The Effect And Mechanism Of Exosomes Derived From Mesenchymal Stem Cells On Neointimal Formation After Vascular Injury

Objective: Intravascular restenosis is the pathological process of stenosis and obstruction of the lumen again after vascular endothelial trauma after percutaneous coronary intervention.Although the incidence of restenosis has been reduced from the initial 10%-30% to the current 5% with the emergence of drug stents and drug balloons,the prognosis of patients with coronary heart disease has been greatly improved,and the treatment of coronary heart disease has undergone a landmark change.However,no breakthrough progress has been made in the prevention and treatment of restenosis,especially in diabetes and complex lesions such as small blood vessels,diffuse lesions,bifurcation lesions,and the restenosis rate reaches more than 20%.In addition,drug-eluting stents or balloons cannot be used to treat all neointimal plaque formation.Therefore,solving intravascular restenosis has become an important and urgent task.Mesenchymal stem cells(MSC)are a kind of pluripotent stem cells.They can be obtained from a large number of connective tissues,such as bone marrow,fat and umbilical cord blood.They have the characteristics of self-renewal and can differentiate into osteoblasts,adipocyte cells,nerve cells and other types of cells.MSC multipotent differentiation and the characteristic easy culture and expansion have attracted much attention in MSC therapy research.As a bridge for transferring functional substances between adjacent cells,exosomes have received more and more attention in the microenvironment of cell-cell interaction.Exosomes,as membrane-enclosed vesicles secreted by MSC,not only have the characteristic of MSC,but also greatly reduce the risk of MSC transplantation,such as immune rejection,teratogenic tumors and pulmonary embolism,and are more stable,easy to store,low immunogenicity and low risk.As a result,exosomes derived from mesenchymal stem cells(MSC-Exo)are increasing in research related to repairing damaged organs and immune regulation.MSC derived from bone marrow have been shown to inhibit neointimal formation by promoting the re-endothelialization of damaged blood vessels.Human MSC have therapeutic effects by reducing inflammation.MSC-Exo has been shown to mediate cell-to-cell communication between cells and its target cells,and mi R-125 b derived from MSC-Exo inhibits neointimal formation after vascular injury.However,the effect and exact mechanism of MSC-Exo on neointimal formation after vascular injury have not yet been fully elaborated.In order to analyze the effect and mechanism of MSC-Exo on neointimal formation after vascular injury,MSC-Exo was introduced in this study to analyze the effect and mechanism of MSC-Exo on proliferation and migration of endothelial cells(EC)in vitro.The rat carotid artery injury model is established to understand whether exogenous injection of MSC-Exo can directionally aggregate to the vascular injury area and target homing after vascular injury,and how it affects the neointimal formation after vascular injury and its possible mechanism of action.Methods: 1.Extraction and biological characteristics of MSC-Exo MSC are isolated from rat bone marrow and identified by microscopic observation and flow cytometry to analyze cell surface antigens.Exosomes are isolated by using exosome purification kit from MSC culture supernatant and analyzed by using transmission electron microscopy.The biological characteristics of extracted exosomes are identified by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and western blot.PKH67-labeled exosomes are co-cultured with EC,and the uptake of exosomes is detected by confocal electron microscope.2.The effect and mechanism of MSC-Exo on the proliferation and migration of EC In order to detect the effect of MSC-Exo on the proliferation of EC,EC are divided into control group,MSC culture supernatant group(MSC-CM),EC culture supernatant group(EC-CM),MSC-Exo group and MSC culture supernatant group without MSC-Exo(CM-Exo-free).Cell immunofluorescence is used to detect their effects on proliferation of EC.By diluting MSC-Exo to 0?g/m L,0.1?g/m L,1?g/m L,10?g/m L and 100?g/m L co-cultivation with EC,cell counting kit-8(CCK8)is used to detect the effects of proliferation of EC.The effect of MSC-Exo on the expression of proliferation-related proteins: proliferating cell nuclear antigen(PCNA)and cyclin D1(Cyclin D1)is detected by western blot.In order to detect the effect of MSC-Exo on migration of EC,EC are divided into control group and MSC-Exo group,and the effect of MSC-Exo on migration of EC is detected by cell scratch.The effect of MSC-Exo on the expression of migration-related proteins: vimentin,matrix metalloproteinase 2(matrix metalloproteinase,MMP2)and matrix metalloproteinase 9(matrix metalloproteinase,MMP9)is detected by western blot.In order to further analyze the effect of Erk1/2 signaling pathway on the proliferation and migration of EC,EC were divided into MSC-Exo group,MSC-Exo + DMSO group and MSC-Exo + SCH772984(Erk1/2 inhibitor)group,and the proliferation and migration of EC are detected by CCK8,cell immunofluorescence and cell scratch.The expression of PCNA,Cyclin D1,Vimentin,MMP2,MMP9,extracellular regulated protein kinases 1/2(Erk1/2)and phosphorylated extracellular regulated protein kinases 1/2(P-Erk1/2)in EC is detected by western blot.3.The effect and mechanism of MSC-Exo on neointimal formation after vascular injury By establishing a balloon-induced rat carotid artery injury model,rats are divided into sham group,normal saline group and MSC-Exo group.MSC-Exo or normal saline are injected into the tail vein,while the sham group is not treated.The injection cycle is for 2 weeks or 4 weeks.Evans blue staining is used to detect the effect of MSC-Exo on re-endothelialization of injured blood vessels.Hematoxylin-eosin(H&E)staining and Masson staining are used to detect the effect of MSC-Exo on neointimal formation after vascular injury.Immunohistochemistry and immunofluorescence are used to detect the effect of MSC-Exo on the expression of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31),von willebrand factor(v WF)and alpha-smooth muscle actin(?-SMA).Results: 1.Biological characteristics of MSC-Exo The expression of CD29,CD90 and CD11 b is analyzed by flow cytometry.The results show that the positive rate of CD29 is 99.6%,the positive rate of CD90 is 99.8%,while the positive rate of CD11 b is 0.17%.Therefore,it is determined that the extracted cells are MSC.It is found that the size of the extracted exosomes was between 30-150 nm,mainly concentrated around 110 nm by NTA.The shape is elliptical and disc by TEM.Western blot results show that CD81,CD63,tumor susceptibility gene 101(TSG101)and heat shock proteins 70(HSP70)are expressed in the extracted exosomes,but no expression of calnexin.The results show that the extraction of MSC-Exo is successful.PKH67 labeled MSC-Exo appear around the nucleus of EC by the analysis of exosome uptake experiments,indicating that MSC-Exo could be taken up by EC.2.MSC-Exo promote the proliferation and migration of EC CCK8 results show that MSC culture supernatant could promote the proliferation of EC,and time-dependent increase,while EC treated by EC supernatant is almost the same as control group,and has no effect on the proliferation of EC.Between 0-100?g/ m L,MSC-Exo could promote the proliferation of EC,and MSC-Exo has the best effect on promoting the proliferation at a concentration of 10?g/m L.Compared with the control group,MSC-Exo could promote the proliferation of EC,while the supernatant of MSC cultured without exosomes has almost no difference with the control group,and has no effect on the proliferation of EC.Western blot results show that MSC-Exo could up-regulate the expression of PCNA and Cyclin D1.At the same time,immunofluorescence shows that MSC-Exo increase the positive rate of Ki67 in EC.Cell scratch results show that MSC-Exo promote the migration of EC.Further analysis of the expression of migration-related proteins,western blot results show that MSC-Exo significantly up-regulated the expression of Vimentin,MMP2 and MMP9.3.MSC-Exo activate the Erk1/2 signaling pathway Western blot results show that MSC-Exo could significantly up-regulate the expression of P-Erk1/2 and down-regulate the expression of Erk1/2.Therefore,MSC-Exo activate the Erk1/2 signaling pathway.In addition,western blot results show that SCH772984 could block the Erk1/2 signaling pathway.CCK8 results show that the addition of SCH772984 significantly inhibits the proliferation and migration of MSC-Exo on EC compared with MSC-Exo and MSC-Exo + DMSO groups.Immunofluorescence results also show that the increased Ki67 positive rate of MSC-Exo is reversed by SCH772984.Western blot results show that the addition of SCH772984 could significantly down-regulate the expression of PCNA,Cyclin D1,MMP2,MMP9 and Vimentin compared with the MSC-Exo and MSC-Exo + DMSO groups.4.MSC-Exo inhibit the neointimal formation after vascular injury Evans blue staining results show that MSC-Exo could accelerate re-endothelialization of injured vascular compared with saline group after two-week continuous injection.After four-week continuous injection,there is almost no difference on the endothelialization rate between the MSC-Exo group and the saline group.H&E staining results show that the saline treatment group significantly increases the ratio of neointimal to media(I/M),neointimal area,media area and collagen area compared with the sham operation group.The ratio of I/M,neointimal area,media area and collagen area are larger after four-week treatment.MSC-Exo could greatly reduce the neointimal hyperplasia,such as: I/M,neointimal area,media area and collagen area,and the effect is better after four-weeks treatment.Immunohistochemical results show that compared with the sham operation group,the saline treatment group significantly increases the expression of ?-SMA,down-regulating the expression of CD31 and v WF,and the expression of ?-SMA is significantly down-regulated,while the expression CD31 and of v WF is significantly up-regulated after MSC-Exo treatment.Immunofluorescence results show that the fluorescence intensity of CD31 and v WF in the saline group is significantly reduced,but after MSC-Exo treatment,the fluorescence intensity of CD31 and v WF is greatly enhanced compared with the sham operation group,indicating that MSC-Exo could significantly increase the expression of CD31 and v WF.Conclusion: In vitro experiments have shown that MSC-Exo could promote the proliferation and migration of EC by activating the Erk1/2 signaling pathway,and up-regulate the expression of proliferation and migration-related proteins;in vivo experiments show that MSC-Exo could promote the re-endothelialization of injured blood vessels and up-regulate the expression of CD31 and v WF,down-regulate the expression of ?-SMA,inhibiting the neointimal formation.In summary,MSC-Exo could inhibit the neointimal formation by activating Erk1/2 signaling pathway after vascular injury.