**4.** *H. pylori* **infection and endothelial dysfunction**

#### **4.1** *H. pylori* **infection and endothelial dysfunction in patients**

Endothelial cells play a critical role in maintaining the integrity of vascular structure and function. Endothelial dysfunction is an important contributing factor to the pathogenesis of CVDs including HTN and atherosclerosis [4]. Early studies with small patient samples suggested that there was no clear association between chronic infections, including infection with Chlamydia pneumoniae, cytomegalovirus, Epstein–Barr virus, and *H. pylori*, and decreased endothelial function [48]. A small study with a total of 53 pediatric patients using Doppler ultrasonography of the brachial artery showed that percent ratio of the change in systolic diameters during hyperemic phase to the basal diameter (endothelium-dependent) was not significantly different between *H. pylori*-negative and -positive groups in pediatric population [49].

However, accumulating data clearly supports the concept that *H. pylori* infection could lead to significant endothelial dysfunction in patients. Using high-frequency ultrasonographic imaging of the brachial artery, it was found that endotheliumdependent flow-mediated vasodilation (FMD) was significantly lower in the subjects with seropositive antibodies to *H. pylori* than in the ones with seronegative antibodies to *H. pylori*, while endothelium-independent nitroglycerin-induced vasodilation was similar in both groups [50]. Similarly, another study with patients with chronic gastritis associated with *H. pylori* infection demonstrated that the level of FMD in patients with positive *H. pylori* infection was significantly lower than those with negative *H. pylori* infection and the healthy control group [51]. Studies also showed that the levels of C-reactive protein and soluble intercellular adhesion molecule-1 (ICAM-1) were significantly higher in subjects with seropositive antibodies to *H. pylori* than in those with seronegative antibodies to *H. pylori* [50]. The levels of endothelial dysfunction biomarkers, including endothelin-1 (ET-1), E-selectin, and ICAM-1, were found to be significantly higher in *H. pylori* (+) patients than in *H. pylori* (−) subjects [52].

One of the important questions is whether endothelial dysfunction associated with *H. pylori* infection is reversible. In a study in 2011, vascular function measurements (ankle brachial index and flow-mediated diameter percent change) were made in patients with *H. pylori* infection at the time of study enrollment and 3 months afterwards with *H. pylori* eradication. Subjects with *H. pylori* infection were treated with standard triple antibiotics therapy. It was found that *H. pylori*positive subjects had severe endothelial dysfunction that improved significantly after *H. pylori* eradication with triple antibiotics. Subjects without *H. pylori* infection also had endothelial dysfunction, however, that was not improved after treatment with triple antibiotics. These data suggests that endothelial dysfunction in patients with *H. pylori* infection appear to be reversible [53].

In a recent study, the investigators carefully selected 18 young patients (both male and female) with *H. pylori* infection without any known risk factors for endothelial dysfunction to evaluate endothelium-dependent FMD of the brachial artery with ultrasound. A group of 13 age- and sex-matched young healthy volunteers served as the controls. The diagnosis of *H. pylori* infection was confirmed with gastric endoscopic biopsy and 13C urea breath test for each patient. No other confounding variables except the conditions listed in the exclusion criteria were considered for subject selection. Young patients were recruited to minimize the risk factors for endothelial dysfunction. Patients were excluded from the study if any of the following conditions was present: 1) history of *H. pylori* eradication, 2) use of any medications including antibiotics, proton pump inhibitors, or H2-receptor blockers 3 months before the study, 3) age < 18 or > 35 years, 4) connective tissue diseases or immunological diseases, 5) mental disorders, 6) asthma or COPD, 7) hematological disorders, 8) thyroid diseases, 9) malignancies, 10) recent (within 3 months) or chronic infection except *H. pylori* infection, 11) congestive heart failure, 12) abnormal renal or liver function; 13) congenital heart diseases, 14) hypertension, 15) smoking, 16) diabetes mellitus, 17) lipid abnormalities, 18) stroke, 19) obesity, 20) sedentary life style, 21) alcohol use, 22) any use of energy drinks or coffee or tea within 48 hours, and 23) unresponsive to anti-*H. pylori* therapy. After fasting for 8 to 12 hours, brachial artery FMD was evaluated for patients and control subjects, and presented as percent change in post-ischemia diameter over baseline. The data showed that patients with *H. pylori* infection exhibited a significant reduction in endothelium-dependent vasodilatation compared with the controls (**Figure 1A**). When patients with *H. pylori* infection were treated with BIS-based quadruple oral anti-*H. pylori* therapy (100 mg furazolidone, 100 mg doxycycline, 5 mg ilaprazole, and 220 mg colloidal bismuth tartrate, twice a day for 2 weeks) [54], their endothelium-dependent FMD of the brachial artery was effectively restored (**Figure 1B**) [55]. The effectiveness of *H. pylori* eradication

#### **Figure 1.**

H. pylori *infection significantly impairs endothelium-dependent flow-mediated dilatation (FMD) in human subjects and endothelium-dependent vascular relaxation in mice. Patients with* H. pylori *infection and healthy control subjects were evaluated for endothelium-dependent flow-mediated dilatation (***FMD***) of the brachial artery with ultrasound. The diagnosis of* H. pylori *infection was confirmed with gastric endoscopic biopsy and 13C urea breath test for each patient. Patients with* H. pylori *infection (n = 18 patients) displayed a significant reduction in their endothelium-dependent FMD compared with the controls (n = 13 subjects) (A). Eradication of* H. pylori *infection with anti-*H. pylori *therapy effectively restored the endothelium-dependent FMD in patients with* H. pylori *infection (n = 10 patients with anti-*H. pylori *therapy) (B). Mice infected with CagA<sup>+</sup>* H. pylori *for 1 week significantly decreased acetylcholine (***Ach***)-induced endothelium-dependent relaxation of thoracic aorta without change in nitroglycerin (NTG)-induced endothelium-independent vasorelaxation (***data not shown***) after sub-maximal contraction with phenylephrine (***PE***) (10−6 M). The impaired achinduced endothelium-dependent vasorelaxation persisted for as long as the infection was present for at least 24 weeks (***C***) without change in NTG-induced endothelium-independent vasorelaxation (***data not shown***). Eradication of* H. pylori *infection with anti-*H. pylori *therapy effectively restored ach-induced endotheliumdependent vasorelaxation in mice with 12 weeks of chronic CagA+* H. pylori *infection, # p < 0.05 (compared to CagA+* H. pylori *+ treatment mice); \*\*p < 0.01 (compared to NC mice) (C), n = 8 mice for each group.* **NC***: Normal control;* **Ach***: Acetylcholine;* **NTG***: Nitroglycerin. Data were presented as mean ± SE. [adopted and modified from (55) with permission].*

with antimicrobial therapies was confirmed using 13C urea breath test for the study patients. These data confirms that endothelial dysfunction in patients with *H. pylori* is indeed reversible (very likely within 1 year of infection).

#### **4.2** *H. pylori* **infection and endothelial dysfunction in animal models**

In the same recent study, the investigators used specific-pathogen-free male C57BL/6 mice to establish a mouse *H. pylori* infection model to determine if impaired endothelial function in human subjects with *H. pylori* infection could be re-produced in animal model using the *H. pylori* bacteria isolated from patients. Since the vast majority (>90%) of *H. pylori* infection patients in East or Southeast Asian countries are infected with CagA<sup>+</sup> *H. pylori* [56, 57], and CagA is considered to be involved in the extragastric diseases associated with *H. pylori* infection [44, 58–60], CagA<sup>+</sup> *H. pylori* bacteria isolated from gastric ulcer patients were prepared, characterized, and used for the animal experiments with phosphate buffer solution (PBS) as control. After fasting overnight, mice were infected with *H. pylori* inoculum in PBS by intragastric gavage once per day for 3 days to achieve 100% infection rate. Successful infection with CagA<sup>+</sup> *H. pylori* in mice was confirmed with both positive Rapid Urease Test (RUT) and Giemsa staining as described [61]. A 100% infection rate was achieved in C57BL/6 mice with this method. Control mice received the same volume of PBS by intragastric gavage.

Thoracic aorta was collected to evaluate endothelium-dependent relaxation to acetylcholine (Ach) and endothelium-independent relaxation to nitroglycerin (NTG) at week 1, 8, 12, and 24 after *H. pylori* infection to determine if there was a significant difference in endothelial dysfunction after acute (1 week) and chronic (24 weeks) *H. pylori* infection. Indeed, Ach-induced endothelium-dependent relaxation was significantly reduced in mice 1 week after *H. pylori* infection without Helicobacter pylori *Infection and Endothelial Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.97260*

change in NTG-induced endothelium-independent relaxation. The impaired Achinduced endothelium-dependent relaxation persisted for as long as the infection was present for at least 24 weeks in the infected mice without change in vascular contraction to either phenylephrine or potassium chloride (**Figure 1C**), while NTGinduced endothelium-independent relaxation remained intact [55]. These data demonstrated that *H. pylori* infection selectively impairs endothelium-dependent relaxation, not endothelium-independent relaxation, of thoracic aorta in mice that are similar to the findings in human subjects with *H. pylori* infection.

Efforts were made to examine if eradication of *H. pylori* infection could improve endothelium-dependent vasodilation to confirm if *H. pylori* infection was indeed the reason for endothelial dysfunction. As expected, elimination of *H. pylori* infection in mice with anti-*H. pylori* therapy (123.3 mg/Kg bismuth potassium citrate, 102.75 mg/kg tinidazole, and 51.38 mg/kg clarithromycin once daily for 2 weeks via intragastric gavage) significantly improved Ach-induced endotheliumdependent vasorelaxation without change in NTG-induced endotheliumindependent relaxation (**Figure 1C**). For the control group, *H. pylori* infected mice were given the same volume of normal saline. The effectiveness of *H. pylori* eradication with antimicrobial therapies in mice was confirmed using RUT and Giemsa staining [55, 61]. These findings confirm that impairment of endotheliumdependent vasodilation associated with *H. pylori* infection is reversible in mouse model, similar to the observations in human subjects.
