankle systolic a negative HP (proven by a bipressure. An ABI was calculated by dividing the ankle opsy) and served as the control group. All HP-positive systolic blood pressure by the greater of the 2 upper-exsubjects were symptomatic: 6 (19%) had coronary artery tremity systolic blood pressures. An ABI between 0.9 and disease, 10 (32%) had hypertension, 5 (16%) had diabetes 1.3 is considered normal. An ABI below 0.9 is indicative of mellitus, 2 (6%) had atrial fibrillation, and 9 (29%) were peripheral artery disease. An ABI below 0.5 is indicative of smokers. All HP-negative subjects were symptomatic: 1 severe arterial stenosis. (9%) subject had coronary artery disease, 3 (27%) had All measurements of the FMD% were performed early in hypertension, 2 (18%) had diabetes mellitus, none had atrial the morning, in a quiet and dark room and at controlled fibrillation, and 2 (18%) were smokers. ambient temperatures between 20°C and 26°C, and the The study was approved by the Ethics Committee of the patient was still fasting after an overnight fast of at least 10 hospital, and an informed consent for participation in the hours (water was permitted), with the subjects supine and study was obtained from all subjects. All procedures were after 10 minutes of rest. The subject’s right arm was comperformed early in the morning, when patients were still fortably immobilized in the extending position, allowing for fasting. After signing a consent form, every patient underultrasound scanning of the brachial artery 5-10 cm above went upper gastrointestinal endoscopy with gastric (antral) the antecubital fossa. In each examination, recording of biopsy. The biopsies were examined using CUTest (CUTvessel images was followed by inflation of a cuff to supraCampilobacter [Helicobacter]-Urease-Test): in vitro diagsystolic pressure (40-50 mm Hg above systolic pressure) for nostic for the rapid detection of bacterial urease activity in 5 minutes. Then the cuff was deflated and the brachial artery samples of gastric or duodenal mucosa (Temmler Pharma diameter was imaged and recorded for 3 minutes. FMD% GmbH & Co., Marburg, Germany) to prove the existence of more than 10% is considered a normal response; FMD% HP in the stomach. All rapid urease tests were approved by lower than 10% reflects endothelial dysfunction, which histopathologic examination. Clinical parameters included means a high likelihood of developing a cardiovascular weight, height, and waist circumference. Vascular measureevent in the future. Subjects with a negative FMD% result ments included the flow-mediated diameter percent change (the artery is constricted after stress and not dilated as (FMD%) (brachial artery method) and the ankle brachial expected) have the worst prognosis. index (ABI). HP-positive subjects were treated with triple The body mass index (BMI) is a standardized measure of therapy (2 kinds of antibiotics and a proton pump inhibitor) the relationship of body mass to height. The BMI is calcufor 10 days. Vascular studies (ABI and FMD%) were perlated by dividing the weight in kilograms by the height in formed again 3 months after the first visit for all participatmeters squared (kg/m2). ing subjects. Subjects that were negative for HP were invited for a second visit 3 months after the first visit. The RESULTS same vascular measurements were done early in the morning 3 months after the first visit for all subjects that took part Thirty-one subjects had documented HP-positive biopsies and were treated with a triple therapy for 10 days. Their in the study.

Blum et al Table 1

Helicobacter Pylori Eradication Can Reverse Endothelial Dysfunction Clinical Characteristics

Number Age (years) Weight (kg) Height (m) BMI W. Circ.

H. Pylori⫹

H. Pylori⫺

P Value

31 50.8 ⫾ 16.7 79.6 ⫾ 14.8 1.70 ⫾ 0.1 27.5 ⫾ 4.4 97.6 ⫾ 16.7

11 55.4 ⫾ 9.3 83.4 ⫾ 16.8 1.68 ⫾ 0.1 29.6 ⫾ 6.1 104.4 ⫾ 13.7

.27 .51 .50 .30 .20

was 8.4 ⫾ 17.5 pg/mL for HP-positive subjects and following HP eradication (3 months after the first measurement) it was 13.5 ⫾ 30.7 pg/mL (P ⫽ .45). IL-6 level on admission for HP negative subjects was 5.6 ⫾ 1.8 pg/mL and 4.7 ⫾ 1.3 pg/mL after 3 months (P ⫽ .27) (Table 2).

DISCUSSION

BMI ⫽ body mass index (kg/m2); W. Circ. ⫽ waist circumference.

average age was 50.8 ⫾ 16.7 years. Sixteen were men (48%); 6 (19%) had coronary artery disease, 10 (32%) were hypertensive, 5 (16%) had diabetes mellitus, 2 (6%) had atrial fibrillation, and 9 (29%) were active smokers. Average weight was 79.6 ⫾ 14.8 kg, height was 1.70 ⫾ 0.1 m, BMI was 27.5 ⫾ 4.4, and average waist circumference was 97.6 ⫾ 16.7 cm (Table 1). Average age of the 11 HP-negative subjects (5 men, 6 women) was 55.4 ⫾ 9.3 years old; only 1 (9%) had coronary artery disease, 3 (27%) had hypertension, 2 (18%) had diabetes mellitus, no one had atrial fibrillation, and only 2 (18%) were active smokers. Their average weight was 83.4 ⫾ 16.8 kg, height was 1.68 ⫾ 0.1 meters, BMI 29.6 ⫾ 6.1, and waist circumference was 104.4 ⫾ 13.7 cm (Table 1). No statistical difference was observed in relation with age (P ⫽ .27), weight (P ⫽ .51), height (P ⫽ .50), BMI (P ⫽ .30), and waist circumference (P ⫽ .20) (Table 1). HP-positive subjects had severe endothelial dysfunction (⫺1.26 ⫾ 8.4%) that improved significantly after 3 months (8.4 ⫾ 9.0%) (P ⫽ .001) following HP eradication (Table 2). HP-negative subjects had endothelial dysfunction (1.9 ⫾ 9.7%) that was not significantly changed after 3 months (5.6 ⫾ 8.3%) (P ⫽ .41) (Table 2). ABI was not affected by HP infection. Among HPpositive subjects, it was 1.2 ⫾ 0.2 and did not change after HP eradication (1.2 ⫾ 0.2) (P ⫽ .46). In HP-negative subjects, the ABI was 1.25 ⫾ 0.2 when they entered the study and it was 1.31 ⫾ 0.2 after 3 months (P ⫽ .51) (Table 2). Soluble IL-6 levels in serum were not elevated before and after treatment for HP. On entry to the study, IL-6 level

Table 2

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Our study has demonstrated that symptomatic HP-positive subjects had severe endothelial dysfunction that was significantly improved to normal values after HP eradication. HP-negative subjects had endothelial dysfunction on entry to the study and continued to have endothelial dysfunction 3 months afterwards. Unlike some (but not all) previous studies, we did not find an increase in inflammatory markers, and the level of the proinflammatory marker that we studied (IL-6) was not changed following HP eradication. A study that evaluated inflammation in HP-positive subjects found that C-reactive protein (CRP) level was increased in subjects who were seropositive for HP, with a positive correlation with anti-HP antibody titer.11 Intercellular cell adhesion molecule 1 also was increased in seropositive subjects, along with endothelial dysfunction. This association remained significant after adjusting for age, BMI, blood pressure, serum lipids, glucose, and CRP.11 Paraoxonase 1 activity was significantly inhibited in HPpositive subjects who also had thicker carotid intima-media layer compared with HP-negative subjects.12 Vasoreactivity studies found that the arterial stiffness of young HP-positive subjects was higher compared with HP-negative subjects; however, this difference was not observed in elderly individuals.13 Another prospective study found an association between HP infection and decreased high-density lipoprotein cholesterol and increased apoB14 that can support the mechanistic association between HP seropositivity and atherosclerosis and future cardiovascular events. In vitro studies have shown that endothelial function can be attributed to vacuolating cytotoxin A, an HP-secreted virulence factor that elicits its effects by modulating plasma and mitochondrial membrane ion permeability15 as well as altering intracellular vesicular trafficking leading to vacuole formation,16 with vacuolating cytotoxin A-dependent reduction in endothelial nitric oxide.17 Another association was found in a

Vascular Reactivity and Markers of Inflammation H. Pylori⫹

H. Pylori⫺

Before Rx After FMD% P-value ABI P-value IL-6 (pg/mL) P-value

3 Months After

⫺1.26 ⫾ 8.4

8.4 ⫾ 9.0

Before Rx 1.9 ⫾ 9.7

.001 1.2 ⫾ 0.2

1.2 ⫾ 0.2

1.25 ⫾ 0.2

1.31 ⫾ 0.2 .51

13.5 ⫾ 30.7 .45

5.6 ⫾ 8.3 .41

.46 8.4 ⫾ 17.5

3 Months After

5.6 ⫾ 1.8

4.7 ⫾ 1.3 .27

ABI ⫽ ankle brachial index; FMD% ⫽ flow-mediated diameter (nitric oxide-dependent) percent change; sIL-6 ⫽ soluble interleukin-6.

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study that measured asymmetric dimethylarginine (ADMA) (a potent endogenous nitric oxide synthase inhibitor) before and after eradication of HP by conventional triple therapy. A significant reduction of ADMA was found after HP eradication; thus, HP can cause atherosclerosis (through high ADMA levels that inhibit endothelial nitric oxide synthase), and eradicating HP may decrease the risk of atherosclerosis and future cardiovascular events.18 We found that HP-positive subjects had endothelial dysfunction; however, we could not detect an inflammatory effect measured by IL-6 in the serum. Apparently, we are not the only ones who did not find such an effect. A study that measured IL-6 level in HP-positive patients with peptic ulcer disease and in HP-positive patients with gastric cancer found that only cancer patients had high IL-6 levels, while patients with peptic ulcer disease had the same levels as the control group.19 Among 42 dyspeptic patients that underwent antral histopathologic examination and Giemsa stain for HP detection, 30 were diagnosed as having an active HP. No difference in IL-6, IL-8, and tumor necrosis factor-␣ was observed between the HP-positive and HP-negative subjects.20 IL-6 levels were measured in coronary artery disease patients before coronary intervention, and no correlation was found between antibodies against HP and IL-6 or CRP levels.21 There are inconsistent data concerning markers of systemic inflammation in HP-positive subjects. It could be that there are different ethnic groups that may develop inflammatory reactions in a different way, or there could be a sex effect. Another explanation could be that there are different stages of inflammatory response that develop over time, and some people ask for medical advice earlier than others, and this time difference may explain the difference in levels of inflammatory markers detected in the serum of HP-positive subjects.

Study Limitations The relatively small population studied is a limiting factor and in order to confirm our results, another study should be done with larger populations.

CONCLUSIONS The novel finding of our study was that HP eradication improved endothelial dysfunction significantly, and thus, may prevent atherosclerosis and future cardiovascular events.

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