Helicobacter Pylori Infection Causes Persistent Platelet Activation In Vivo Through Enhanced Lipid Peroxidation
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Center of Excellence on Aging (G.D., M.N., A.F., D.F., T.T., G.C., F.C.), Fondazione Università "G. d’Annunzio," and Departments of Medicine and Drug Sciences, University of Chieti "G. d’Annunzio" Schools of Medicine and Pharmacy, Italy; and Departments of Medical Therapy (S.B.) and Pharmacology (C.P.), University of Rome "La Sapienza," Italy.
Reprint requests to: Carlo Patrono, MD, Center of Excellence on Aging, Fondazione Università "G. d’Annunzio," Via Colle dell’Ara, 66013, Chieti, Italy. E-mail cpatrono@unich.it
Abstract
Objective— We aimed at investigating the relationship between Helicobacter pylori infection and in vivo lipid peroxidation and platelet activation, as reflected by urinary 8-iso-prostaglandin (PG)F2 and 11-dehydro-thromboxane (TX)B2, respectively, in otherwise healthy dyspeptic subjects.
Methods and Results— We measured urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion in 40 dyspeptic subjects with a positive 13C-urea breath test and 38 dyspeptic individuals with a negative test. Moreover, we investigated the effects of H pylori eradication on prostanoid metabolite excretion in 23 H pylori–positive subjects. We also measured prostanoid metabolite excretion before and after selective cyclooxygenase-2 inhibition with rofecoxib in 4 H pylori–positive subjects. Urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion was significantly higher in the H pylori–positive individuals than in controls. A significant direct correlation was found between the degree of positivity to the13C-urea breath test and urinary 8-iso-PGF2 excretion. The latter was linearly correlated with urinary 11-dehydro-TXB2. Successful eradication of H pylori infection led to a significant reduction in both 8-iso-PGF2 and 11-dehydro-TXB2. Furthermore, their levels were unaffected after treatment with rofecoxib.
Conclusions— Our study provides evidence of enhanced in vivo lipid peroxidation and platelet activation in association with H pylori infection and suggests a novel mechanism by which an infectious agent could contribute to atherothrombosis.
Urinary excretion of 8-iso-PGF2a and 11-dehydro-TXB2 was significantly higher in Helicobacter pylori–positive individuals than in controls, with direct correlations between the degree of positivity to the 13C-urea breath test and urinary 8-iso-PGF2 and between the 2 metabolites. Successful H pylori eradication led to a reduction in these indexes of lipid peroxidation and platelet activation. We suggest a novel mechanism by which an infectious agent could contribute to atherothrombosis.
Key Words: risk factors ? oxidant stress ? platelets ? infection ? inflammation
Introduction
Inflammatory mechanisms have been implicated in the pathogenesis of atherosclerosis, and a significant association between infectious burden and the extent and long-term prognosis of atherosclerosis has been reported.1
Helicobacter pylori infection represents one of the most widespread human infectious diseases.2 Several studies have reported an association between infection with H pylori and incidence of vascular disease, particularly coronary heart disease (CHD).3 However, most of these data derived from cross-sectional and retrospective studies and potential confounders, such as socioeconomic status or smoking pattern,3,4 may complicate their interpretation. Analysis restricted to the available prospective studies in socioeconomically homogeneous populations provides limited evidence for an association between H pylori and CHD.5 However, a case-control and sibling pairs study of early-onset myocardial infarction reported strong evidence for an association between H pylori infection and CHD, suggesting that the potential proinflammatory effects of H pylori might be of greater importance at younger ages.6
Experimental studies have shown that H pylori induces platelet aggregation in gastric mucosal microcirculation.7,8 However, no previous study has examined the occurrence and mechanism(s) of platelet activation in vivo that might be associated with H pylori infection in humans.
Several cardiovascular risk factors are associated with low-grade inflammation, increased oxidant stress, and lipid peroxidation.9 F2-isoprostanes, a family of bioactive prostaglandin (PG)F2-like compounds10 produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen-free radicals on cell membranes and low-density lipoprotein (LDL) particles,9 represent a reliable marker of in vivo lipid peroxidation. Among F2-isoprostanes, of particular interest is 8-iso-PGF2, which induces vasoconstriction and modulates function of human platelets.11 Measurement of urinary F2-isoprostanes has been used extensively in clinical settings putatively associated with increased oxidant stress.9
In the present study, we tested the hypothesis that low-grade inflammation associated with H pylori infection3 would induce increased in vivo lipid peroxidation with generation of 8-iso-PGF2 and other biologically active isoeicosanoids and that these compounds would in turn contribute to platelet activation in this setting. The latter was evaluated by measuring urinary excretion of 11-dehydro-thromboxane (TX)B2, a stable enzymatic derivative of TXA2, a labile eicosanoid that amplifies platelet activation in response to other stimuli and induces irreversible platelet aggregation.12
Thus, the aim of our study was to investigate the causal relationship between H pylori infection and the rates of in vivo lipid peroxidation and platelet activation in otherwise healthy dyspeptic subjects through biochemical measurements and pharmacological interventions.
Methods
Study Participants
We initially studied 28 dyspeptic men aged <60 years who had a first-time positive 13C-urea breath test (UBT) for H pylori infection and had not received antibiotic treatment previously. Twenty-eight age-matched dyspeptic men who had a negative UBT for H pylori infection were recruited as a control group. To investigate potential gender-related differences, we performed a second cross-sectional study in 12 dyspeptic women aged <60 years with a first-time positive UBT who had not been treated previously. Ten age-matched dyspeptic women who had a negative UBT were enrolled as a control group. Because the results of the 2 studies were internally consistent, the data are presented as a whole. The clinical characteristics of study subjects are summarized in the Table .
Baseline Characteristics of H Pylori–Positive and –Negative Subjects
To avoid confounding by other determinants of oxidant stress and platelet activation, subjects were excluded if they had a history or evidence of atherothrombotic diseases, diabetes mellitus, cigarette smoking, dyslipidemia, and arterial hypertension, as well as current or recent (<3 months) systemic or localized infections. Moreover, dyspeptic subjects were excluded if they were taking low-dose aspirin, nonsteroidal anti-inflammatory drugs, or vitamin supplements. Informed consent was obtained from each participating subject, and the protocol was approved by the University of Chieti ethics committee.
Study Design
A cross-sectional comparison of urinary 8-iso-PGF2 and 11-dehydro-TXB2, a major enzymatic metabolite of TXA2,13 was performed between subjects positive and negative for H pylori infection. All participants were studied as outpatients after a 12-hour fast. Each subject performed an overnight urine collection. Urine samples were added with the antioxidant 4-hydroxy-Tempo (1 mmol/L; Sigma) and stored at –20°C until extraction.
To examine the effects of H pylori eradication on indexes of lipid peroxidation and platelet activation, an open pilot intervention study was performed in H pylori–positive subjects. Thus, we investigated the effects of a standard antibiotic therapy on urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion in 15 of the 28 H pylori–positive men and in 8 of the 12 H pylori–positive women. Entry criteria were a clear indication of antibiotic therapy, lack of contraindications, and willingness to participate in this additional study. These subjects were given 1000 mg amoxicillin twice daily plus 500 mg clarithromycin twice daily for 7 days. A standard regimen of a proton pump inhibitor (20 mg omeprazole twice daily) was also given.14 Before and after treatment, participants were instructed to perform an overnight urine collection. At least 4 weeks after completing the triple therapy, the UBT was repeated to assess eradication. In case of treatment failure (11 subjects), a new treatment regimen was prescribed. Five subjects who were found still infected after the first course of therapy were treated with 500 mg clarithromycin twice daily and 500 mg tinidazole twice daily for 7 days. A dosage of 400 mg ranitidine bismuth citrate twice daily was also given. Six subjects who failed to respond to the first treatment regimen were lost to follow-up.
Because H pylori infection is associated with cyclooxygenase-2 (COX-2) expression in the gastric mucosa15–17 and 8-iso-PGF2 and TXA2 can be formed by human monocytes through a COX-2–dependent mechanism,18 a third study was performed to evaluate whether selective inhibition of COX-2 activity had any influence on 8-iso-PGF2 in subjects positive for H pylori infection. For this purpose, 4 of the H pylori–positive subjects were given 12.5 mg rofecoxib, a highly selective COX-2 inhibitor,19 once daily for 7 days. Participants collected overnight urine samples at the beginning and the end of the rofecoxib treatment for measurement of 8-iso-PGF2 and 11-dehydro-TXB2 excretion.
Analytical Measurements
The presence of H pylori was determined by UBT. UBT consisted of a baseline breath sample and a second sample 30 minutes after administration of 75 mg of 13C-labeled urea (Isotec) dissolved in orange juice.20 Subjects were fasted overnight. The 13C-enrichment in breath was analyzed using an isotope ratio mass spectrometer (ABCA; Europa Scientific) The UBT was considered positive when the difference with baseline at 30' (DOB30; ie, the difference between -value at 30' and -value at baseline) was 5%. This noninvasive test is as accurate in predicting H pylori status as invasive tests and is the recommended test for diagnosis of H pylori infection.21 Urinary 8-iso-PGF2 and 11-dehydro-TXB2 were measured by previously described and validated radioimmunoassay methods.22,23
Statistical Analysis
Data were analyzed by nonparametric methods to avoid assumptions about the distribution of the measured variables. An ANOVA was performed with the Kruskall–Wallis method. Subsequent pairwise comparisons were made with the Mann–Whitney U test. Differences between baseline and post-treatment values were analyzed with the Wilcoxon signed-rank test. Moreover, the association of eicosanoid measurements with other biochemical parameters was assessed by the Spearman rank correlation test. A multiple linear regression analysis was performed to further quantify the relationship between 11-dehydro-TXB2 excretion and the other variables in the cross-sectional studies.
The cross-sectional and intervention studies had a >80% power to detect an H pylori–related difference in urinary 11-dehydro-TXB2 excretion of 1 SD between groups with a 2-tailed of 0.05.
All values are reported as median (range). P values <0.05 were regarded as statistically significant. All tests were 2-tailed, and analyses were performed using a computer software package (Statistica 1999 edition; StatSoft; or Statistical Package for the Social Sciences, version 12.0; SPSS).
Results
Urinary 8-iso-PGF2 excretion was significantly higher in the 40 H pylori–positive than in the 38 H pylori–negative subjects (357 [154 to 645] pg/mg versus 189 [85 to 306] pg/mg creatinine; median (range) P=0.0001; Figure 1). Urinary 11-dehydro-TXB2 excretion rate was also significantly increased in H pylori–positive compared with H pylori–negative subjects (868 [339 to 1630] pg/mg versus 378 [212 to 690] pg/mg creatinine; P=0.0001; Figure 1). In the 40 H pylori–positive individuals, a statistically significant direct correlation was found between DOB30 in breath samples and urinary 8-iso-PGF2 excretion rates (=0.505; P=0.0016). The latter index of lipid peroxidation was in turn linearly correlated with the rate of TX biosynthesis, as reflected by urinary 11-dehydro-TXB2 excretion (=0.52; P=0.0012).
Figure 1. Urinary excretion of 8-iso-PGF2 (top) and 11-dehydro-TXB2 (bottom) in H pylori–negative vs H pylori–positive subjects. Error bars represent minimum and maximum values; , median values; boxes, interquartile range.
A multiple regression analysis performed in 78 subjects showed that DOB30 in breath samples (regression coefficient 0.44; SE 0.11; P=0.000128), male sex (regression coefficient 0.39; SE 0.08; P=0.0150), and urinary 8-iso-PGF2 excretion rates (regression coefficient 0.39; SE 0.11; P=0.000456) independently correlated with 11-dehydro-TXB2 excretion.
Effects of Eradication Therapy
We also investigated the effects of H pylori eradication on urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion to test the hypothesis of a cause–effect relationship between H pylori infection and enhanced lipid peroxidation and platelet activation in this setting. Thus, we evaluated the effects of eradication therapy in 15 H pylori–positive men and 8 H pylori–positive women.
Successful eradication, achieved in 12 of the 23 treated patients, was associated with a statistically significant reduction in 8-iso-PGF2 (from 400 [233 to 566] pg/mg to 247 [176 to 348] pg/mg creatinine; P=0.0022) and 11-dehydro-TXB2 urinary excretion (from 1049 [606 to 1630] pg/mg to 600 [303 to 822] pg/mg creatinine; P=0.0022). Excretion rates of the 2 metabolites remained substantially unchanged in association with unsuccessful eradication in the other 11 subjects (Figure 2). The coefficient of variation for 3 repeated measurements of urinary 8-iso-PGF2 and 11-dehydro-TXB2, obtained from 7 subjects who failed eradication therapy, averaged 21.3±10.3% and 18.2±10.5%, respectively.
Figure 2. Effects of antibiotic treatment on urinary 8-iso-PGF2 (top) and 11-dehydro-TXB2 (bottom) in H pylori–positive subjects. Dots and lines connecting dots represent individual measurements; solid symbols and continuous lines, values of subjects with successful eradication; open symbols and broken lines, values of subjects with unsuccessful eradication; error bars, minimum and maximum values; , median values; boxes, interquartile range, represented on the left and on the right side of each panel.
In 5 of the 11 subjects in whom H pylori failed to be eradicated, a different treatment regimen was prescribed after a period of 10±2 weeks. This second cycle of therapy led to successful eradication in 3 subjects, with a consistent reduction in 8-iso-PGF2 (from 371 [331 to 483] pg/mg to 285 [132 to 288] pg/mg creatinine) and 11-dehydro-TXB2 excretion (from 1040 [680 to 1200] pg/mg to 327 [254–375] pg/mg creatinine), whereas urinary levels of both metabolites remained unchanged in the 2 subjects who failed to be eradicated (data not shown).
In the 15 subjects who eventually achieved successful eradication (12 after the first regimen plus 3 after the second regimen), the reduction in DOB30 was associated with a fall in TX biosynthesis, the average extent of which showed a remarkably good fitting with the linear relationship between DOB30 values and 11-dehydro-TXB2 excretion rates, as established in the whole group of H pylori–positive subjects at baseline (Figure 3).
Figure 3. Correlation between DOB30 and urinary 11-dehydro-TXB2 excretion in H pylori–positive subjects. The 40 data points represent the baseline measurements for the 28 and 12 dyspeptic men and women, respectively. Of these, 15 and 8 were subjected to treatment. Twelve subjects responded to the first treatment regimen, and the average results for these 12 before treatment are shown by the . The average results for these 12 responding subjects after treatment are shown by the .
Effects of Selective COX-2 Inhibition
Urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion rates were not affected after 1 week of selective COX-2 inhibition achieved with rofecoxib (from 280 [247 to 338] pg/mg to 303 [259 to 320] pg/mg, and from 553 [463 to 578] pg/mg to 579 [363 to 606] pg/mg creatinine, respectively). This finding is consistent with a non-COX–dependent mechanism of F2-isoprostane formation in association with H pylori infection. Moreover, these results demonstrate that enhanced TX biosynthesis is not a byproduct of COX-2 expression in response to H pylori infection.
Discussion
Atherothrombotic events do occur among individuals without readily apparent cardiovascular risk factors.24 In recent years, inflammation has been suggested to play a key role in the initiation and progression of the atherosclerotic process.25 Circulating markers of persistent low-grade inflammation, such as C-reactive protein, can predict recurrence of major vascular events in patients with established ischemic heart disease,26–28 as well as the risk of a first myocardial infarction in apparently healthy subjects.29 Persistent infections may represent a potential trigger of systemic inflammation, and evidence for a link between total infectious burden and atherosclerotic severity has been provided.1,30,31 A weak and controversial association between H pylori infection and CHD has been described.32 H pylori, a primary pathogen for peptic ulcer disease, gastric cancer, and lymphoma2 is a potential source of inflammatory cytokines possibly contributing to the atherosclerotic process.33 It has been hypothesized that H pylori infection might modify serum lipid concentrations, thus increasing the risk for cardiovascular disease.34 Moreover, H pylori is independently associated with increased fibrinogen levels in healthy subjects,35 and an association between an IgG antibody response to multiple pathogens, including H pylori, and endothelial dysfunction has been reported.36 Finally, it has been suggested that chronic atrophic gastritis induced by H pylori, causing malabsorption of vitamin B12 and folate, may lead to increased plasma levels of homocysteine, a known risk factor for vascular disease.37 H pylori–specific DNA has been detected recently in atheromatous plaques of patients with severe coronary artery disease, supporting the hypothesis of direct involvement of the bacterium in the progression and instability of atherosclerotic lesions.38
An important link has been shown between coronary artery disease and infection with H pylori, and its eradication significantly attenuated the reduction in coronary lumen after coronary angioplasty.39 Moreover, H pylori eradication increased high-density lipoprotein cholesterol and decreased C-reactive protein, thrombin–antithrombin complexes, and lipoprotein(a) levels in type 1 diabetic patients.40
In the present study, we have identified a novel mechanism through which H pylori infection may enhance cardiovascular risk (ie, persistent platelet activation). Biochemical evidence of enhanced platelet activation in vivo in association with H pylori infection was obtained through noninvasive measurements of TX metabolite excretion that avoid artifactual platelet activation during and after blood sampling.41 The exclusion criteria used to recruit dyspeptic otherwise healthy subjects avoided confounding by traditional cardiovascular risk factors that can affect the rate of platelet activation. It should be emphasized that TXA2 biosynthesis measured in H pylori–positive subjects in the present study is comparable to that reported previously in association with traditional cardiovascular risk factors such as hypercholesterolemia,42 diabetes mellitus,43 obesity,44 and hypertension.45
Furthermore, we characterized a putative biochemical link between H pylori infection and platelet activation by investigating the in vivo formation of F2-isoprostanes, as reflected by the urinary excretion of the PGF2 isomer 8-iso-PGF2. This family of bioactive isoeicosanoids is produced through free radical–catalyzed peroxidation of arachidonic acid that can occur on cell membranes and LDL particles.10 Measurement of unmetabolized F2-isoprostanes in plasma and urine has proved to be a valuable approach to assess the actual rate of lipid peroxidation in vivo.9,10 Repeated measurements of 8-iso-PGF2 in those subjects in whom H pylori eradication failed demonstrated a persistent abnormality with limited intrasubject variability over time. Evidence for a cause-and-effect relationship between H pylori infection and enhanced lipid peroxidation was provided by the linear correlation between the 13C-UBT and urinary of 8-iso-PGF2 excretion rates as well as by the statistically significant reduction in F2-isoprostane formation after successful eradication (Figure 2). The nonenzymatic nature of 8-iso-PGF2 production in this setting was confirmed by the failure of rofecoxib, a highly selective COX-2 inhibitor,19 to reduce its urinary excretion to any detectable extent.
Enhanced formation of 8-iso-PGF2 in H pylori–positive subjects correlated with increased TXA2 biosynthesis, as reflected by 11-dehydro-TXB2 excretion. Although the systemic concentrations of 8-iso-PGF2 may be too low to trigger platelet activation, this autacoid can synergize with subthreshold concentrations of other agonists in inducing platelet adhesion and aggregation.46,47 Moreover, it is likely that enhanced formation of 8-iso-PGF2 as a consequence of H pylori infection is associated with the release of other bioactive isoeicosanoids formed through the same mechanism of oxygen radical–catalyzed peroxidation of arachidonic acid. To assess the potential contribution to TXA2 biosynthesis of COX-2 expressed by inflammatory and epithelial cells in response to H pylori infection, we investigated the short-term effects of rofecoxib on 11-dehydro-TXB2 excretion in H pylori–positive subjects. The results of this intervention study are consistent with COX-1 being the main COX isoform involved in TXA2 biosynthesis in this setting and make it unlikely that the reduction in 11-dehydro-TXB2 excretion associated with successful H pylori eradication is a reflection of reduced COX-2 expression in the gastric mucosa.16
The findings in H pylori–positive patients extend similar observations made in other clinical settings such as hypercholesterolemia,42 diabetes mellitus,43 cigarette smoking,48,49 homozygous homocystinuria,50 visceral obesity,44 and renovascular hypertension.51 Thus, regardless of the mechanism(s) responsible for enhanced lipid peroxidation, there is quite convincing evidence from studies in such diverse conditions that enhanced generation of bioactive isoeicosanoids may transduce the oxidant signal associated with a variety of cardiovascular risk factors into a functional platelet response, possibly contributing to enhanced thrombotic risk.
In conclusion, the present study provides biochemical evidence of enhanced in vivo lipid peroxidation and platelet activation in dyspeptic individuals with H pylori infection and identifies a novel mechanism through which an infectious agent could contribute to development of atherothrombosis. Reversibility of the hemostatic abnormality after successful eradication of H pylori may have clinical implications for cardiovascular risk management.
Received July 9, 2004; accepted September 17, 2004.
References
Espinola-Klein C, Rupprecht HJ, Blankenberg S, Bickel C, Kopp H, Rippin G, Victor A, Hafner G, Schlumberger W, Meyer J; AtheroGene Investigators. Impact of infectious burden on extent and long-term prognosis of atherosclerosis. Circulation. 2002; 105: 15–21.
Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med. 2002; 347: 1175–1186.
Ridker PM. Inflammation, infection, and cardiovascular risk. How good is the clinical evidence? Circulation. 1998; 97: 1671–1674.
Murray LJ. Helicobacter pylori infection: relation with cardiovascular risk factors, ischaemic heart disease, and social class. Br Heart J. 1995; 74: 497–501.
Ridker PM. A prospective study of Helicobacter pylori seropositivity and the risk for future myocardial infarction among socioeconomically similar US men. Ann Intern Med. 2001; 135: 184–188.
Danesh J, Youngman L, Clark S, Parish S, Peto R, Collins R. Helicobacter pylori infection and early onset myocardial infarction: case-control and sibling pairs study. BMJ. 1999; 319: 1157–1162.
Kalia N, Jacob S, Brown NJ, Reed MW, Morton D, Bardhan KD. Studies on the gastric mucosal microcirculation. 2. Helicobacter pylori water soluble extracts induce platelet aggregation in the gastric mucosal microcirculation in vivo. Gut. 1997; 41: 748–752.
Elizalde JI, Gomez J, Panes J, Lozano M, Casadevall M, Ramirez J, Pizcueta P, Marco F, Rojas FD, Granger DN, Pique JM. Platelet activation in mice and human Helicobacter pylori infection. J Clin Invest. 1997; 100: 996–1005.
Davì G, Falco A, Patrono C. Determinants of F2-isoprostane biosynthesis and inhibition in man. Chem Phys Lipids. 2004; 128: 149–163.
Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalysed mechanism. Proc Natl Acad Sci U S A. 1990; 87: 9383–9387.
Patrono C, FitzGerald GA. Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler Thromb Vasc Biol. 1997; 17: 2309–2315.
Patrono C, Davì G, Ciabattoni G. Thromboxane biosynthesis and metabolism in relation to cardiovascular risk factors. Trends Cardiovasc Med. 1992; 2: 15–20.
Ciabattoni G, Pugliese F, Davì G, Pierucci A, Simonetti BM, Patrono C. Fractional conversion of thromboxane B2 to urinary 11-dehydro-thromboxane B2 in man. Biochim Biophys Acta. 1989; 992: 66–70.
Malfertheiner P, Mégraud F, O’Morain C, Hungin AP, Jones R, Axon A, Graham DY, Tytgat G; European Helicobacter Pylori Study Group (EHPSG). Current concepts in the management of Helicobacter pylori infection—the Maastricht 2–2000 Consensus Report. Aliment Pharmacol Ther. 2002; 16: 167–180.
Sawaoka H, Kawano S, Tsuji S, Tsuji M, Sun W, Gunawan ES, Hori M. Helicobacter pylori infection induces cyclooxygenase-2 expression in human gastric mucosa. Prostaglandins Leukotrienes Essent Fatty Acids. 1998; 59: 313–316.
McCarthy CJ, Crofford LJ, Greenson J, Scheiman JM. Cyclooxygenase-2 expression in gastric antral mucosa before and after eradication of Helicobacter pylori infection. Am J Gastroenterol. 1999; 94: 1218–1223.
Fu S, Ramanujam KS, Wong A, Fantry GT, Drachenberg CB, James SP, Meltzer SJ, Wilson KT. Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology. 1999; 116: 1319–1329.
Patrignani P, Santini G, Panara MR, Sciulli MG, Greco A, Rotondo MT, di Giamberardino M, Maclouf J, Ciabattoni G, Patrono C. Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation. Br J Pharmacol. 1996; 118: 1285–1293.
FitzGerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med. 2001; 345: 433–442.
Dominguez-Munoz JE, Leodolter A, Sauerbruch T, Malfertheiner P. A citric acid solution is an optimal test drink in the 13C-urea breath test for the diagnosis of Helicobacter pylori infection. Gut. 1997; 40: 459–462.
Cutler AF, Havstad S, Ma CK, Blaser MJ, Perez-Perez GI, Schubert TT. Accuracy of invasive and noninvasive tests to diagnose Helicobacter pylori infection. Gastroenterology. 1995; 109: 136–141.
Wang Z, Ciabattoni G, Créminon C, Lawson J, Fitzgerald GA, Patrono C, Maclouf J. Immunological characterization of urinary 8-epi-PGF2 excretion in man. J Pharmacol Exp Ther. 1995; 275: 94–100.
Ciabattoni G, Maclouf J, Catella F, Fitzgerald GA, Patrono C. Radioimmunoassay of 11-dehydro-TXB2 in human plasma and urine. Biochim Biophys Acta. 1987; 918: 293–297.
Braunwald E. Shattuck lecture: cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med. 1997; 337: 1360–1369.
Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
De Beer FC, Hind CR, Fox KM, Allan RM, Maseri A, Pepys MB. Measurement of serum C-reactive protein concentration in myocardial ischemia and infarction. Br Heart J. 1982; 47: 239–243.
Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB; European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet. 1997; 349: 462–466.
Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, Flaker GC, Braunwald E; Cholesterol and Recurrent Events (CARE) Investigators. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation. 1998; 98: 839–844.
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1996; 336: 973–979.
Danesh J, Whincup P, Walker M, Lennon L, Thomson A, Appleby P, Gallimore JR, Pepys MB. Low grade inflammation and coronary heart disease: prospective study and updated meta-analysis. BMJ. 2000; 321: 199–204.
Ridker PM. On evolutionary biology, inflammation, infection and the causes of atherosclerosis. Circulation. 2002; 105: 2–4.
Mendall MA, Goggin PM, Molineaux N, Levy J, Toosy T, Strachan D, Camm AJ, Northfield TC. Relation of Helicobacter pylori infection and coronary heart disease. Br Heart J. 1994; 71: 437–439.
Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-, interleukin-1 ?, and interleukin-8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol. 1994; 29: 425–429.
Niemela S, Karttunen T, Korhonen T, Laara E, Karttunen R, Ikaheimo M, Kesaniemi YA. Could Helicobacter pylori infection increase the risk of coronary heart disease by modifying serum lipid concentrations? Heart. 1996; 75: 573–575.
Patel P, Carrington D, Strachan DP, Leatham E, Goggin P, Northfield TC, Mendall MA. Fibrinogen: a link between chronic infection and coronary heart disease. Lancet. 1994; 343: 1634–1635.
Prasad A, Zhu J, Halcox JP, Waclawiw MA, Epstein SE, Quyyumi AA. Predisposition to atherosclerosis by infections: role of endothelial dysfunction. Circulation. 2002; 106: 184–190.
Tamura A, Fujioka T, Nasu M. Relation of Helicobacter pylori infection to plasma vitamin B12, folic acid, and homocysteine levels in patients who underwent diagnostic coronary arteriography. Am J Gastroenterol. 2002; 97: 861–866.
Kowalski M, Rees W, Konturek PC, Grove R, Scheffold T, Meixner H, Brunec M, Franz N, Konturek JW, Pieniazek P, Hahn EG, Konturek SJ, Thale J, Warnecke H. Detection of Helicobacter pylori specific DNA in human atheromatous coronary arteries and its association to prior myocardial infarction and unstable angina. Dig Liver Dis. 2002; 34: 398–402.
Kowalski M, Konturek PC, Pieniazek P, Karczewska E, Kluczka A, Grove R, Kranig W, Nasseri R, Thale J, Hahn EG, Konturek SJ. Prevalence of Helicobacter pylori infection in coronary artery disease and effect of its eradication on coronary lumen reduction after percutaneous coronary angioplasty. Dig Liver Dis. 2001; 33: 222–229.
de Luis DA, Garcia Avello A, Lasuncion MA, Aller R, Martin de Argila C, Boixeda de Miquel D, de la Calle H. Improvement in lipid and haemostasis patterns after Helicobacter pylori infection eradication in type 1 diabetic patients. Clin Nutr. 1999; 18: 227–231.
FitzGerald GA, Pedersen AK, Patrono C. Analysis of prostacyclin and thromboxane biosynthesis in cardiovascular disease. Circulation. 1983; 67: 1174–1177.
Davì G, Alessandrini P, Mezzetti A, Minotti G, Bucciarelli T, Costantini F, Cipollone F, Bon GB, Ciabattoni G, Patrono C. In vivo formation of 8-epi-prostaglandin F2 is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1997; 17: 3230–3235.
Davì G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S, Pennese E, Vitacolonna E, Bucciarelli T, Costantini F, Capani F, Patrono C. In vivo formation of 8-iso-prostaglandin F2 and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation. 1999; 99: 224–229.
Davì G, Guagnano MT, Ciabattoni G, Basili S, Falco A, Marinopiccoli M, Nutini M, Sensi S, Patrono C. Platelet activation in obese women: role of inflammation and oxidant stress. J Am Med Assoc. 2002; 288: 2008–2014.
Minuz P, Patrignani P, Gaino S, Seta F, Capone ML, Tacconelli S, Degan M, Faccini G, Fornasiero A, Talamini G, Tommasoli R, Arosio E, Santonastaso CL, Lechi A, Patrono C. Determinants of platelet activation in human essential hypertension. Hypertension. 2004; 43: 64–70.
Praticò D, Smyth EM, Violi F, FitzGerald GA. Local amplification of platelet function by 8-epi-prostaglandin F2 is not mediated by thromboxane receptor isoforms. J Biol Chem. 1996; 271: 14916–14924.
Minuz P, Andrioli G, Degan M, Gaino S, Ortolani R, Tommasoli R, Zuliani V, Lechi A, Lechi C. The F2-isoprostane 8-epiprostaglandin F2 increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO. Arterioscler Thromb Vasc Biol. 1998; 18: 1248–1256.
Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM, Shyr Y, Strauss WE, Oates JA, Roberts LJ II. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers: smoking as a cause of oxidative damage. N Engl J Med. 1995; 332: 1198–1203.
Reilly M, Delanty N, Lawson JA, Fitzgerald GA. Modulation of oxidant stress in vivo in chronic cigarette smokers. Circulation. 1996; 94: 19–25.
Davì G, Di Minno G, Coppola A, Andria G, Cerbone AM, Madonna P, Tufano A, Falco A, Marchesani P, Ciabattoni G, Patrono C. Oxidative stress and platelet activation in homozygous homocystinuria. Circulation. 2001; 104: 1124–1128.
Minuz, P, Patrignani P, Gaino S, Degan M, Menapace L, Tommasoli R, Seta F, Capone ML, Tacconelli S, Palatresi S, Bencini C, Del Vecchio C, Mansueto G, Arosio E, Santonastaso CL, Lechi A, Morganti A, Patrono C. Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease. Circulation. 2002; 106: 2800–2805.(Giovanni Davì; Matteo Ner)
Reprint requests to: Carlo Patrono, MD, Center of Excellence on Aging, Fondazione Università "G. d’Annunzio," Via Colle dell’Ara, 66013, Chieti, Italy. E-mail cpatrono@unich.it
Abstract
Objective— We aimed at investigating the relationship between Helicobacter pylori infection and in vivo lipid peroxidation and platelet activation, as reflected by urinary 8-iso-prostaglandin (PG)F2 and 11-dehydro-thromboxane (TX)B2, respectively, in otherwise healthy dyspeptic subjects.
Methods and Results— We measured urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion in 40 dyspeptic subjects with a positive 13C-urea breath test and 38 dyspeptic individuals with a negative test. Moreover, we investigated the effects of H pylori eradication on prostanoid metabolite excretion in 23 H pylori–positive subjects. We also measured prostanoid metabolite excretion before and after selective cyclooxygenase-2 inhibition with rofecoxib in 4 H pylori–positive subjects. Urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion was significantly higher in the H pylori–positive individuals than in controls. A significant direct correlation was found between the degree of positivity to the13C-urea breath test and urinary 8-iso-PGF2 excretion. The latter was linearly correlated with urinary 11-dehydro-TXB2. Successful eradication of H pylori infection led to a significant reduction in both 8-iso-PGF2 and 11-dehydro-TXB2. Furthermore, their levels were unaffected after treatment with rofecoxib.
Conclusions— Our study provides evidence of enhanced in vivo lipid peroxidation and platelet activation in association with H pylori infection and suggests a novel mechanism by which an infectious agent could contribute to atherothrombosis.
Urinary excretion of 8-iso-PGF2a and 11-dehydro-TXB2 was significantly higher in Helicobacter pylori–positive individuals than in controls, with direct correlations between the degree of positivity to the 13C-urea breath test and urinary 8-iso-PGF2 and between the 2 metabolites. Successful H pylori eradication led to a reduction in these indexes of lipid peroxidation and platelet activation. We suggest a novel mechanism by which an infectious agent could contribute to atherothrombosis.
Key Words: risk factors ? oxidant stress ? platelets ? infection ? inflammation
Introduction
Inflammatory mechanisms have been implicated in the pathogenesis of atherosclerosis, and a significant association between infectious burden and the extent and long-term prognosis of atherosclerosis has been reported.1
Helicobacter pylori infection represents one of the most widespread human infectious diseases.2 Several studies have reported an association between infection with H pylori and incidence of vascular disease, particularly coronary heart disease (CHD).3 However, most of these data derived from cross-sectional and retrospective studies and potential confounders, such as socioeconomic status or smoking pattern,3,4 may complicate their interpretation. Analysis restricted to the available prospective studies in socioeconomically homogeneous populations provides limited evidence for an association between H pylori and CHD.5 However, a case-control and sibling pairs study of early-onset myocardial infarction reported strong evidence for an association between H pylori infection and CHD, suggesting that the potential proinflammatory effects of H pylori might be of greater importance at younger ages.6
Experimental studies have shown that H pylori induces platelet aggregation in gastric mucosal microcirculation.7,8 However, no previous study has examined the occurrence and mechanism(s) of platelet activation in vivo that might be associated with H pylori infection in humans.
Several cardiovascular risk factors are associated with low-grade inflammation, increased oxidant stress, and lipid peroxidation.9 F2-isoprostanes, a family of bioactive prostaglandin (PG)F2-like compounds10 produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen-free radicals on cell membranes and low-density lipoprotein (LDL) particles,9 represent a reliable marker of in vivo lipid peroxidation. Among F2-isoprostanes, of particular interest is 8-iso-PGF2, which induces vasoconstriction and modulates function of human platelets.11 Measurement of urinary F2-isoprostanes has been used extensively in clinical settings putatively associated with increased oxidant stress.9
In the present study, we tested the hypothesis that low-grade inflammation associated with H pylori infection3 would induce increased in vivo lipid peroxidation with generation of 8-iso-PGF2 and other biologically active isoeicosanoids and that these compounds would in turn contribute to platelet activation in this setting. The latter was evaluated by measuring urinary excretion of 11-dehydro-thromboxane (TX)B2, a stable enzymatic derivative of TXA2, a labile eicosanoid that amplifies platelet activation in response to other stimuli and induces irreversible platelet aggregation.12
Thus, the aim of our study was to investigate the causal relationship between H pylori infection and the rates of in vivo lipid peroxidation and platelet activation in otherwise healthy dyspeptic subjects through biochemical measurements and pharmacological interventions.
Methods
Study Participants
We initially studied 28 dyspeptic men aged <60 years who had a first-time positive 13C-urea breath test (UBT) for H pylori infection and had not received antibiotic treatment previously. Twenty-eight age-matched dyspeptic men who had a negative UBT for H pylori infection were recruited as a control group. To investigate potential gender-related differences, we performed a second cross-sectional study in 12 dyspeptic women aged <60 years with a first-time positive UBT who had not been treated previously. Ten age-matched dyspeptic women who had a negative UBT were enrolled as a control group. Because the results of the 2 studies were internally consistent, the data are presented as a whole. The clinical characteristics of study subjects are summarized in the Table .
Baseline Characteristics of H Pylori–Positive and –Negative Subjects
To avoid confounding by other determinants of oxidant stress and platelet activation, subjects were excluded if they had a history or evidence of atherothrombotic diseases, diabetes mellitus, cigarette smoking, dyslipidemia, and arterial hypertension, as well as current or recent (<3 months) systemic or localized infections. Moreover, dyspeptic subjects were excluded if they were taking low-dose aspirin, nonsteroidal anti-inflammatory drugs, or vitamin supplements. Informed consent was obtained from each participating subject, and the protocol was approved by the University of Chieti ethics committee.
Study Design
A cross-sectional comparison of urinary 8-iso-PGF2 and 11-dehydro-TXB2, a major enzymatic metabolite of TXA2,13 was performed between subjects positive and negative for H pylori infection. All participants were studied as outpatients after a 12-hour fast. Each subject performed an overnight urine collection. Urine samples were added with the antioxidant 4-hydroxy-Tempo (1 mmol/L; Sigma) and stored at –20°C until extraction.
To examine the effects of H pylori eradication on indexes of lipid peroxidation and platelet activation, an open pilot intervention study was performed in H pylori–positive subjects. Thus, we investigated the effects of a standard antibiotic therapy on urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion in 15 of the 28 H pylori–positive men and in 8 of the 12 H pylori–positive women. Entry criteria were a clear indication of antibiotic therapy, lack of contraindications, and willingness to participate in this additional study. These subjects were given 1000 mg amoxicillin twice daily plus 500 mg clarithromycin twice daily for 7 days. A standard regimen of a proton pump inhibitor (20 mg omeprazole twice daily) was also given.14 Before and after treatment, participants were instructed to perform an overnight urine collection. At least 4 weeks after completing the triple therapy, the UBT was repeated to assess eradication. In case of treatment failure (11 subjects), a new treatment regimen was prescribed. Five subjects who were found still infected after the first course of therapy were treated with 500 mg clarithromycin twice daily and 500 mg tinidazole twice daily for 7 days. A dosage of 400 mg ranitidine bismuth citrate twice daily was also given. Six subjects who failed to respond to the first treatment regimen were lost to follow-up.
Because H pylori infection is associated with cyclooxygenase-2 (COX-2) expression in the gastric mucosa15–17 and 8-iso-PGF2 and TXA2 can be formed by human monocytes through a COX-2–dependent mechanism,18 a third study was performed to evaluate whether selective inhibition of COX-2 activity had any influence on 8-iso-PGF2 in subjects positive for H pylori infection. For this purpose, 4 of the H pylori–positive subjects were given 12.5 mg rofecoxib, a highly selective COX-2 inhibitor,19 once daily for 7 days. Participants collected overnight urine samples at the beginning and the end of the rofecoxib treatment for measurement of 8-iso-PGF2 and 11-dehydro-TXB2 excretion.
Analytical Measurements
The presence of H pylori was determined by UBT. UBT consisted of a baseline breath sample and a second sample 30 minutes after administration of 75 mg of 13C-labeled urea (Isotec) dissolved in orange juice.20 Subjects were fasted overnight. The 13C-enrichment in breath was analyzed using an isotope ratio mass spectrometer (ABCA; Europa Scientific) The UBT was considered positive when the difference with baseline at 30' (DOB30; ie, the difference between -value at 30' and -value at baseline) was 5%. This noninvasive test is as accurate in predicting H pylori status as invasive tests and is the recommended test for diagnosis of H pylori infection.21 Urinary 8-iso-PGF2 and 11-dehydro-TXB2 were measured by previously described and validated radioimmunoassay methods.22,23
Statistical Analysis
Data were analyzed by nonparametric methods to avoid assumptions about the distribution of the measured variables. An ANOVA was performed with the Kruskall–Wallis method. Subsequent pairwise comparisons were made with the Mann–Whitney U test. Differences between baseline and post-treatment values were analyzed with the Wilcoxon signed-rank test. Moreover, the association of eicosanoid measurements with other biochemical parameters was assessed by the Spearman rank correlation test. A multiple linear regression analysis was performed to further quantify the relationship between 11-dehydro-TXB2 excretion and the other variables in the cross-sectional studies.
The cross-sectional and intervention studies had a >80% power to detect an H pylori–related difference in urinary 11-dehydro-TXB2 excretion of 1 SD between groups with a 2-tailed of 0.05.
All values are reported as median (range). P values <0.05 were regarded as statistically significant. All tests were 2-tailed, and analyses were performed using a computer software package (Statistica 1999 edition; StatSoft; or Statistical Package for the Social Sciences, version 12.0; SPSS).
Results
Urinary 8-iso-PGF2 excretion was significantly higher in the 40 H pylori–positive than in the 38 H pylori–negative subjects (357 [154 to 645] pg/mg versus 189 [85 to 306] pg/mg creatinine; median (range) P=0.0001; Figure 1). Urinary 11-dehydro-TXB2 excretion rate was also significantly increased in H pylori–positive compared with H pylori–negative subjects (868 [339 to 1630] pg/mg versus 378 [212 to 690] pg/mg creatinine; P=0.0001; Figure 1). In the 40 H pylori–positive individuals, a statistically significant direct correlation was found between DOB30 in breath samples and urinary 8-iso-PGF2 excretion rates (=0.505; P=0.0016). The latter index of lipid peroxidation was in turn linearly correlated with the rate of TX biosynthesis, as reflected by urinary 11-dehydro-TXB2 excretion (=0.52; P=0.0012).
Figure 1. Urinary excretion of 8-iso-PGF2 (top) and 11-dehydro-TXB2 (bottom) in H pylori–negative vs H pylori–positive subjects. Error bars represent minimum and maximum values; , median values; boxes, interquartile range.
A multiple regression analysis performed in 78 subjects showed that DOB30 in breath samples (regression coefficient 0.44; SE 0.11; P=0.000128), male sex (regression coefficient 0.39; SE 0.08; P=0.0150), and urinary 8-iso-PGF2 excretion rates (regression coefficient 0.39; SE 0.11; P=0.000456) independently correlated with 11-dehydro-TXB2 excretion.
Effects of Eradication Therapy
We also investigated the effects of H pylori eradication on urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion to test the hypothesis of a cause–effect relationship between H pylori infection and enhanced lipid peroxidation and platelet activation in this setting. Thus, we evaluated the effects of eradication therapy in 15 H pylori–positive men and 8 H pylori–positive women.
Successful eradication, achieved in 12 of the 23 treated patients, was associated with a statistically significant reduction in 8-iso-PGF2 (from 400 [233 to 566] pg/mg to 247 [176 to 348] pg/mg creatinine; P=0.0022) and 11-dehydro-TXB2 urinary excretion (from 1049 [606 to 1630] pg/mg to 600 [303 to 822] pg/mg creatinine; P=0.0022). Excretion rates of the 2 metabolites remained substantially unchanged in association with unsuccessful eradication in the other 11 subjects (Figure 2). The coefficient of variation for 3 repeated measurements of urinary 8-iso-PGF2 and 11-dehydro-TXB2, obtained from 7 subjects who failed eradication therapy, averaged 21.3±10.3% and 18.2±10.5%, respectively.
Figure 2. Effects of antibiotic treatment on urinary 8-iso-PGF2 (top) and 11-dehydro-TXB2 (bottom) in H pylori–positive subjects. Dots and lines connecting dots represent individual measurements; solid symbols and continuous lines, values of subjects with successful eradication; open symbols and broken lines, values of subjects with unsuccessful eradication; error bars, minimum and maximum values; , median values; boxes, interquartile range, represented on the left and on the right side of each panel.
In 5 of the 11 subjects in whom H pylori failed to be eradicated, a different treatment regimen was prescribed after a period of 10±2 weeks. This second cycle of therapy led to successful eradication in 3 subjects, with a consistent reduction in 8-iso-PGF2 (from 371 [331 to 483] pg/mg to 285 [132 to 288] pg/mg creatinine) and 11-dehydro-TXB2 excretion (from 1040 [680 to 1200] pg/mg to 327 [254–375] pg/mg creatinine), whereas urinary levels of both metabolites remained unchanged in the 2 subjects who failed to be eradicated (data not shown).
In the 15 subjects who eventually achieved successful eradication (12 after the first regimen plus 3 after the second regimen), the reduction in DOB30 was associated with a fall in TX biosynthesis, the average extent of which showed a remarkably good fitting with the linear relationship between DOB30 values and 11-dehydro-TXB2 excretion rates, as established in the whole group of H pylori–positive subjects at baseline (Figure 3).
Figure 3. Correlation between DOB30 and urinary 11-dehydro-TXB2 excretion in H pylori–positive subjects. The 40 data points represent the baseline measurements for the 28 and 12 dyspeptic men and women, respectively. Of these, 15 and 8 were subjected to treatment. Twelve subjects responded to the first treatment regimen, and the average results for these 12 before treatment are shown by the . The average results for these 12 responding subjects after treatment are shown by the .
Effects of Selective COX-2 Inhibition
Urinary 8-iso-PGF2 and 11-dehydro-TXB2 excretion rates were not affected after 1 week of selective COX-2 inhibition achieved with rofecoxib (from 280 [247 to 338] pg/mg to 303 [259 to 320] pg/mg, and from 553 [463 to 578] pg/mg to 579 [363 to 606] pg/mg creatinine, respectively). This finding is consistent with a non-COX–dependent mechanism of F2-isoprostane formation in association with H pylori infection. Moreover, these results demonstrate that enhanced TX biosynthesis is not a byproduct of COX-2 expression in response to H pylori infection.
Discussion
Atherothrombotic events do occur among individuals without readily apparent cardiovascular risk factors.24 In recent years, inflammation has been suggested to play a key role in the initiation and progression of the atherosclerotic process.25 Circulating markers of persistent low-grade inflammation, such as C-reactive protein, can predict recurrence of major vascular events in patients with established ischemic heart disease,26–28 as well as the risk of a first myocardial infarction in apparently healthy subjects.29 Persistent infections may represent a potential trigger of systemic inflammation, and evidence for a link between total infectious burden and atherosclerotic severity has been provided.1,30,31 A weak and controversial association between H pylori infection and CHD has been described.32 H pylori, a primary pathogen for peptic ulcer disease, gastric cancer, and lymphoma2 is a potential source of inflammatory cytokines possibly contributing to the atherosclerotic process.33 It has been hypothesized that H pylori infection might modify serum lipid concentrations, thus increasing the risk for cardiovascular disease.34 Moreover, H pylori is independently associated with increased fibrinogen levels in healthy subjects,35 and an association between an IgG antibody response to multiple pathogens, including H pylori, and endothelial dysfunction has been reported.36 Finally, it has been suggested that chronic atrophic gastritis induced by H pylori, causing malabsorption of vitamin B12 and folate, may lead to increased plasma levels of homocysteine, a known risk factor for vascular disease.37 H pylori–specific DNA has been detected recently in atheromatous plaques of patients with severe coronary artery disease, supporting the hypothesis of direct involvement of the bacterium in the progression and instability of atherosclerotic lesions.38
An important link has been shown between coronary artery disease and infection with H pylori, and its eradication significantly attenuated the reduction in coronary lumen after coronary angioplasty.39 Moreover, H pylori eradication increased high-density lipoprotein cholesterol and decreased C-reactive protein, thrombin–antithrombin complexes, and lipoprotein(a) levels in type 1 diabetic patients.40
In the present study, we have identified a novel mechanism through which H pylori infection may enhance cardiovascular risk (ie, persistent platelet activation). Biochemical evidence of enhanced platelet activation in vivo in association with H pylori infection was obtained through noninvasive measurements of TX metabolite excretion that avoid artifactual platelet activation during and after blood sampling.41 The exclusion criteria used to recruit dyspeptic otherwise healthy subjects avoided confounding by traditional cardiovascular risk factors that can affect the rate of platelet activation. It should be emphasized that TXA2 biosynthesis measured in H pylori–positive subjects in the present study is comparable to that reported previously in association with traditional cardiovascular risk factors such as hypercholesterolemia,42 diabetes mellitus,43 obesity,44 and hypertension.45
Furthermore, we characterized a putative biochemical link between H pylori infection and platelet activation by investigating the in vivo formation of F2-isoprostanes, as reflected by the urinary excretion of the PGF2 isomer 8-iso-PGF2. This family of bioactive isoeicosanoids is produced through free radical–catalyzed peroxidation of arachidonic acid that can occur on cell membranes and LDL particles.10 Measurement of unmetabolized F2-isoprostanes in plasma and urine has proved to be a valuable approach to assess the actual rate of lipid peroxidation in vivo.9,10 Repeated measurements of 8-iso-PGF2 in those subjects in whom H pylori eradication failed demonstrated a persistent abnormality with limited intrasubject variability over time. Evidence for a cause-and-effect relationship between H pylori infection and enhanced lipid peroxidation was provided by the linear correlation between the 13C-UBT and urinary of 8-iso-PGF2 excretion rates as well as by the statistically significant reduction in F2-isoprostane formation after successful eradication (Figure 2). The nonenzymatic nature of 8-iso-PGF2 production in this setting was confirmed by the failure of rofecoxib, a highly selective COX-2 inhibitor,19 to reduce its urinary excretion to any detectable extent.
Enhanced formation of 8-iso-PGF2 in H pylori–positive subjects correlated with increased TXA2 biosynthesis, as reflected by 11-dehydro-TXB2 excretion. Although the systemic concentrations of 8-iso-PGF2 may be too low to trigger platelet activation, this autacoid can synergize with subthreshold concentrations of other agonists in inducing platelet adhesion and aggregation.46,47 Moreover, it is likely that enhanced formation of 8-iso-PGF2 as a consequence of H pylori infection is associated with the release of other bioactive isoeicosanoids formed through the same mechanism of oxygen radical–catalyzed peroxidation of arachidonic acid. To assess the potential contribution to TXA2 biosynthesis of COX-2 expressed by inflammatory and epithelial cells in response to H pylori infection, we investigated the short-term effects of rofecoxib on 11-dehydro-TXB2 excretion in H pylori–positive subjects. The results of this intervention study are consistent with COX-1 being the main COX isoform involved in TXA2 biosynthesis in this setting and make it unlikely that the reduction in 11-dehydro-TXB2 excretion associated with successful H pylori eradication is a reflection of reduced COX-2 expression in the gastric mucosa.16
The findings in H pylori–positive patients extend similar observations made in other clinical settings such as hypercholesterolemia,42 diabetes mellitus,43 cigarette smoking,48,49 homozygous homocystinuria,50 visceral obesity,44 and renovascular hypertension.51 Thus, regardless of the mechanism(s) responsible for enhanced lipid peroxidation, there is quite convincing evidence from studies in such diverse conditions that enhanced generation of bioactive isoeicosanoids may transduce the oxidant signal associated with a variety of cardiovascular risk factors into a functional platelet response, possibly contributing to enhanced thrombotic risk.
In conclusion, the present study provides biochemical evidence of enhanced in vivo lipid peroxidation and platelet activation in dyspeptic individuals with H pylori infection and identifies a novel mechanism through which an infectious agent could contribute to development of atherothrombosis. Reversibility of the hemostatic abnormality after successful eradication of H pylori may have clinical implications for cardiovascular risk management.
Received July 9, 2004; accepted September 17, 2004.
References
Espinola-Klein C, Rupprecht HJ, Blankenberg S, Bickel C, Kopp H, Rippin G, Victor A, Hafner G, Schlumberger W, Meyer J; AtheroGene Investigators. Impact of infectious burden on extent and long-term prognosis of atherosclerosis. Circulation. 2002; 105: 15–21.
Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med. 2002; 347: 1175–1186.
Ridker PM. Inflammation, infection, and cardiovascular risk. How good is the clinical evidence? Circulation. 1998; 97: 1671–1674.
Murray LJ. Helicobacter pylori infection: relation with cardiovascular risk factors, ischaemic heart disease, and social class. Br Heart J. 1995; 74: 497–501.
Ridker PM. A prospective study of Helicobacter pylori seropositivity and the risk for future myocardial infarction among socioeconomically similar US men. Ann Intern Med. 2001; 135: 184–188.
Danesh J, Youngman L, Clark S, Parish S, Peto R, Collins R. Helicobacter pylori infection and early onset myocardial infarction: case-control and sibling pairs study. BMJ. 1999; 319: 1157–1162.
Kalia N, Jacob S, Brown NJ, Reed MW, Morton D, Bardhan KD. Studies on the gastric mucosal microcirculation. 2. Helicobacter pylori water soluble extracts induce platelet aggregation in the gastric mucosal microcirculation in vivo. Gut. 1997; 41: 748–752.
Elizalde JI, Gomez J, Panes J, Lozano M, Casadevall M, Ramirez J, Pizcueta P, Marco F, Rojas FD, Granger DN, Pique JM. Platelet activation in mice and human Helicobacter pylori infection. J Clin Invest. 1997; 100: 996–1005.
Davì G, Falco A, Patrono C. Determinants of F2-isoprostane biosynthesis and inhibition in man. Chem Phys Lipids. 2004; 128: 149–163.
Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalysed mechanism. Proc Natl Acad Sci U S A. 1990; 87: 9383–9387.
Patrono C, FitzGerald GA. Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler Thromb Vasc Biol. 1997; 17: 2309–2315.
Patrono C, Davì G, Ciabattoni G. Thromboxane biosynthesis and metabolism in relation to cardiovascular risk factors. Trends Cardiovasc Med. 1992; 2: 15–20.
Ciabattoni G, Pugliese F, Davì G, Pierucci A, Simonetti BM, Patrono C. Fractional conversion of thromboxane B2 to urinary 11-dehydro-thromboxane B2 in man. Biochim Biophys Acta. 1989; 992: 66–70.
Malfertheiner P, Mégraud F, O’Morain C, Hungin AP, Jones R, Axon A, Graham DY, Tytgat G; European Helicobacter Pylori Study Group (EHPSG). Current concepts in the management of Helicobacter pylori infection—the Maastricht 2–2000 Consensus Report. Aliment Pharmacol Ther. 2002; 16: 167–180.
Sawaoka H, Kawano S, Tsuji S, Tsuji M, Sun W, Gunawan ES, Hori M. Helicobacter pylori infection induces cyclooxygenase-2 expression in human gastric mucosa. Prostaglandins Leukotrienes Essent Fatty Acids. 1998; 59: 313–316.
McCarthy CJ, Crofford LJ, Greenson J, Scheiman JM. Cyclooxygenase-2 expression in gastric antral mucosa before and after eradication of Helicobacter pylori infection. Am J Gastroenterol. 1999; 94: 1218–1223.
Fu S, Ramanujam KS, Wong A, Fantry GT, Drachenberg CB, James SP, Meltzer SJ, Wilson KT. Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology. 1999; 116: 1319–1329.
Patrignani P, Santini G, Panara MR, Sciulli MG, Greco A, Rotondo MT, di Giamberardino M, Maclouf J, Ciabattoni G, Patrono C. Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation. Br J Pharmacol. 1996; 118: 1285–1293.
FitzGerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med. 2001; 345: 433–442.
Dominguez-Munoz JE, Leodolter A, Sauerbruch T, Malfertheiner P. A citric acid solution is an optimal test drink in the 13C-urea breath test for the diagnosis of Helicobacter pylori infection. Gut. 1997; 40: 459–462.
Cutler AF, Havstad S, Ma CK, Blaser MJ, Perez-Perez GI, Schubert TT. Accuracy of invasive and noninvasive tests to diagnose Helicobacter pylori infection. Gastroenterology. 1995; 109: 136–141.
Wang Z, Ciabattoni G, Créminon C, Lawson J, Fitzgerald GA, Patrono C, Maclouf J. Immunological characterization of urinary 8-epi-PGF2 excretion in man. J Pharmacol Exp Ther. 1995; 275: 94–100.
Ciabattoni G, Maclouf J, Catella F, Fitzgerald GA, Patrono C. Radioimmunoassay of 11-dehydro-TXB2 in human plasma and urine. Biochim Biophys Acta. 1987; 918: 293–297.
Braunwald E. Shattuck lecture: cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med. 1997; 337: 1360–1369.
Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
De Beer FC, Hind CR, Fox KM, Allan RM, Maseri A, Pepys MB. Measurement of serum C-reactive protein concentration in myocardial ischemia and infarction. Br Heart J. 1982; 47: 239–243.
Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB; European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet. 1997; 349: 462–466.
Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, Flaker GC, Braunwald E; Cholesterol and Recurrent Events (CARE) Investigators. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation. 1998; 98: 839–844.
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1996; 336: 973–979.
Danesh J, Whincup P, Walker M, Lennon L, Thomson A, Appleby P, Gallimore JR, Pepys MB. Low grade inflammation and coronary heart disease: prospective study and updated meta-analysis. BMJ. 2000; 321: 199–204.
Ridker PM. On evolutionary biology, inflammation, infection and the causes of atherosclerosis. Circulation. 2002; 105: 2–4.
Mendall MA, Goggin PM, Molineaux N, Levy J, Toosy T, Strachan D, Camm AJ, Northfield TC. Relation of Helicobacter pylori infection and coronary heart disease. Br Heart J. 1994; 71: 437–439.
Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-, interleukin-1 ?, and interleukin-8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol. 1994; 29: 425–429.
Niemela S, Karttunen T, Korhonen T, Laara E, Karttunen R, Ikaheimo M, Kesaniemi YA. Could Helicobacter pylori infection increase the risk of coronary heart disease by modifying serum lipid concentrations? Heart. 1996; 75: 573–575.
Patel P, Carrington D, Strachan DP, Leatham E, Goggin P, Northfield TC, Mendall MA. Fibrinogen: a link between chronic infection and coronary heart disease. Lancet. 1994; 343: 1634–1635.
Prasad A, Zhu J, Halcox JP, Waclawiw MA, Epstein SE, Quyyumi AA. Predisposition to atherosclerosis by infections: role of endothelial dysfunction. Circulation. 2002; 106: 184–190.
Tamura A, Fujioka T, Nasu M. Relation of Helicobacter pylori infection to plasma vitamin B12, folic acid, and homocysteine levels in patients who underwent diagnostic coronary arteriography. Am J Gastroenterol. 2002; 97: 861–866.
Kowalski M, Rees W, Konturek PC, Grove R, Scheffold T, Meixner H, Brunec M, Franz N, Konturek JW, Pieniazek P, Hahn EG, Konturek SJ, Thale J, Warnecke H. Detection of Helicobacter pylori specific DNA in human atheromatous coronary arteries and its association to prior myocardial infarction and unstable angina. Dig Liver Dis. 2002; 34: 398–402.
Kowalski M, Konturek PC, Pieniazek P, Karczewska E, Kluczka A, Grove R, Kranig W, Nasseri R, Thale J, Hahn EG, Konturek SJ. Prevalence of Helicobacter pylori infection in coronary artery disease and effect of its eradication on coronary lumen reduction after percutaneous coronary angioplasty. Dig Liver Dis. 2001; 33: 222–229.
de Luis DA, Garcia Avello A, Lasuncion MA, Aller R, Martin de Argila C, Boixeda de Miquel D, de la Calle H. Improvement in lipid and haemostasis patterns after Helicobacter pylori infection eradication in type 1 diabetic patients. Clin Nutr. 1999; 18: 227–231.
FitzGerald GA, Pedersen AK, Patrono C. Analysis of prostacyclin and thromboxane biosynthesis in cardiovascular disease. Circulation. 1983; 67: 1174–1177.
Davì G, Alessandrini P, Mezzetti A, Minotti G, Bucciarelli T, Costantini F, Cipollone F, Bon GB, Ciabattoni G, Patrono C. In vivo formation of 8-epi-prostaglandin F2 is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1997; 17: 3230–3235.
Davì G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S, Pennese E, Vitacolonna E, Bucciarelli T, Costantini F, Capani F, Patrono C. In vivo formation of 8-iso-prostaglandin F2 and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation. 1999; 99: 224–229.
Davì G, Guagnano MT, Ciabattoni G, Basili S, Falco A, Marinopiccoli M, Nutini M, Sensi S, Patrono C. Platelet activation in obese women: role of inflammation and oxidant stress. J Am Med Assoc. 2002; 288: 2008–2014.
Minuz P, Patrignani P, Gaino S, Seta F, Capone ML, Tacconelli S, Degan M, Faccini G, Fornasiero A, Talamini G, Tommasoli R, Arosio E, Santonastaso CL, Lechi A, Patrono C. Determinants of platelet activation in human essential hypertension. Hypertension. 2004; 43: 64–70.
Praticò D, Smyth EM, Violi F, FitzGerald GA. Local amplification of platelet function by 8-epi-prostaglandin F2 is not mediated by thromboxane receptor isoforms. J Biol Chem. 1996; 271: 14916–14924.
Minuz P, Andrioli G, Degan M, Gaino S, Ortolani R, Tommasoli R, Zuliani V, Lechi A, Lechi C. The F2-isoprostane 8-epiprostaglandin F2 increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO. Arterioscler Thromb Vasc Biol. 1998; 18: 1248–1256.
Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM, Shyr Y, Strauss WE, Oates JA, Roberts LJ II. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers: smoking as a cause of oxidative damage. N Engl J Med. 1995; 332: 1198–1203.
Reilly M, Delanty N, Lawson JA, Fitzgerald GA. Modulation of oxidant stress in vivo in chronic cigarette smokers. Circulation. 1996; 94: 19–25.
Davì G, Di Minno G, Coppola A, Andria G, Cerbone AM, Madonna P, Tufano A, Falco A, Marchesani P, Ciabattoni G, Patrono C. Oxidative stress and platelet activation in homozygous homocystinuria. Circulation. 2001; 104: 1124–1128.
Minuz, P, Patrignani P, Gaino S, Degan M, Menapace L, Tommasoli R, Seta F, Capone ML, Tacconelli S, Palatresi S, Bencini C, Del Vecchio C, Mansueto G, Arosio E, Santonastaso CL, Lechi A, Morganti A, Patrono C. Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease. Circulation. 2002; 106: 2800–2805.(Giovanni Davì; Matteo Ner)