Helicobacter pylori infection selectively attenuates endothelial function in male mice via exosomes-mediated ROS production

Abstract

Background: Substantial sex differences exist in atherosclerosis. Excessive reactive oxygen species (ROS) formation could lead to endothelial dysfunction which is critical to atherosclerosis development and progression. Helicobacter pylori (H. pylori) infection has been shown to attenuate endothelial function via exosomes-mediated ROS formation. We have demonstrated that H. pylori infection selectively increases atherosclerosis risk in males with unknown mechanism(s). The present study was to test the hypothesis that H. pylori infection impaired endothelial function selectively in male mice through exosome-mediated ROS formation.

Methods and results: Age-matched male and female C57BL/6 mice were infected with CagA+ H. pylori to investigate sex differences in H. pylori infection-induced endothelial dysfunction. H. pylori infection attenuated acetylcholine (ACh)-induced endothelium-dependent aortic relaxation without changing nitroglycerine-induced endothelium-independent relaxation in male but not female mice, associated with increased ROS formation in aorta compared with controls, which could be reversed by N-acetylcysteine treatment. Treatment of cultured mouse brain microvascular endothelial cells with exosomes from H. pylori infected male, not female, mice significantly increased intracellular ROS production and impaired endothelial function with decreased migration, tube formation, and proliferation, which could be prevented with N-acetylcysteine treatment.

Conclusions: H. pylori infection selectively impairs endothelial function in male mice due to exosome-mediated ROS formation.

Keywords: Helicobacter pylori; atherosclerosis; endothelial dysfunction; reactive oxygen species; sex difference.

Figure 1

H. pylori infection selectively resulted in significant endothelial dysfunction in male C57BL/6 mice. Endothelium-dependent aortic relaxation to ACh was significantly attenuated in male C57BL/6 mice (A, B), not in females (C, D) with acute (1 week) or chronic (12 weeks) H. pylori infection over the control. NC, normal control; Ach, acetylcholine; Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 using t-test, n=8-10 mice for each group at each time point.

Figure 2

H. pylori infection selectively impairs endothelial function in male mice via excessive ROS formation. Representative fluorescent images (A) and quantitative analysis of aortic ROS (B) in male and female C57BL/6 mice with H. pylori infection or PBS control (**P<0.01 by t-test). Treatment with NAC effectively prevented aortic ROS production (C, D) (*P<0.05, **P<0.01 by one-way ANOVA with Bonferroni’s test or Kruskal-Wallis test with Dunn’s post hoc test) and preserved aortic relaxation to Ach (E, F) in male C57BL/6 mice with acute (1 week) or chronic (12 weeks) H. pylori infection, without change in endothelium-independent aortic relaxation to NTG (G, H). *P<0.05, **P<0.01 (vs NC), ^# P<0.05 (vs H. pylori + NAC); ^$ P<0.05, ^$$ P<0.01 (vs NAC) by one-way ANOVA with Bonferroni’s test or Kruskal-Wallis test with Dunn’s post hoc test. NC, normal control; Hp, H. pylori; Ach, acetylcholine; NAC, N-acetylcysteine; NTG, nitroglycerin. Data are shown as mean ± SEM; n=8-10 mice for each group at each time point.

Figure 3

Exosomes from the serum of male mice with H. pylori infection increased intracellular ROS level in vitro. Exosomes isolated from mice with H. pylori infection exhibited typical exosome characteristics with unique morphologies as shown on transmission electron microscopy (A) and size distribution (B, C)There was no significant difference in serum exosomes protein levels between male and female mice infected with H. pylori; (D, E) Co-culture of bEND.3 cells with exosomes from the serum of male mice, not from the serum of female mice, with H. pylori infection significantly enhanced the levels of intracellular ROS in bEND.3 cells. NC, negative control; Hp, H. pylori; Exo, Exosomes; Exo-NC, Exosomes from C57BL/6 mice with PBS gavage; Exo-H. pylori, Exosomes from C57BL/6 mice with H. pylori infection. **P < 0.01 by t-test. Data are shown as mean ± SEM; n=7-8 independent experiments for every measurement. Scale bars = 100 μm.

Figure 4

Exosomes from the serum of male mice with H. pylori infection attenuated endothelial function in vitro. Treatment of bEND.3 with serum exosomes from H. pylori infected male C57BL/6 mice, but not female mice significantly inhibited the endothelial function with decreased migration (A), tube formation (B), and proliferation (C). NC, negative control; Hp, H.pylori; Exo, Exosomes; Exo-NC, Exosomes from C57BL/6 mice with PBS gavage; Exo-H. pylori, Exosomes from C57BL/6 mice with H. pylori infection. *P< 0.05, **P < 0.01, ***P < 0.001 by t-test. Data are shown as mean ± SEM; n=6-7 independent experiments for every measurement. Scale bars (A) = 25 μm; Scale bars (B) = 10; Scale bars (C) = 100 μm.

Figure 5

NAC treatment effectively prevented ROS formation and preserved endothelial function in vitro. Treatment of bEND.3 cells with NAC effectively attenuated the levels of intracellular ROS (A, B) and maintained the function of bEND.3 cells (C–E) co-cultured with serum exosomes from H. pylori infected male mice. Exo-NC, exosomes from control male mouse serum; Exo-Hp, Exosomes from H. pylori infected male mouse serum; NAC, N-acetylcysteine. Data are shown as mean ± SEM; *P<0.05; **P<0.01; ***P<0.001 by one-way ANOVA. n=8 independent experiments for every measurement. Scale bar (A) = 100 μm.