Change Of Plasma Exosomes Mi Rna In Type 2 Diabetic Atherosclerosis Patients And Repair Effect Of Endothelial Progenitor Cells-derived Exosomes On Endothelial Cells Of Type 2diabetic Atherosclerosis Mice

Background:Diabetes,especially type 2 diabetes,has become a worldwide public health problem.Diabetic large vascular complications are not only become the main causes of disability,death in patients with type 2 diabetes,but also brought huge economic and social burden to the world.Therefore,it is very important to explore the mechanism of the development of atherosclerosis in type 2 diabetes.At present,the vascular endothelial cell damage caused by inflammatory reaction and oxidative stress is the initial stage of atherosclerosis,after that the vascular endothelial barrier function is impaired.Vascular endothelial cells are a semi-selective permeable barrier that covers the surface of the endovascular cavity,which is composed of single-layer continuous cells and plays a role of vascular barrier.Endothelial progenitor cells are progenitor cells of vascular endothelial cells.Under different stress and stimulation conditions,endothelial progenitor cells are mobilized to repair damaged blood vessels in peripheral blood and secrete a variety of substances to promote the formation of blood vessels.Exosome is a lipid bilayer membrane-derived vesicle that is secreted by various cells,and also one of the important substances secreted by the endothelial progenitor cells.Exosome contains mRNA,miRNA,various kinds of proteins,and participate in important physiological processes such as cell communication,tumor cell growth and redevelopment of blood vessels.In recent years,the relationship between diabetes and exosome has attracted wide attention both in the domestic and overseas.Some experts predict that exosomes can not only be used as biomarkers for the development of diabetes,but also as a target for diabetes treatment.However,there are few studies on the effect of exosomes on atherosclerosis of diabetes.Objectives:1.To observe the change of plasma exosomes miRNA in atherosclerosis（AS）patients complicating with type 2 diabetes mellitus（T2DM）;2.To investigate the influence of the normal mice endothelial progenitor cells-derived exosomes（EPC-Exos）on the expression levels of apoptosis and proliferation of vascular endothelial cells in type 2 diabetic AS mice.3.To explore the influence of normal mice EPC-Exos on the endothelium-dependent vasodilatation function of thoracic aorta in type 2 diabetic AS mice.Methods:1.The plasma of type 2 diabetic AS and normal control patients was collected.The plasma exosomes RNA was extracted and detected by second-generation sequencing technology.Then the difference of expression of plasma exosomes miRNA in two groups was compared.2.The db/db mice were fed with high fat food to establish the type 2 diabetic AS mice model while the C57 mice with basic food as the control group.After 14 weeks feeding,every group was randomly separated into two groups including EPC-Exos intervention group and PBS control group.Four groups including AS+Exos group,AS+PBS group,CON+Exos group and CON+PBS group were used in our experiments.Mice were killed after two-week intervention.Vascular endothelial mark proteins including CD31,Bax,Caspase-3 and Mac-2 in different groups were compared by immunohistochemistry.The expression levels of proliferating cell nuclear antigen（PCNA）were also compared by immunofluorescence.Moreover,the response of tension in the thoracic aortic rings to vasoconstrictor and vasodilator were compared between different groups.Results:1.MicroRNA sequencing:Compared with the NC group,the expression of plasma exosomes miRNA in type 2 diabetic AS group was reduced.There are 7 miRNAs involved in the AS pathways.2.Type 2 diabetic AS mice model:The blood glucose of all AS group mice was in the diabetes standard while that of all CON group was in the normal range.HE staining showed there was no obvious formation of atherosclerotic plaques in the arterial endothelial of CON+PBS and CON+Exos group,while AS+PBS group mice had marked atherosclerotic plaque formation,which meant the successful construction of the type 2 diabetic AS mice model.More importantly,atherosclerotic plaques in the arterial endothelial of AS+Exos group decreased markedly.3.The influence of EPC-Exos on proliferation and apoptosis of the endothelial cells of thoracic aorta in AS mice.3.1 The influence of EPC-Exos on the expressions of caspase-3 and Bax of thoracic aorta endothelial cells in AS mice.The expression levels of caspase-3 and Bax of vascular endothelial cells in CON+PBS and CON+Exos groups were low.Compared with the CON+PBS group,the expression levels of caspase-3 and Bax of AS+PBS group were significant increased.However,compared with the AS+PBS group,the expressions of caspase-3and Bax of AS+Exos group were significantly decreased.3.2 The influence of EPC-Exos on the expressions of Galectin-3 of thoracic aorta endothelial cells in AS mice.The expression levels of Galectin-3 of vascular endothelial cells in CON+PBS and CON+Exos groups were low.Compared with the CON+PBS group,the expression of Galectin-3 of AS+PBS group was significantly increased.The expression of Galectin-3of AS+Exos group was obviously reduced compared with the AS+PBS group.3.3 The influence of EPC-Exos on the expressions of CD31 of vascular endothelial cells.The expression of CD31 of vascular endothelial cells in CON+PBS and CON+Exos groups were visible.Compared with the CON+PBS group,the expression of CD31 of vascular endothelial cells of AS+PBS group was obviously decreased.However,compared with the AS+PBS group,the expression of CD31 of AS+Exos group was markedly increased.3.4 The influence of EPC-Exos on the expressions of PCNA of vascular endothelial cellsCompared with the CON+PBS group,the expression of PCNA of vascular endothelial cells of AS+PBS group had an obvious reduction.However,compared with the AS+PBS group,the expression of PNCA of AS+Exos group was remarkably increased.3.5The influence of EPC-Exos on the thoracic aorta vasoconstriction of AS mice3.5.1 The influence of EPC-Exos on vascular contraction n by 60 mM KCLCompared with the CON+PBS group,vascular contraction of the AS+PBS group weakened obviously（P=0.012）.However,compared with the AS+PBS group,vascular contraction of the AS+Exos group was significantly enhanced,resuming to normal level of control group（P<0.001）.3.5.2 The influence of EPC-Exos on the vasodilation by acetyl-choline（ACh）The vasodilation effect of AS+PBS group caused by ACh was significantly weaker than that of the other three groups.Moreover,vasodilation effect of the AS+Exosgroup was stronger than that of AS+PBS group at the 6 concentration points among 10^-4.5-10^-3mmol/L,whereas there was no significance difference between CON+PBSgroup and CON+Exos group.3.5.3 The influence of EPCs on the vasodilation by sodium nitroprusside（SNP）Exogenous nitric oxide provided by SNP could dilate the vascular smooth muscle cell which could lead to the vasodilation of vessels.To investigate the change of vasodilation of the vascular smooth muscle cells,thoracic aorta from each group was extracted to measure the tension.It was shown that SNP could dilate the vascular smooth muscle cells and cause the endothelium-independent vasodilation.There was no statistic difference in SNP vasodilation function between the four groups.Conclusions:1.The expression of plasma exosomes miRNA of type 2 diabetic AS patient is reduced.There are 7 miRNA involved in the AS pathway.2.EPC-Exos could inhibit cell apoptosis and promote the proliferation of vascular endothelial cells in type 2 diabetic AS mice.3.EPC-Exos could improve endothelium-dependent vasodilation of thoracic aorta in AS mice.