A Systematic Review and Meta-Analysis on the Relation Between Helicobacter Pylori Infection and Atherosclerosis in the Iranian Population

Background: Various evidences have recently been provided indicating the relationship between infection with Helicobacter pylori and the extra-gastric complication, especially atherosclerosis. Atherosclerosis is the most important predisposing factor for cardiovascular disorders due to the high prevalence of cardiovascular disorders in developing countries (areas where a high population of people are infected with Helicobacter pylori), in particular in Iran. Methods: In a current systematic review and meta-analysis study, we collected all studies of the isolation of Helicobacter pylori from atherosclerotic plaques in Iranian cardiovascular patients to investigate the presence or absence of a relationship between Helicobacter pylori infection and susceptibility to atherosclerosis. The present meta-analysis was done by Comprehensive Meta-Analysis software (CMA-Ver. 2.0). This software has the ability to combine studies and impact sizes into the results and is highly acceptable. Results: Overall, the frequency of H. pylori in atherosclerotic plaques in the coronary artery disease (CAD) patients was estimated to be 41.30%. However, the highest and lowest frequency of infection with Helicobacter pylori were 80% and 0%, respectively. Conclusions: However generally, regarding the previous studies, we collected all the Iranian published articles and showed that the infection with Helicobacter pylori was signicantly related with the atherosclerosis, but further complementary and more extensive studies are required to conrm this hypothesis.


Background
Cardiovascular disease (CVD) is the most common cause of mortality worldwide (50% of global mortality), with an unprecedented increase in the rate of coronary artery disease (CAD) mortality in the last two decades and it has become one of the most signi cant concerns of the global health system [1][2][3]. Atherosclerosis is undoubtedly the most important predisposing factor for cardiovascular disorders with a high prevalence in the developed as well as the developing countries [2][3][4][5]. Atherosclerosis is a chronic procedure with several factors being involved in its formation [6]. Based on the available evidence, traditional risk factors such as smoking, obesity, hypertension, hypercholesterolemia, and host genome polymorphisms have not had important impacts on the pathogenesis of atherosclerosis, and recent studies suggest that in ammatory diseases play a central role in this respect [7][8]. Infectious agents are considered as the most important in ammatory triggers in the body as well as a risk factor for atherosclerosis due to the stimulating the in ammatory procedure and damaging vascular endothelial cells [9][10]. According to the review of the literature, the most important infectious agents that are associated with atherosclerosis include: Chlamydia pneumoniae, Hepatitis C virus, Human Immunode ciency virus, Epstein-Barr virus, Hepatitis B virus, Human T lymphotropic virus type I, Cytomegalovirus, Mycobacterium tuberculosis and Helicobacter pylori [11][12][13][14][15][16][17]. Helicobacter pylori (H. pylori) is a gram-negative, of microaerophilic and spiral form that resides in the human gastric mucosa layer and is an etiologic agent of chronic gastritis, gastric ulcer, duodenal ulcer, and gastric cancer [18].
Recent studies have shown that H. pylori is also isolated from dental plaques, human saliva, duodenum, feces and atherosclerotic plaques and is strongly related with the extra-gastrointestinal disorders such as idiopathic thrombocytopenic purpurae, neurological disorders (stroke events), psychiatric, gynecological, pre-Eclampsia, infertility, glaucoma, dermatologic complications, lung cancer, iron de ciency anemia, and atherosclerosis [19][20][21][22][23][24][25][26][27][28][29][30]. Unfortunately, the rate of colonization with H. pylori is high worldwide, especially in Asia (from 25-50% in the developed countries to 90% in the developing countries), and despite the eradication of H. pylori infection due to the destruction of antibiotics in acidic stomach conditions, the in uence of the bacterium under the gastric mucus and the antibiotic resistance has been reduced in the recent years [31][32][33]. According to the review of the literature, the rate of gastric cancer in Asia is higher than in the western countries, and many researchers attribute this to the high rate of colonization with H. pylori in this geographical region [34][35]. Moreover, the rate of atherosclerosis in the developing countries (where the rate of colonization with Helicobacter pylori is 90%) is higher than in the developed countries [4,[36][37]. The hypothesis of the correlation between H. pylori and atherosclerosis was rst suggested by Mendall et al. in 1994 [38]. Despite extensive studies about the correlation of infection with H. pylori and atherosclerosis, it seems that one of the most important reasons for increasing the rate of atherosclerosis in the developing countries may be the high prevalence of colonization with H. pylori in these regions, especially since H. pylori has been isolated from atherosclerotic plaques of the patients [39][40][41]. However, some resources have rejected the relationship between H. pylori and atherosclerosis, and there is no precise response to the question. Thus, more extensive studies are required in this respect. [42][43][44]. The rate of atherosclerosis and cardiovascular disorders in Iran is high and based on the Iranian Society of Atherosclerosis, 300 deaths occur each day due to cardiovascular diseases in Iran. Moreover, considering that the high rate of colonization with H. pylori in Iran (estimated to be more than 85%), Iran is regarded as one of the most appropriate places to investigate the possible association between H. pylori infection and atherosclerosis, and the present study was conducted to investigate the relationship between the colonization with H. pylori and susceptibility to atherosclerosis in the Iranian patients.

Search strategy
The present comprehensive meta-analysis was ful lled based on the PRISMA guideline proposed by Liberati et al. [45]. t was intended in this study to evaluate the relation between colonization with Helicobacter pylori in cardiovascular patients with atherosclerotic plaques. For this purpose, the related articles the systematic literature searches in PubMed, Scopus, Google scholar databases and the domestic databases such as Iranmedex, SID, ISC and Magiran were collected considering the combination keywords including Helicobacter pylori, Iran, atherosclerosis, atherosclerotic plaques, cardiovascular diseases, DNA, Infection, Coronary artery disease and coronary arteries; in addition original articles and congress abstracts were studied independently by two the authors, without considering the range of the required time. Finally, the duplicate articles were eliminated and the abstract and title of the studies were selected based on the eligible criteria (Fig. 1).

Selection criteria
The screening process of the studies was performed independently by two the authors. The titles and abstracts were initially evaluated; inclusion criteria included 1) studies on the isolation of H. pylori from atherosclerosis plaques, 2) the patients with cardiovascular disorders, 3) the studies limited to Iran, 4) cross-sectional, cohort and case-control studies, 5) method of Helicobacter infection diagnosis consisting of culture, PCR and serology (detection of IgG), and 6) studies in English and Farsi languages. However, the exclusion criteria were: 1) in vitro studies and lab animals, 2) studies containing inadequate or inadequate method or results, 3) case reports and review articles, 4) simultaneous co-infection with Helicobacter pylori and other infectious agents, 5) duplicate articles, and 6) not-free articles. It is to note that a study included in our study, despite being a letter to the editor, since it was subject to the eligible criteria.

Quality assessment and data extraction
The Joanna Briggs Institute (JBI) critical appraisal checklist (Data not shown) was used to evaluate the quality of the studies based on eligibility of criteria. Information such as the author, publishing year, province, diagnostic methods and frequency of H. pylori isolates were listed in Table 1 among CAD patients with atherosclerosis plaques.

Data analysis
The possible relationship between Helicobacter pylori infection and atherosclerosis in atherosclerotic plaque samples of the Iranian CAD patients was evaluated and reported using the odds ratio (OR) at 95% con dence intervals (95% Cis) (random effects models when the heterogeneity was high; heterogeneity was assessed using I 2 statistic and Cochrane Q statistic; I 2 > 25% and P-value < 0.1). The statistical analysis for this study was done by Comprehensive Meta-Analysis (CMA) software-Ver. 2.0 (Biostat, Englewood, NJ) [46], and the publication bias were also measured by the asymmetry of the funnel plot.

Results
A total of 94 articles were considered from the databases of PubMed, Scopus, Google scholar, and Iranian databases. After omitting the duplicate studies, the rest of the articles were entered in Endnote software, and the title, abstract and full-text of the articles were reviewed and collected on the basis of eligible criteria for the cross-sectional, case-control and cohort studies regarding the isolation of Helicobacter pylori from the atherosclerotic plaques in the Iranian CAD patients. Ultimately, 12 studies consisting of the original articles and congress abstracts included in the comprehensive meta-analysis ( Fig. 1).
Population distribution, characteristics of studies and diagnostic methods of the studies were summarized in Table 1. Since the forms of the cardiovascular disease were not mentioned in a signi cant number of the studies, the patients were not categorized in the subgroups and the frequency of Helicobacter pylori infection was calculated simultaneously for all the CAD patients. Overall, the frequency of H. pylori in atherosclerotic plaques in the CAD patients was estimated to be 41.30%. However, the highest and lowest frequency of infection with Helicobacter pylori were 80% and 0%, respectively. In addition, the odds ratio was used to evaluate the relationship between infection with Helicobacter pylori and atherosclerosis. Based on the available evidence, the odds ratio appeared to be more reliable, in terms of the considered outcomes, than the relative risk method in the low populations and in the unavailability of incidences within the community to investigate the signi cant relations and therefore we used the odds ratio method for our purpose [58]. The present meta-analysis was done by Comprehensive Meta-Analysis software (CMA-Ver. 2.0). This software has the ability to combine studies and impact sizes into the results and is highly acceptable and commonly used [59]. Statistical analysis revealed that infection with Helicobacter pylori was signi cantly related with the atherosclerosis (The ORs 95% CI: 2.50 (1.83,3.42); P-value < 0.00) and the infection with H. pylori is predisposed to atherosclerosis and cardiovascular disorders in Iran (Fig. 2). Also, the asymmetry of the funnel plot demonstrated the potential publication bias in the present study (Fig. 3). However, the present study was also a comprehensive document con rming the relationship between the infection with Helicobacter pylori in atherosclerotic plaques formation and the susceptibility to CAD.

Discussion
Atherosclerosis is the most common cause of cardiovascular diseases, especially the ischemic heart disease and stroke, and is among the top four causes of death worldwide [60]. Atherosclerosis is a compound Greek word for athero meaning gruel or paste and sclerosis meaning hardness; it is a disorder of large and medium arteries that begins with damage to the vascular endothelial cells and changes in the blood circulation and subsequent to the formation of the atherosclerosis plaques including the necrotic cores, calci ed region, lipid particles, smooth muscle cells (SMCs), endothelial cells (ECs), polymorphonuclear cells (PMNs) and foamy cells (alternative macrophages) [60][61]. According to the available evidence, the most prominent stimulants for the formation and development of the atherosclerotic plaques are processes of vascular endothelium damage (especially the intima) and the chronic in ammation [60][61][62]. Infectious agents, especially Chlamydia pneumoniae, Mycobacterium tuberculosis and Helicobacter pylori, are among the most important infections that can cause chronic in ammation to escape the immune system and appear to be involved in the formation of the atherosclerotic plaques [20]. The hypothesis of the role of Helicobacter pylori infection and atherosclerosis was rst reported by Menall et al. and the track of Helicobacter pylori infection has been observed in atherosclerotic plaques of coronary, carotid and aortic arteries [38,63]. In particular, the eradication of H. pylori infection decreased the CRP and proin ammatory response and improved the endothelial dysfunction and protective effect on early stages of the atherosclerosis formation [64][65][66]. In the present study, we collected and analyzed all the published articles about Helicobacter pylori infection in the Iranian cardiovascular patients, since the prevalence of atherosclerosis is high in Iran and a signi cant population in Iran is infected with H. pylori. Thus, the studies about the Iranian patients can provide very valuable results regarding the impact of H. pylori infection and the susceptibility to cardiovascular disorders. According to the present comprehensive meta-analysis, the rate of colonization with Helicobacter pylori and the formation of atherosclerotic plaques in the Iranian CAD patients are signi cantly related and may make the individuals susceptible to the cardiovascular disorders. According to the literature review, Helicobacter pylori contributes to the formation of the atherosclerotic plaques and CAD through the chronic in ammation, injuries and endothelial dysfunction, as well as the impaired body metabolism (Fig. 4). Studies have shown that chronic in ammation during atherosclerosis stimulates Th1 activity and causes the production of pro-in ammatory responses that result in calling and recruiting the in ammatory cells especially PMNs subsequent to the IL-1, IL-6, IFN-ϒ and TNF-α production, and macrophages also enter into the location in response to MCP1 / 2, inducing the in ammatory reactions that result in the destruction and dysfunction of the endothelial cells [61][62][63][67][68]. Since H. pylori is capable of inducing the chronic in ammatory and Th1 response, it seems that it may be involved in the pathogenesis of atherosclerosis [67][68]. Furthermore, H. pylori can in some cases of chronic gastritis lead to atrophy and malabsorption of vitamin B12 and folic acid by stopping the gastric acid production, resulting in high levels of homocysteine, which increases the damage to the vessel wall through the stimulation of nitric oxide (NO) production and in ammation, and its serum level is elevated in a large number of patients affected by atherosclerosis and CAD [69][70][71][72]. According to existing studies, the people infected with H. pylori develop dyslipidemia [73]. Hoffmeister et al. demonstrated that the CAD patients infected with Helicobacter pylori had higher levels of cholesterol, LDL, triglyceride, and apolipoprotein-B as compared to the CAD patients infected with chlamydia pneumonia or cytomegalovirus, and the HDL levels of these patients were also lower [74]. However, similar studies have shown that after eradication of the infection with H. pylori, the serum levels of HDL and apolipoprotein-AI / AII increase and cholesterol, triglyceride, while that of the LDL levels decrease. It seems that helicobacter pylori increase the fatty acid by disrupting the fatty acid metabolism and causes it to be deposited on the vessel wall [73][74][75]. Metabolic disorders can also provide the of incidence of atherosclerosis [76]. Gillum et al. showed that there was a signi cant relationship between H. pylori seropositivity and CAD in the diabetic patients [77]. Moreover, de Luis showed that the rate of CAD and cerebrovascular disorders between the diabetic patients infected with H. pylori is high [78]. In his studies, Polyzos et al. proved that infection with H. pylori is associated with resistance to insulin, and perhaps the infection with H. pylori via the impact on the metabolism (particularly glucose) of the body might lead to atherosclerosis, especially since the glucose resistance is improved subsequent to the eradication of infection with H. pylori and the level of the adiponectin (a factor for preventing metabolic disorders) is increased [79][80]. Processes such as hypertension and arterial stiffness have been implicated in the process of atherosclerosis, and studies have shown that there is a signi cant relationship between infection with helicobacter pylori and these factors [81][82]. Recently, the effect of Helicobacter pylori virulence factors has also been studied on atherosclerosis, and a previous report has shown a signi cant relationship between the infection with cytotoxin-associated gene A (CagA) positive strains and carotid plaque [83]. Also, Bastiani et al. showed that H. pylori CagA seropositivity was very high in the patients who encountered stroke [84]. It has been found in this respect that CagA strains induce atherosclerosis by the destruction of the vascular endothelial cells, modi cation of the oxidized LDL and stimulating the in ammation, and eradicating H. pylori CagA infection has stopped the above processes having a protective effect in the patients [85][86][87].
In addition, it has nowadays been suggested that CagA and HSPs stimulate the production of autoantibodies and endothelial dysfunction that ultimately lead to atherosclerosis [88][89]. There have been numerous reports about the relationship between Helicobacter pylori infection and the cardiovascular disease (CAD) [63,89]. According to the literature review, Longo-Mbenza et al. evaluated the cardiovascular risk factors, in a longitudinal (provident) study, in 205 patients for a ten-year follow-up and showed that H. pylori IgG titers were high in a substantial population of CAD patients [90]. In their studies on a population of 2029 in South Korea, Park et al. demonstrated that there was a signi cant relationship between H. pylori seropositivity and the CAD patients (ORs: 1.23; p = 0.049) [91]. Other casecontrol studies conducted in India, Turkey, and Japan con rmed the results of previous studies [91][92][93][94].
Our study also con rmed the relationship between Helicobacter pylori infection and atherosclerosis in the CAD patients. Isolation of H. pylori from the atherosclerotic plaques is one of the most important evidence for approval of the role of this microorganism in the atherosclerosis and CAD [63,89]. Extensive studies are so far ful lled in this regard. For instance, the rate of isolation of H. pylori from atherosclerosis was 27.2-33.5% in the study by Jha et al. [92]. In another study in Argentina, the carotid plaque isolation was about 83% [95]. In a cross-sectional study in Turkey, Kilic et [101]. However, in another meta-analysis, 13 studies were reviewed and no signi cant relationship was found between H. pylori and the stroke [102].

Conclusions
We had limitations in this study, the rst of which was the limited number of studies that required more extensive studies. Moreover, in some studies, the diagnosis of H. pylori was based on the evaluation of H. pylori IgG seropositivity, but this test also yielded false positive results after the treatments. Thirdly, studies were restricted to geographic regions and this led to the selection bias. Fourthly, the type of CAD was not considered in several studies (e.g. coronary disease, ischemia, stroke, etc.). Fifthly, the studies were of case-control and cross-sectional basis, while the provident (longitudinal) studies were more appropriate for investigations of this study. Sixth, the virulence factors such as CagA and VacA were not suggested, and the seventh point was about the publication bias of the studies. However generally, regarding the previous studies, we collected all the Iranian published articles and showed that the infection with Helicobacter pylori was signi cantly related with the atherosclerosis, but further complementary and more extensive studies are required to con rm this hypothesis.

List Of Abbreviations
Helicobacter

Declarations
Ethics approval and consent to participate The manuscript methods was based on researching on global databases.

Consent for publication
All authors have consent for publication of data Availability of data and materials All data generated or analyzed during this study are included in this published article

Competing interests
The authors declare that they have no competing interests    Funnel plot of standard error by Log odds ratio (OR).