Bone Marrow Mesenchymal Stem Cell Exosome MiR-638/Sp1 Regulate EMT Signal Pathway To Inhibit The Proliferative Phenotypic Switch Of Vascular Smooth Muscle Cells In Aortic Dissection

Background Aortic dissection(AD)is a fatal macrovascular disease.The death rate of untreated aortic dissection is as high as 90%.The treatment methods of AD mainly include drug therapy,interventional therapy(endovascular repair therapy),and surgical treatment.Although these treatments have made great progress,the complications such as the high difficulty of surgery and the influence of multiple organ function after surgery cannot be completely avoided.AD is still a serious life-threatening disease,and intervention is needed as soon as possible after clinical diagnosis.At present,the treatment of AD,especially the treatment of Stanford type A aortic dissection,is still a difficult problem in the field of surgical treatment.In order to explore better treatment methods,it is essential to clarify the pathogenesis of AD from the molecular level for the gene targeted therapy in the future.Vascular smooth muscle cells(VSMC)exist in two phenotypes: proliferative and contractive.They change from proliferative and synthetic phenotypes in embryonic tissues to static and contractive phenotypes in adult blood vessels.In healthy blood vessels,most VSMC present contractive phenotype.Due to the continuous stimulation of pathological damage(hypertension stimulation,hypoxia,inflammation),most VSMC in contractive state can be converted into proliferative phenotype.Some literature reports show that the hyperproliferative phenotypic switch of VSMC is considered as one of the landmark pathological changes of AD.Therefore,the study of VSMC phenotypic switch may reveal the pathogenesis of AD.Exosomes participate in the physiological and pathological mechanism of cardiovascular diseases.As an important signal molecular carrier for information transmission between cells,they contain RNA(miRNA,circ RNA,linc RNA),proteins,lipids and other biological active molecules,which play a regulatory role in related signal pathways.Among them,miRNA carried by exosomes is the hotspot of current researches.More and more studies show that miRNA plays an important role in cell growth,differentiation,metabolism,apoptosis and other pathophysiological processes,and miRNA plays a significant role in regulating gene expression.Some studies have shown that some exosomes produced by miRNA transfection into stem cells partially inhibit cell proliferation by down-regulating the expression of thioredoxin interaction protein(TXNIP)gene.After the exosomes are taken up by VSMC,they can regulate cell proliferation and migration,leading to endothelial inflammation and atherosclerosis.Therefore,by increasing the exosome level of exosomes or combining exosomes into VSMC through exogenous gene modification,it may help to inhibit its excessive proliferation and reduce the incidence and progression of aortic dissection.Mesenchymal stem cell(MSC)exosome has been the research focus of clinical gene targeted therapy because of its easy availability,multi-directional differentiation and strong self-renewal ability.Many studies have reported that MSC has many biological functions such as regulating inflammatory reaction and immunosuppression.Recent studies have shown that the therapeutic effect of MSC mainly depends on its paracrine function,and the substances secreted by MSC mainly include exosomes,which is used to play a therapeutic role.To sum up,we propose a scientific hypothesis that MSC exosomes may inhibit the proliferation and migration of VSMC and inhibit the over-proliferative phenotypic switch of VSMC to achieve the therapeutic effect on AD.This experiment is intended to establish a cell model of VSMC hyperproliferative phenotypic switch in the aorta,explore the effect of MSC exosome on VSMC hyperproliferative phenotypic switch,proliferation and migration,and preliminarily explore its mechanism of inhibiting VSMC hyperproliferative phenotypic switch,inhibiting abnormal proliferation and migration,and further inhibiting vascular proliferation in the middle layer,It lays a theoretical foundation for the further development and application of the clinical treatment method of MSC exosome inhibiting the proliferation of vascular mesothelial cells.Objective Over-proliferative phenotypic switch of VSMC in the middle layer of aorta is an important factor in the occurrence of aortic dissection,and its mechanism has not been fully clarified.The phenotypic switch of VSMC is one of the important factors which affect cell proliferation and migration.Exosomes have been reported to inhibit VSMC proliferation,inhibit the expression of adhesion molecules and oxidative stress,and maintain normal lipid metabolism.However,there are few reports on the proliferative phenotypic switch of VSMC in the middle layer of aorta.This experiment is to study the effect of miR-638/Sp1 axis on VSMC proliferative phenotypic switch in aortic dissection,and explore whether MSC exosome has therapeutic effect on VSMC proliferative phenotypic switch.Through the use of MSC exosome,it can inhibit VSMC hyperproliferative phenotype conversion and inhibit the proliferation of arterial middle layer cells,thus achieving the early diagnosis of aortic dissection,helping the treatment of aortic dissection,and even preventing the occurrence of aortic dissection to a certain extent.Method1.According to gene sequencing and bioinformatics analysis,screen differentially expressed miRNAs,and determine the target miRNA as miR-638.The database screen target genes(Sp1)with binding sites with miR-638.Collect the tissue samples of ascending aorta of AD and non-AD,extract the RNA,and carry out PCR and Western blot identification to verify the differential expression of miR-638 and Sp1.2.Angiotensin II(Ang II)was used to establish a cell model of VSMC proliferative phenotypic switch.Ang Ⅱ can promote the proliferative phenotypic switch,cell proliferation and migration of VSMC.Set the concentration gradient as0.1umol/L,0.5umol/L,1.0umol/L,1.5umol/L and 2.0umol/L respectively,and set the time gradient as 6h,12 h,18h,24 h and 36 h respectively.After treating VSMC with Ang Ⅱ of different concentration/time,use PCR,Western Blot,CCK-8,Transwell,scratch test,Ed U test and other methods to detect the phenotypic switch,proliferation and migration of VSMC,Determine the optimal concentration and time for Ang Ⅱ to promote VSMC hyperproliferative phenotypic switch,and establish a cell model of VSMC in the follow-up study.3.To explore the effect of MSC exosome on VSMC phenotypic switch.In this study,MSC exosome were co-cultured with VSMC,and the effects of MSC exosome on VSMC hyperproliferative phenotypic switch,proliferation and migration were verified by PCR,Western Blot and Transwell methods.The exosome in the supernatant of MSC were collected by differential centrifugation after ultrahigh speed centrifugation,and the characteristics of the exosome were verified by light microscopy and Western blot.In the cell model in vitro,the isolated VSMC was treated with MSC exosome,and the proliferative phenotypic switch,proliferation and migration of VSMC were detected by PCR,Western Blot,CCK-8 and Transwell methods,to verify the inhibitory effect of MSC exosome on the proliferative phenotypic switch and proliferation and migration of VSMC in aortic dissection.4.To explore the effect of miR-638/Sp1 regulate EMT signal pathway on VSMC proliferative phenotypic switch.Select miR-638 as the target miRNA through gene chip analysis.Using RNA transfection technology(lipo3000),in vitro cell model,VSMC was transfected with miR-638 plasmid and RNA interferon to detect the changes in the expression of miR-638 and Sp1.At the same time,the remediation experiment was carried out by transfecting Sp1 plasmid and interferon.The proliferation phenotypic switch of VSMC was detected by q PCR,Western Blot and other methods,and the proliferation and migration of cells were detected by CCK-8method,Ed U method,Transwell method and scratch test.At the same time,the binding site of miR-638 and Sp1 was further verified by double luciferase assay.5.It was confirmed that the exosome of MSC contained miR-638,and its effect on VSMC proliferative phenotypic switch was verified.Extract miRNA from MSC exosome,and detect miR-638 in the exosome by PCR and other methods.MSC was transfected with miR-638 plasmid overexpression and RNA interferon,and the treated stem cell exosome were treated with VSMC cell model in vitro.The proliferative phenotypic switch of VSMC was detected by q PCR,Western Blot and other methods.The proliferation and migration of VSMC were detected by CCK-8,Ed U,Transwell and scratch test.6.The proliferative phenotypic switch and cell proliferation of VSMC were detected by constructing animal models,subcutaneous injection of Ang II to induce proliferative phenotypic switch model,injection of MSC exosome through tail vein,HE staining,immunohistochemistry,PCR,Western blot and other methods to further verify the experimental results in vitro.Results1.The ascending aorta tissue removed from patients with aortic dissection(non hereditary)during operation was taken as the experimental group,and the aorta tissue removed from patients with coronary artery bypass surgery during ascending aortic anastomosis was taken as the control group for RNA extraction.The results of PCR and Western blot showed that the expression of miR-638 in aortic dissection was decreased,while the expression of Sp1 was increased.2.The induction of VSMC by 1 umol/L Ang II for 24 hours can promote the proliferation and migration of VSMC to the maximum extent,and promote the switch of VSMC proliferative phenotype.The results are stable and can be used as the induction conditions for subsequent detection and molecular biological experiments.3.The effect of MSC exosome on VSMC phenotypic switch.MSC exosomes were collected by differential centrifugation,and the existence of exosomes was confirmed by electron microscopy and Western blot.In the in vitro cell model induced by Ang II,MSC exosome were added to co-culture.Through the detection of VSMC proliferative and functional phenotypic conversion markers,it was found that with the addition of exosome,the level of SMA,MYH11,SM22,CAL markers in the cell model increased.Combined with the results of CCK-8,Transwell,and scratch test,the cell proliferation and migration ability decreased,indicating that the proliferative ability and hyperproliferative phenotypic conversion of VSMC were inhibited.4.The effect of miR-638/Sp1 regulate EMT signal pathway on VSMC phenotypic switch.In both tissue and blood exosome samples,the expression of miR-638 is decreased.In the Ang II-induced cell model in vitro,miR-638 in VSMC was overexpressed and knocked down by RNA interference technology.It was found that the level of SMA,MYH11,SM22 and CAL markers in the cell model decreased with the decrease of miR-638 expression;When miR-638 is overexpressed,the levels of SMA,MYH11,SM22,and CAL markers increase.Combined with the results of CCK-8,Transwell,and scratch test,the results show that when miR-638 is overexpressed,the cell proliferation and migration ability is enhanced,and the cell proliferative phenotypic switch is more significant,indicating that the proliferative ability and proliferative phenotypic switch of VSMC are promoted.The above phenomenon can be reversed by the remedy of low expression of Sp1.Similarly,when miR-638 is overexpressed,The proliferative phenotypic switch of VSMC was inhibited,and the result could be remedied by the reversal of Sp1 overexpression,which proved that miR-638 had an inhibitory effect on the proliferation and proliferative phenotypic switch of VSMC,and that miR-638 could combine with Sp1.Therefore,miR-638/Sp1 has an inhibitory effect on VSMC proliferation and proliferative phenotype transformation by regulating the EMT signaling pathway.5.Extract miRNA from MSC exosome,and detect miR-638 in exosome by PCR and other methods.After co-culturing the MSC exosome transfected with miR-638 plasmid and RNA interferon with the cell model,it was found that when co-culturing the MSC exosome with high expression of miR-638,the levels of SMA,MYH11,SM22 and CAL markers in the cell model increased.When co-culturing the MSC exosome with low expression of miR-638,the levels of markers in the cell model did not change significantly.Combined with the results of CCK-8,Transwell and scratch test,It shows that MSC exosomes play a role in VSMC’s proliferative ability and proliferative phenotypic switch through its miR-638.6.Through animal experiments,combined with the results of immunohistochemistry,PCR,Western blot and so on,while in the same animal model injected with Ang II,in the animals injected with MSC exosome,the proliferative phenotypic switch of VSMC was significantly inhibited,and the levels of SMA,MYH11,SM22,CAL markers were increased,which further confirmed the inhibitory effect of MSC exosome miR-638 on the proliferative phenotypic switch and cell proliferation and migration of VSMC treated with Ang II.Conclusion1.In patients with aortic dissection,the VSMC in the middle layer of ascending aorta underwent hyperproliferative phenotypic switch.2.Ang II induced VSMC at 1 umol/L for 24 hours,which was the most reasonable condition to simulate VSMC proliferative phenotype conversion cell model in ascending aorta of patients with aortic dissection.3.The miR-638 secreted by MSC exosome can inhibit VSMC hyperproliferative phenotypic switch and inhibit VSMC proliferation and migration.4.MiR-638/Sp1 can regulate EMT signal pathway to inhibit or even reverse the hyperproliferative phenotypic switch and hyperproliferative migration of VSMC in aortic dissection.5.The exosome of MSC contains miR-638,which is absorbed by VSMC and inhibits VSMC hyperproliferative phenotypic switch and hyperproliferative migration through miR-638/Sp1 axis.6.In vivo experiments confirmed that the MSC exosome miR-638 had an inhibitory effect on the proliferative phenotypic switch of aortic VSMC.In conclusion,the expression of miR-638 in VSMC of aortic dissection aortic wall is decreased,and the combination with Sp1 is reduced,which increases the proliferative phenotype conversion ability of VSMC,proliferation and migration ability.The exosome of MSC miR-638 inhibits the proliferative phenotype conversion of VSMC through miR-638/Sp1 axis regulation,and reduces the proliferative and migration ability of VSMC.This study provides a new theoretical basis for the molecular pathway and pathogenesis of miRNA in the pathogenesis of aortic dissection.More importantly,it also provides new ideas and research directions for the early diagnosis of aortic dissection and even the target of drug treatment.