The Effect Of Cytotoxin-Associated Gene A~+ Helicobacter Pylori Infection On Endothelial Dysfunction

Background and Objective:Helicobacter pylori(H.pylori)is well recognized as the primary pathogen involved in peptic ulcer,chronic gastritis,and gastric cancer.Recent data indicate that H.pylori infection,especially Cytotoxin-associated gene A positive H.pylori(Cag A~+H.pylori)infection,could also contribute to important extragastrointestinal diseases,such as cardiovascular diseases(including atheroselerosis and coronary heart disease),neurological disorders,hematological diseases(especially idiopathic thrombocytopenia)and metabolic disorders.However,studies show inconsistent results regarding the relationship between H.pylori infection and cardiovascular diseases and how H.pylori infection could lead to atherosclerosis is unknown at this point.Endothelial cells play a key role in maintaining the structural and functional integrity of vasculature,and endothelial dysfunction is considered the initial event leading to the development of atherosclerosis.Endothelial dysfunction is believed to play a critical role on the mechanism of atherosclerosis pathogenesis.Studies shown that H.pylori itself and Cag A protein,a virulence factor secreted by H.pylori,could induce the production of Reactive oxygen species(ROS)in cells,resulting in excessive accumulation of ROS and oxidative stress response,ultimately lead to cell apoptosis and DNA damage.The increasing ROS cause the inflammatory response and apoptosis of vascular endothelial cells,and affect the vasomotor and antithrombotic effects of endothelial cells,and then affect the elastic function and structure of blood vessels.Oxidative stress is considered to be the main cause of endothelial dysfunction.Exosomes play an important role in cell-to-cell communications through transport of a wide spectrum of bioactive constituents.Recent study shown that Cag A existed in serum-derived exosomes from patients with Cag A~+H.pylori infection,and in vitro experiments confirmed that exosomes carrying Cag A could internalize into gastric epithelial cells and induce morphological changes.Exosomes are known to be critically involved in cell-to-cell communication and cell functions through various mechanisms including regulation of extra-and intracellular redox states via direct and/or indirect modification of ROS content.This suggests that Cag A~+H.pylori virulence factor Cag A protein is most likely to enter the blood circulation through exosomes,a carrier secreted by gastric epithelial cells,and internalizes into endothelial cells to stimulate the oxidative stress response of blood vessels,and then leads to abnormal structure and function of vascular endothelial cells,eventually leads to the occurrence and development of cardiovascular diseases such as atherosclerosis.The present study was designed to test the hypothesis that H.pylori infection,especially Cag A~+H.pylori infection impaires endothelial function through exosome-mediated ROS formation.It will provide new ideas and new targets for clinical prevention and treatment of H.pylori infection related cardiovascular diseases.Methods:1.The relationship between Cag A~+H.pylori infection and vascular endothelial function:A total of 30 patients with H.pylori infection from the department of Gastroenterology,Xiangya Third Hospital of Central South University and 30 subjects without H.pylori infection were recruited.The Flow-mediated vasodilatation(FMD)of brachial artery was measured by Doppler ultrasonography in all subjects,and the vascular endothelial function of the two groups was compared.The endothelial function of aorta in C57BL/6 mice infected with Cag A~+H.pylori or Cag A~-H.pylori were detected.In order to further clarify the relationship between H.pylori infection and endothelial dysfunction,patients with H.pylori infection and infected mice were treated with anti-H.pylori therapy,and vascular endothelial function was tested again after H.pylori eradication.2.Mechanism of endothelial dysfunction induced by Cag A~+H.pylori infection:(1)The role of oxidative stress in endothelial dysfunction induced by Cag A~+H.pylori infection:Firstly,vascular ROS of Cag A~+H.pylori and Cag A~-H.pylori infected mice were detected.Western Blot was used to detect the expression of antioxidant enzymes,including Catalase and Glutathione peroxidase-1(Gpx-1)and Superoxide dismutase-1(SOD-1),in the vascular of Cag A~+H.pylori infected mice.To further clarify the mechanism of ROS involvement in vascular endothelial dysfunction caused by Cag A~+H.pylori infection,we used antioxidant transgenic mouse model(overexpression of SOD-1,SOD-3 and Gpx-1)and antioxidant N-acetylcysteine(NAC)to treat C57BL/6 mouse model and and then detected endothelial function of the two Cag A~+H.pylori infected mice model,respectively.(2)The role of exosomes in endothelial dysfunction induced by Cag A~+H.pylori infection:Firstly,exosomes from Cag A~+H.pylori infected patients,mice and human gastric epithelial cell line(GES-1)were isolated by ultracentrifugation and identified by Transmission electron microscope(TEM),particle size analyzer and Western Blot.Cag A protein was detected in exosomes by Western Blot.Then,vascular endothelial cell lines and C57BL/6 mice were used to detect whether exosomes from Cag A~+H.pylori infection(Exo-Cag A~+H.pylori)could internalize into vascular endothelial cells in vitro and in vivo,and further detected endothelial function of cell lines and mice with the stimulation of Exo-Cag A~+H.pylori.Finally,to further clarify the role of exosomes in endothelial dysfunction induced by Cag A~+H.pylori infection,we treated mouse models with exosomes inhibitor GW4869 and then detected the changes in endothelial function in mice.(3)Cag A~+H.pylori infection impairs endothelial function through exosomes-mediated ROS formation:Firstly,to confirm that exosomes can stimulate the formation of vascular ROS,vascular endothelial cell lines and C57BL/6 mice were used to detect the ROS formation under the stimulation of Exo-Cag A~+H.pylori in vitro and in vivo.Then in order to confirm that exosomes lead to endothelial dysfunction by increasing ROS formation,vascular endothelial cell lines and C57BL/6 mice were treated with antioxidant NAC to inhibit ROS formation and then detected the endothelial function of cell lines and mice under the stimulation of Exo-Cag A~+H.pylori.Finally,in order to clarify the mechanism of the endothelial dysfunction caused by Cag A~+H.pylori infection through exosomes-mediated ROS formation,the mouse model was treated with exosomes inhibitor GW4869 and then detected the changes of ROS expression and vascular endothelial function of mice after inhibition of exosomes production and release.Results:1.The relationship between Cag A~+H.pylori infection and vascular endothelial function:We recruited 30 young patients with H.pylori infection and without known risk factors for endothelial dysfunction to evaluate endothelium-dependent FMD of the brachial artery.A group of30 young healthy volunteers served as control.No significant difference in age,gender,hypertension,hyperlipidemia,blood glucose and smoking history was present between the infected-patients and control subjects.Patients with H.pylori infection exhibited a significant reduction in FMD compared with controls.Cag A~+H.pylori colonized gastric mucosa more effectively than Cag A~-H.pylori in mice.Ex vivo Acetylcholine(ACh)-induced endothelium dependent relaxation of aortic rings was significantly decreased in mice with Cag A~+H.pylori infection at both 1week and 12 weeks post-infection compared with controls.Endothelium-independent relaxation to Nitroglycerin(NTG)was unchanged between the groups.In contrast,no significant changes in ACh-induced relaxation or NTG-induced relaxation were observed in mice with Cag A~-H.pylori infection compared with controls.When patients with H.pylori infection were treated with anti-H.pylori therapy,their endothelium-dependent FMD of brachial artery was effectively restored.H.pylori elimination in mice with anti-H.pylori therapy also significantly improved Ach-induced vasorelaxation without change in NTG-induced relaxation.2.Mechanism of endothelial dysfunction induced by Cag A~+H.pylori infection(1)The role of oxidative stress in endothelial dysfunction induced by Cag A~+H.pylori infection:Cag A~+H.pylori infection significantly increased aortic ROS production compared with their controls.In contrast,Cag A~-H.pylori infection had no significant effect on ROS levels compared with controls.NAC treatment effectively blocked excessive ROS production in aortic segments and preserved ACh-induced relaxation in mice after 1 or 12 weeks of Cag A~+H.pylori infection,without differences in NTG induced relaxation.Cag A~+H.pylori infection significantly decreased the protein levels of SOD-1 and Gpx-1 in aortas from C57BL/6 mice 1-week post-infection as compared to controls,while no significant difference was observed in the protein level of catalase in the same aortic extracts.To determine if decreased expression of SOD-1and Gpx-1 was indeed responsible for the increase of aortic ROS production in mice with Cag A~+H.pylori infection,a transgenic(TG)mouse model on C57BL/6 background that concomitantly overexpresses an antioxidant enzymes network(AON)composed of human SOD-1,SOD-3,and Gpx-1 was used.Overexpression of antioxidant enzymes effectively prevented Cag A~+H.pylori infection-induced ROS production in the aorta of mice and preserved ACh-induced endothelium-dependent aortic relaxation without changes in NTG-induced relaxation.(2)The role of exosomes in vascular endothelial dysfunction induced by Cag A~+H.pylori infection:Serum exosomes were prepared from human or mice with Cag A~+H.pylori and Cag A~-H.pylori infection.Exosomes from conditioned medium of GES-1 cultured with Cag A~+H.pylori and with Cag A~-H.pylori were prepared.Exosomes were characterized using TEM,particle size analysis,and Western Blot.Western blot analysis demonstrated that exosomes from human serum,mice serum and GES-1 infected with Cag A~+H.pylori contained the unique Cag A protein,whereas exosomes from those infected with Cag A~-H.pylori had no Cag A protein.We also observed that a detectable amount of PKH67-labeled(green)exosomes were present in HUVEC using 3D confocal microscopy at 12 hours after incubation with the labeled exosomes.A significant amount of PKH26-labeled(red)exosomes were observed in aortic en face endothelium of C57BL/6 mice after tail injection.Treatment with serum exosomes from human and mice infected with Cag A~+H.pylori(Exo-serum-Cag A~+H.pylori)and CM exosomes from GES-1 infected with Cag A~+H.pylori(Exo-CM-Cag A~+H.pylori)significantly inhibited the function of endothelial cells in vitro with decreases in migration,tube formation and proliferation compared with control exosomes.Culture with serum exosomes from mice and CM exosomes from GES-1 infected with Cag A~-H.pylori had no significant effect on endothelial function.Intravenous administration of Exo-CM-Cag A~+H.pylori also suppressed ACh-induced endothelium-dependent relaxation of mice,without effect on NTG-induced endothelium-independent relaxation.Inhibition of exosome secretion with GW4869 effectively preserved endothelial function in mice with Cag A~+H.pylori infection.(3)Cag A~+H.pylori infection impairs endothelial function through exosomes-mediated ROS formation:Serum exosomes from mice or CM exosomes from GES-1 infected with Cag A~+H.pylori,not Cag A~-H.pylori infection,significantly increased intracellular ROS formation in endothelial cells compared to exosomes from non-infected controls.Intravenous administration of Exo-CM-Cag A~+H.pylori also significantly increased aortic ROS levels of mice.NAC treatment decreased intracellular ROS production,and preserved function of endothelial cell in the presence of Exo-Cag A~+H.pylori.Treatment with GW4869significantly reduced serum exosomes levels,prevented excessive aortic ROS production and preserved ACh-induced relaxation in mice with Cag A~+H.pylori infection at both 1 week and 12 weeks post-infection.Conclusion:(1)Cag A~+H.pylori infection induced endothelial dysfunction;Cag A~-H.pylori infection had no significant effect on endothelial function.(2)Oxidative stress and exosomes play an important role of endothelial dysfunction induced by Cag A~+H.pylori.(3)Cag A~+H.pylori infection impaired endothelial function through exosomes-mediated ROS formation.