**5. Role of exosomes in mediating the effect of** *H. pylori* **infection on endothelial function**

*H. pylori* do not enter the blood circulation themselves because of the gastric tissue barrier and a unique survival and growth environment [67]. However, *H. pylori* virulence factor CagA and *H. pylori* DNA are present in human atherosclerotic lesions and human aorta, carotid and coronary arteries [44, 68–70]. Many cells are known to release extracellular vesicles with unique biophysical and biochemical properties [71, 72], that are referred as exosomes (with diameters from 30 to 200 nm) [73]. Exosomes are found in various body fluids including blood, urine, saliva, and breast milk, and play an important role in cell-to-cell communications through transport of a wide spectrum of bioactive constituents including proteins, lipids, and miRNAs [74, 75]. Recent studies have demonstrated that exosomes are critically involved in the transfer of proteins during infections like prion protein in neurodegenerative disease [76], human immunodeficiency virus-related proteins [77], and human T-cell leukemia virus type-1 proteins [78]. Indeed, it is shown that *H. pylori* infection increases the expression of miR-25 in gastric epithelial cells and is associated with increased levels of exosometransmitted miR-25 in peripheral blood in human subjects. Further studies demonstrate that Kruppel-like factor 2 (KLF2) is a direct target of exosometransmitted miR-25 in vascular endothelial cells. MiR-25/KLF2 axis is involved in the regulation of NF-κB signaling pathway, resulting in increased expression of IL-6, monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and ICAM-1 [79].

To determine how *H. pylori* infection impairs endothelial function, a recent study tested the hypothesis that *H. pylori* could interact with gastric epithelial cells (GES-1), leading to the release of CagA-containing exosomes into the circulation that in turn impair endothelial function [55]. Indeed, Western blotting analysis and immunofluorescence staining demonstrated that the unique *H. pylori* virulence factor CagA entered into human GES-1 after incubation with CagA+ *H. pylori* (**Figure 2A** and **B**). Further studies showed that characteristic exosomes were present in the conditioned media of human GES-1cultured with CagA<sup>+</sup> *H. pylori* as defined by specific biomarkers (HSP70 and CD9) using Western blotting, by specific morphologic features using transmission electron microscopy, and by size distribution using a Zetasizer Nano ZS instrument [80]. Western blotting analysis demonstrated that the exosomes from the conditioned media of human GES-1 cultured with CagA<sup>+</sup> *H. pylori* contained the unique CagA protein, while exosomes from the control conditioned media of GES-1 (without culture with *H. pylori*) had no CagA protein (**Figure 2C-E**). When the labeled GES-1-derived exosomes with PKH67 were cultured with HUVECs, a detectable amount of PKH67-labeled exosomes was present in HUVECs using 3-D confocal microscopy after 12 hours of culture (**Figure 2F**), confirming the entry of exosomes into HUVECs. Treatment with human GES-1-derived CagA-containing exosomes significantly inhibited the function of HUVECs with decreased proliferation, migration, and tube formation as compared with the control exosomes (**Figure 2G-I**).

Further studies [55], using the serum exosomes from patients with CagA<sup>+</sup> *H. pylori* infection and from healthy age-and sex-matched volunteers, revealed that serum exosomes from both patients and healthy subjects exhibited the characteristics similar to the exosomes from human gastric epithelial cells GES-1 cultured with CagA+ *H. pylori* in their morphology using transmission electron microscopy, size distribution using a Zetasizer Nano ZS instrument, and unique biomarkers (HSP70 and CD9) using Western blotting. As expected, CagA protein was detected in the serum exosomes from patients with CagA+ *H. pylori* infection, but not from control

Helicobacter pylori *Infection and Endothelial Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.97260*

#### **Figure 2.**

*Exosomes from human gastric epithelial cells GES-1 cultured with CagA+* H. pylori *significantly inhibited endothelial cell function in vitro. Western blotting analysis (A) and immunofluorescence staining (***B***) with 3-D confocal microscope demonstrated that the unique* H. pylori *virulence factor CagA entered into GES-1 after culture with CagA+* H. pylori*. Exosomes isolated from the conditioned media of GES-1 cultured with CagA+* H. pylori *displayed typical features for exosomes including characteristic biomarkers HSP70 and CD9 by western blotting (***C***), morphologies on transmission electron microscopy (***D***), and size using a Zetasizer Nano ZS instrument (***E***). Western blotting analysis confirmed the presence of CagA protein in the exosomes from the conditioned media of GES-1 cultured with CagA+* H. pylori*, but not in the ones from GES-1-conditioned media without CagA+* H. pylori *(***C***). PKH67-labeled GES-1-derived exosomes (***green***) were incubated with* **HUVECs** *(30 μg protein/5 × 104 cells), and a significant amount of PKH67-labeled exosomes were detected inside the HUVECs as visualized using a 3-D confocal microscope (***F***), confirming that the exosomes entered into the cells. Treatment of HUVECs with CagA protein-containing exosomes (50ug/ml) from GES-1-conditioned media for 24 hours significantly inhibited the function of HUVECs with decreased migration (***G,** *scale bars = 200 μm), tube formation (***H,** *scale bars = 200 μm), and proliferation (***I,** *scale bars = 50 μm).* **NC:** *Normal control;* **CagA+ HP:** *CagA+* H. pylori*;* **GES-1***: Human gastric epithelial cells;* **HUVEC:** *Human umbilical vein endothelial cell;* **Exo-CM***: Exosomes derived from conditioned medium. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. Data were presented as mean ± SE, n = 3 independent experiments (experiment was repeated 3 times for every measurement). [adopted from (55) with permission].*

subjects using Western blotting analysis. When labeled human serum exosomes with PKH67 were cultured with HUVECs, a significant amount of PKH67-labeled exosomes was present in HUVECs using 3-D confocal microscopy after 12 hours of culture, confirming the entry of serum exosomes into HUVECs. Treatment with

#### **Figure 3.**

*Inhibition of exosome secretion by GW4869 significantly improved endothelium -dependent vascular relaxation in mice with CagA<sup>+</sup>* H. pylori *infection. Treatment with GW4869 significantly decreased the serum exosome level in the mice with CagA+* H. pylori *infection (A) as reflected by the significantly decreased total exosome protein levels (\*\*\*p < 0.001 by one-way ANOVA with Bonferroni's test), and significantly improved acetylcholine (***ach***)-induced endothelium-dependent relaxation (B) of the aorta in the mice with CagA+* H. pylori *infection without change in nitroglycerin (***NTG***)-induced endothelium-independent relaxation (C). \*p < 0.05 (when CagA<sup>+</sup>* H. pylori *+ GW4869 group compared with CagA<sup>+</sup>* H. pylori *+ DMSO group).* **Ach***: Acetylcholine;* **NTG***: Nitroglycerin;* **DMSO***: Dimethylsulfoxide (solubilizer of GW4869). Data shown were mean ± SE. n = 8 mice for control group and 10 mice for other groups. [adopted from (55) with permission].*

serum-derived CagA-containing exosomes from patients with CagA+ *H. pylori* infection significantly inhibited the function of HUVECs with decreased migration, proliferation, and tube formation. Of note, culture with serum exosomes from healthy control subjects also moderately and yet significantly inhibited endothelial function with decreased migration, tube formation, and proliferation, suggesting that some endogenous substances in the normal serum exosomes could also lead to endothelial dysfunction. However, the serum exosomes from patients with CagA<sup>+</sup> *H. pylori* infection exhibited significantly greater inhibitory effects on endothelial functions than the ones from healthy subjects.

Studies were also performed to determine if blocking exosomes release with GW4869 *in vivo* could improve endothelial function in mice with CagA<sup>+</sup> *H. pylori* infection [55]. Indeed, treatment with GW4869 significantly decreased the level of serum exosomes in the mice with *H. pylori* infection (**Figure 3A**), and effectively preserved Ach-induced endothelium-dependent relaxation of the aorta without change in NTG-induced endothelium-independent relaxation (**Figure 3B** and **C**). These findings suggest that *H. pylori* (especially CagA<sup>+</sup> *H. pylori*) infection could lead to significant endothelial dysfunction in both patients and mice through exosomes-mediated mechanisms.

### **6. Effect of** *H. pylori* **infection on other cardiovascular risk factors**

It is not surprising that *H. pylori* infection increases the risk for atherosclerosis and other CVDs including HTN and stroke. It has been reported that *H. pylori* infection promotes the release of IL-1, IL-6, TNF-ɑ, and other cytokines, and activates local and systemic inflammatory response, thus leading to endothelial dysfunction and atherosclerosis [81–83]. *H. pylori* infection could also lead to malabsorption of vitamin B12, which could increase serum level of homocysteine, and promote the development and progression of atherosclerosis [84]. In addition, *H. pylori* could enhance the oxidation of low-density lipoproteins (LDL) and increase atherosclerotic plaque formation with decreased plaque stability [85, 86]. We also observed that the levels of LDL-cholesterol in patients with *H. pylori* infection were significantly higher than those without *H. pylori* infection, while the level of high-density lipoprotein cholesterol (HDL-C) were significantly decreased in the patients with *H. pylori* infection than those without *H. pylori* infection [47]. Patients with *H. pylori* seropositivity were shown to have increased brachial-ankle

pulse wave velocity (a marker of atherosclerosis), and impaired glucose metabolism [87]. It is believed that *H. pylori* could interact with gastric epithelial cells to upregulate the expression of adhesion molecules, and secrete cytokines, which could activate leukocytes, damage the vascular endothelium, aggravate local and systematic inflammatory responses, and thus promote the development and progression of atherosclerosis and related CVDs.
