Relationship between aneurysm and microorganism : Is Helicobacter pylori a primer agent or has an affinity to the tissue ?

1 Department of Medical Microbiology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey. 2 Thoracic and Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey. 3 Medical Faculty, Sifa University, Izmir, Turkey. 4 Faculty of Engineering, Koc University, Istanbul, Turkey. 5 Department of Microbiology, Forensic Sciences Institute, Istanbul University, Istanbul, Turkey.


INTRODUCTION
There is evidences that certain microorganisms have an etiopathogenic role in the development of aneurysm.Chlamydophila pneumoniae and anaerobic bacteria have been suggested as a possible factors influencing the development and expansion of abdominal aortic aneurysm (AAA) (Mazur et al., 2004;Brook, 2009) .H. pylori which is a Gram-negative rod colonised in the stomachs of humans was considered as a bacteria causing chronic gastritis and playing important roles in peptic ulcer disease, gastric carcinoma and gastric lymphoma.Additionnaly virulence factors, such as cag A, vac A and ice A genes related to pathogenicity, as well as host factors were defined (Dunn et al., 1997;Gatti et al., 2005).According to the findings of previous studies, subjects harbouring Cag A positive strains of H. pylori have been considered to be in risk of carotid atherosclerosis and coronary heart diseases (Nyberg et al., 2008;Ziver et al., 2010).In the pathogenesis of AAA and ascending aortic aneurysm (AsAA), inflammatory response is considered significant and it is also suggested that generally microorganisms may be triggering factors for the initiation of inflammatory process in the host.The most frequently discussed of these microorganisms was initially Chlamydophila pneumoniae as well as herpesviruses (Kotze and Ahmed, 2011).Recently, a number of studies investigating whether Cag A positive H. pylori may increase the risk of atherosclerosis and coronary heart disease have been increased.A case with abdominal aortic aneurysm infected with Helicobacter pylori was presented (Hırose and Kugimiya, 2000) and additionnaly epidemiologic studies detecting H. pylori especially in carotid atherosclerotic plaques were published (Ameriso et al., 2001;Singh et al., 2002;Mayr et al., 2003;Karlsson et al., 2000) but until now, a precise relationship between H. pylori virulence factors and the pathogenesis of aneurysms was not revealed.This study aims to evaluate the role of H.pylori in the etiopathogenesis of AAA and AsAA.

MATERIALS AND METHODS
A total of 50 cases admitted to the department of Cardiovascular Surgery of TR Ministry of Health, Dr. Siyami Ersek Thoracic and Cardiovascular Surgery Training and Research Hospital and diagnosed with AAA and AsAA according to their clinical findings and radiodiagnostic data were enrolled to this study.The study was performed between January 2010 and December 2010 as a crosssectional, case-control study .
Cases with abdominal aortic aneurysm (AAA) consisted of 10 patients having an abdominal aortic diameter larger than 3 cm determined upon radiodiagnostic methods.Three (3) (30%) of them had ruptured AAA, whereas 7 (70%) had non-ruptured AAA, while both groups had an aneurysm diameter of 5 cm.All of the cases with AAA included in the study had a past surgical procedure or had undergone endovascular intervention.
Cases with ascending aortic aneurysm (AsAA) consisted of 40 patients having an ascending aortic diameter larger than 3 cm.Two (2) (5%) of them had ruptured AsAA and from the non ruptured AsAA group, 25 (6.5%) had a minimum of 5 cm aneurysm diameters and 13(32.5%)had maximum 5 cm aneurysm diameters.All patients included in AsAA group had undergone a surgical procedure in the past.
Cases with post stenotic aneurysm consisted of 30 patients.This group was used as a patient control group (PCG).The healthy control groups (HCG) was formed by selecting 47 individuals among people admitted to Computed Tomography (CT) laboratory of the department of radiodiagnostics of Istanbul University (IU) Cerrahpasa Faculty of Medicine (CFM) according to their particular complaint, but not having abdominal or cardiovascular complaints resembling the AAA and AsAA.Abdominal and ascending aortic diameters were within normal limits (<3 cm) in individuals of this group.
The data and results obtained from both groups, such as age, gender, education status, occupation, smoking habits (smokers among the study groups were considered smokers), hypertension, diabetes, cardiovascular disease in family history, total cholesterol, triglyceride, HDL and LDL values, diameter of aneurysm, whether it is ruptured and potential stomach complaints were collected and recorded.Laboratory studies of the research were performed in Serology-ELISA laboratories and molecular biology laboratories of IU CFM department of medical microbiology and in Private Burc Molecular Diagnostic Center.
Blood and aneurysm tissues sampling and test methods were performed by the following procedures.Blood (10 ml) samples were collected by venous puncture from each of the patient and control group cases.The blood samples were centrifuged 5 min at 3000 rpm and the preserved serums were stored at -80°C until laboratory studies.H. pylori specific IgG was determined by ELISA method.Aneurysm tissue samples were stored at -80°C until the test day.
For the determination of H. pylori IgG, 1/101 diluted serum samples were tested using H. pylori ELISA IgG kit (Euroimmun, Lübeck-Germany).A fresh lysate of the standard strain of H. pylori ATCC 43504 was used to perform the test with the kit.A negative test result was evaluated when the absorbance was <0.8.An intermediate test result was evaluated when the absorbances were between 0.8-1.1 and a positive test result was evaluated when the absorbances was >1.1.
Tissue samples were collected during operations from 50 patients with AAA and AsAA and from 30 patients with post-stenotic aneurysm.They are immediately frozen at -70°C until the test day.
For the DNA extraction of H. pylori, collected tissue samples were minced using surgical scalpales under sterile conditions.Genomic DNA was extracted from 25-30 mg of minced tissue using a commercial DNA extraction kit ( High Pure PCR Template Preparation Kit, Roche Diagnostic GmbH Mannheim, Germany) following the manufacturer's instructions.All extracts were stored at -80°C until usage.H. pylori DNA was determined by real-time PCR method using a commercial kit (WAY2GENE ® Helicobacter pylori, Genmar, Turkey) according to the manufacturer's protocol.
Real-time PCR mixture contained 2 µl of sample DNA, 1.6 µl (25 Mm) MgCl2, 14.4 µl agent mix (primers and prob), 2.0 µl enzyme mix in 20 µl finale volume.The assay run on the Roche Light Cycler.The reaction was performed with preliminary denaturation for 10 min at 95°C (slope, 20°C/s), followed by 45 cycles of denaturation at 95°C for 10 s (slope, 20°C/s), annealing at 55°C 10 s (slope, 20°C/s), extension at 72°C for 15 s (slope, 20°C/s).During PCR reaction this process, fluorescences signals was collected in *Corresponding author.E-mail: hrisib@istanbul.edu.tr,hrisibahar@gmail.com.Tel: +90 212 234 34 38 Standard nested PCR protocols were used for amplification of H.pylori. Previously tested clinical samples for H. pylori were used as positive control (urea breath test).Primers choosen from urease A gene region of H.pylori were used for the PCR amplification as previously described (Wang et al., 1993) (Table 1).The PCR mixture contained 0.5 M each primer, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl (pH 8.3), 0.2 mM each dNTP (Fermentase®, Lithuania), and 1.25 U of Taq DNA polymerase (Fermentase®, Lithuania).The final reaction volume was 50 l containing 10 l of DNA sample.Nested PCR was performed by transferring 2 l of the first PCR product to the second round reaction mixture.The cycling conditions used in both rounds consisted of an initial denaturation at 95°C for 2 min, 40 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s followed by a final extension step of 5 min at 72°C.The amplification products were run on a 1.5% agarose gel electrophoresis and visualised on an UV transilluminator.
For genotyping analysis, the cagA, cagE, vacA s1/s2, vacA m1/m2, iceA1/A2, and babA2 genotypes were carried out utilizing PCR assay by using specific primers as described previously (Erzin et al., 2006).Briefly, PCR reaction was performed in a total volume of 25 μl containing approximately 100 ng DNA, 2.5 μl of 10X polymerase buffer, 2.0 mmol/l MgCl2, 0.2 mmol/L dNTPs, 0.4 μmol/l of each primer, and 1 U of Taq polymerase (MBI Fermentas).The PCR program on the icycler™ (BioRad) thermal cycler was as follows: an initial denaturation step at 94°C for 4 min, followed by 40 cycles of 30 s at 94°C, 30 s at 52°C, 1 min at 72°C, and a final extension step of 8 min at 72°C.The amplification products were electrophoresed on 2% agarose gels at 100 Volt for 30 min.The gel and running buffers were 1X TBE (0.89 M Tris-Base, 0.89 M Boric Acid, 20 mM Na2EDTA).The fragments were visualized by ethidium bromide under UV transilluminator.
This study was approved by the ethical committee of Istanbul 1.No Clinical Studies in Deanery of Cerrahpasa Faculty of Medicine by decision no; C-011 on 12.01.2010.

Statistical analysis
SPSS 17.0 statistical program was used for the assessment of the data obtained from the study.Chi-square, Fisher, T test and variance analysis were used in the assessment; p<0.05 was found to be significant.

RESULTS
The patient group with 50 cases ( 10 diagnosed with AAA, and 40 diagnosed with AsAA), the PCG with 30 cases diagnosed with post stenotic aneurysm and the HCG with 47 person were included in this study by being matched in terms of age, gender and smoking habits (Table 2).
In the patient group of 50 cases, 33 were female and 17 were male.Their age were between 34-78 and 12 were smoker and 38 non smoker.In the PCG of 30 case, 11 were female and 19 were male.Their age was between 38-72 and 14 were smoker and 16 non smoker.In the HCG group of 47 individuals, 15 were female and 32 were male.Their age was between 29-67 and 19 were smoker and 28 non smoker.Hypertension was determined in 87% of our total cases diagnosed with aneurysm (AAA,AsAA,PSAG).
The systolic blood pressure of these patients was >160 mmHg, and the diastolic blood pressure was >95 mmHg.89% of our total cases diagnosed with aneurysm were receiving diabetes treatment.Hypertension and diabetes was not detected in individuals of our healthy control group.In 76% of the PG with 50 cases, in 56.7 % of the PCG with 30 cases and in 60% of HCG with 47 person H. pylori IgG was found positive.A statistically significant difference was not found for H. pylori IgG positivity in comparisons between all study and control groups.(p>0.05)(Table 3).
H. pylori DNA was detected in one tissue sample out of 50 patient group with AAA by real-time PCR (Figure 1).This case belong to a 75 years old male patient with AAA secondary to atherosclerosis pathology.The same patient sample was found to be positive with nested PCR method.The size of the PCR product was 360 bp (Figure 2).That positive sample was genotyped by PCR analyses.PCR analysis revealed that the H. pylori strain    has the genotype of VacA S1b/m1 (Figure 3).Other tested virulance genes were negative in our patient.It was reported that type s1/m1 strains produced a higher level of cytotoxin activity in vitro than type s1/m2 strains; none of 19 type s2/m2 strains produced detectable cytotoxin activity (Atherton et al., 1995).H. pylori DNA was not detected in any tissue samples of the other control groups.

DISCUSSION
Atherosclerosis is the mostly emphasized factor among the factors suggested to be related to the pathogenesis in both types of aneurysm.Unlike the risk factors, such as gender, age, weight, smoking habits, hyperlipidemia known to be involved in atherosclerotic plaque development, it is claimed that the trigger factor may be the inflammatory response of microorganisms in vessel tissue (Mehta et al., 1998;Ross, 1993;Alexander, 1994).
It is also known that mycotic aneurysms may develop due to bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Salmonella spp.and Streptococcus spp.reproducing on atherosclerotic plaques (Kumara et al., 2009). In 1997, Saikku, (1997) reported a review particularly the role of Chlamydophila pneumoniae, suggesting that this bacterium may be the trigger factor in the development of atherosclerosis and arterial diseases.
After this introduction, as suggested in Chlamydophila pneumonia atherosclerosis relationship, H. pylori may colonize to the atheromatous tissues instead of normal aorta wall and may cause a lipid accumulation in vessel lumens.There are remarkable studies conducted in recent years on H. pylori suggested to induce aneurysms and to trigger atherosclerosis due to the constant and mild inflammatory response.Acute phase reactants including CRP and fibrinogen, T cell response synthesizing interferon-gamma and proinflammatory cytokine levels are seriously increases by H. pylori in the host, as well as virulence factors such as CagA, VacA, after C. pneumoniae, which are known to be the primary pathogen of gastrointestinal area, particularly gastric tissue (Mendall et al., 1994;Pasceri et al., 1998;Murray et al., 1995;Mendall et al., 1996;Whincup et al., 1996).This probable hypothesis needs to be clarified with new prospective experimental animal studies.Nyberg et al. claimed for the first time until our previous study (Ziver et al., 2010) that a pathogenesis mechanism can play a role between H. pylori and coronary hearth disease and AAA and AsAA.They investigated on a seroepidemiological basis study the fact that H. pylori was associated with coronary heart disease but they did not detected any statisticaly significant differences between their patient and control groups in term of H. pylori-IgG positivity and additionnaly they did not found any connection between H. pylori CagA seropositivity and abdominal aortic aneurysm rupture.According to our H. pylori-IgG results, we did not detect also any statistically significant difference for H. pylori IgG positivity between our study and control groups.This result matches with the result of the seroepidemiological study reported by Nyberg et al. Hovewer, Ziver et al. (2010) detected a statisticaly significant difference for VacA seropositivity but not for CagA between their study and control groups.According to our H. pylori-IgG results of this present study, we can not state that there is a relation between aneurysm and H. pylori .
In molecular based studies concerning aneurysm and H. pylori relationship; Hirose and Kugimiya (2000) reported in a case study, a relevance between AAA and H.pylori, Morre et al. (2000) could not detect evidences supporting the presence of H. pylori in atherosclerotic plaques but detected seropositivity for H. pylori IgG.Hovewer, contrary to these studies, Falkensammer et al. (2007) reported that no H.pylori DNA was presented in tissue samples of 98 AAA cases.Martinez and Martinez, (2002) concluded that H. pylori' may be an indirect agent in atherothrombi development and cardiovascular diseases in their consideration "H.pylori; a new cardiovascular risk factor?". Mannacio et al. (2011) detected no H.pylori DNA in aortic aneurysm samples of 100 cases but detected a significant statistical difference for CagA and VacA positivity between patient and control groups.They detected VacA and the sub-genotype of H. pylori strain was detected as VacA s1m.Hovewer, in the present study, we could not detect H. pylori DNA in 49 AAA and AsAA (40 non-atherosclerotic and 9 atherosclerotic) and 30 post sterotic aneurysm cases, we detected H. pylori DNA in a 75 years old male patient with AAA developed secondary to atherosclerosis.AAA was detected in this patient by abdominal ultrasonography in the cours of the diagnosis of an acute appendicitis, two years ago before his application to our center with AAA.This patient was followed-up by computed tomography (CT) and the diameter of AAA was reached to 63 mm with calcified mural thrombus in 2008.Atheroms were also detected in aort and other arteriols.The genotype of this H. pylori strain was VacA s1b/m1 and any of the cagA, cagE, iceA and babA2 genes were presented.Hovewer, even this case with no significant statistical difference, our results were in accordance with the study of Mannico et al. (2011) andZiver et al. (2010).There was an accordance of our results with the consideration of "VacA may have a role in the pathogenesis of aneurysms" in the seroepidemiological based on the study of Ziver et al. (2010) that was aimed to detect virulance factors.
According to these results, we suggested that, VacA protein which is coded by VacA gene may induct vacuole formation in the membranes of the host cells and also induct apoptosis.VacA protein may also induct the proinflamatuary cytokines and also cause immunosuppression.VacA protein may also induct the activation of CagA protein by activating itself.All of the above suggestions may lead us to think, H. pylori strains harboring VacA may trigger aortic aneurysms based on atherosclerosis in such persistant, chronical H. pylori infections.Hovewer, we can not definitely say, H. pylori is a definitely triggering agent in aneurysm cases based on atherosclerosis and we can only suggest, H. pylori may be a secondary agent which has an affinity to the vascular lesions.
In conclusion, however, VagA s1b1 type H. pylori was detected in vascular tissue of a case with atherosclerotic abdominal aneurysm, these data is not enough to support an etiopathogenetic relationship between aneurysm and H. pylori.Presence of H. pylori DNA in only one case suggests us that H .pylori is not a primer agent and probably has an affinity to the vascular tissues.Hovewer, the detection of this type of H. pylori in a AAA patient with atherosclerosis, suggested us that new, matched, large serial clinical prospective and case-control based seroepidemiological/molecular studies with atherosclerotic patients were needed in order to clarify this relationship.

Figure 1 .
Figure 1.The figure shows amplification curves of positive control and H. pylori positive patient.

Table 1 .
The primer set used in this study.

Table 2 .
Demographic details of Patient and Healthy Control Group.

Table 3 .
Distribution of ELISA results in Patient, Patient Control and HCG cases.