The Role Of Helicobacter Pylori Outer Membrane Vesicles In The Pathogenesis Of Atherosclerosis

Background：Cardiovascular disease is a major cause of death. Currently, the prevalence is in arising phase in China. Atherosclerosis is a chronic inflammatory disease of multifactoretiology, characterized by accumulation of cholesterol and recruitment of macrophages tothe arterial wall. It is also considered as a metabolic or inflammatory disease. The intactendothelial layer is important to keep the proliferation, migration and apoptosis of vascularsmooth muscle cells in the normal range. Therefore, endothelial injury would be involvedin the development of atherosclerosis.Nowadays, the relationship between infection and coronary heart disease has become ahot research topic. Koren et al reported that infection of bacteria from the oral cavity, andperhaps even the gut, may correlate with atherosclerosis. Association studies have beentaken by epidemiology in recent years. However, the mechanism is still unclear. Previousstudies has shown H. pylori DNA and CagA in atherosclerotic plaques. Due to theimpossibility for bacteria to go into the circulation, question about the H. pylori DNA andCagA in vascular walls is arised. H. pylori is a gram-negative bacteria, which could shedouter membrane vesicles (OMVs). It is reported that OMVs contain abundant molecules,including CagA, VacA, lipopolysaccharide (LPS). OMVs could go into the circulation,and plays some role on tissue or organ at a long distance, we wonder whether the OMVsfrom H. pylori DNA and CagA might take place in the pathogenesis of atherosclerosis. Ourrecent studies found that the components in extracellular vesicles could be transferred fromone cell to another by endocytosis or fusion with the recipient cell, and the transferredcomponents in extracellular vesicles could regulate the microRNA and protein expressionof the recipient cells. Other studies also show that when co-culture of OMVs from H. pyloriwith gastric epithelial cells, the characteristic vacuolation in gastric epithelial cells wasfound at3h, the apoptosis was found at24h. In this study, we investigated the function and potential mechanisms of OMVs from H. pylori on human umbilical vein endothelial cells(HUVECs). We also elucidated the critical role of OMVs from H. pylori in pathogenesis ofatherosclerosis.Methods:1. OMVs from H. pylori were isolated through series of ultra-centrifugation steps. Toidentify the OMVs from H. pylori, electronmicroscopic and immunoblotting analysis wereused.2. We examined the proliferation, migration and apoptosis role of OMVs from H.pylori on HUVECs by CCK8, Transwell, TUNEL staining and flow cytometry.3. To explore the mechanisms leading to apoptosis, we studied the expression ofcaspase-3, cleaved-caspase-3, Bax and Bcl2by western blotting.4. To determine the critical role of OMVs from H. pylori that plays in pathogenesis ofatherosclerosis, we injected OMVs from H. pylori into the ApoE-/-mice. Then, the aortaswere isolated under a dissecting microscope; they were then cut longitudinally and stainedwith Oil Red O. The percentage of the plaque area stained by Oil Red O to the total luminalsurface area was determined.Result:1. The electron micrograph showed the outer membrane vesicles from H. pylori wereround, with size about20-300nm. Western blotting showed the presence of CagA and VacA,commonly used markers for OMVs from H. pylori.2. OMVs from H. pylori inhibited the proliferation of HUVECs in a time-andconcentration-dependent manner.3. OMVs from H. pylori inhibited the migration of HUVECs.4. Flow cytometry and TUNEL staining showed that the OMVs from H. pylori inducedapoptosis in HUVECs, and the percentage of apoptotic cells was higher in OMVs from H.pylori group.5. Cleaved-caspase-3expression was increased after treatment with OMVs from H.pylori. Moreover, OMVs form H. pylori increased the expression of pro-apoptotic proteinBax; decreased the expression of the anti-apoptotic protein Bcl-2.6. After injection with OMVs from H. pylori into the ApoE-/-mice for four weeks, OilRed O staining revealed that OMVs-treated ApoE-/-mice had more atherosclerotic plaque than controls.Conclusion:Our present study shows that OMV from H. pylori inhibits proliferation and migrationof HUVECs, increases its apoptosis, which might be taken in the pathogenesis ofatherosclerosis.