| Research background: Cardiac microvascular endothelial cells(CMECs)Play an indisPensable role in regulating and maintaining cardiac function.When the Pressure is overloaded,the myocardial cells become hypertrophic and the cardiac interstitial dePosits increase,which result in cardiac hypertrophy.Whereas,in the hypertrophic myocardial tissue,the microvascular content is relatively insufficient,which leads to the relative ischemia and hypoxia in these tissues,and continued disease progression will lead to heart failure.Studies have found that many biologically active substances and regulatory pathways play an important role in improving the local microenvironment of the myocardium,and exosome mediated paracrine effects have received much attention in recent studies,and exosome are regarded as key regulators of myocardial repair and microvascular regeneration.Exosome contains and Package many biologically active substances,including Proteins,liPids,carbohydrates,Polysaccharides,and cell metabolites,mRNA,miRNA,and ribosomal RNA.Among them,miRNAs are a highly conserved non-coding RNA.It has attracted much attention in recent years.Relevant studies shows that exosome are involved in the Process of cardiac hypertrophy,which transfers miRNAs to adjacent cells and Plays an imPortant role in the communication between cells.Earlier research in this topic found that exosome released by cardiomyocytes which is pretreated by angiotensin II(AngⅡ)can aggravate myocardial hypertrophyin mice after exacerbation of transverse aortic constriction and reduce the density of new blood vessels in hypertrophic myocardial tissues by transmitting high expression miR-29 a.At present,there are no reports about the mechanism of AngⅡ Preconditioning cardiomyocyte-derived exosome miR-29 a regulating CMECs Proliferation,migration and angiogenesis.Therefore,based on the Previous research,this study intends to further establish a stress overload microenvironment in vitro and exPlore the regulatory effect of AngⅡ on the Proliferation,migration and angiogenesis of CMECs derived from cardiomyocyte exosome miR-29 a and its related mechanisms.Part I Study on the effect of CMs-exosome miR-29 a treated byAngⅡ on endothelial cell Proliferation and migration andangiogenesis Objective: To observe the effects of CMs-exosome miR-29 a treated by AngⅡ on endothelial cell Proliferation and migration and angiogenesis.Method:1.Suckling rats Primary myocardial cell culture : using Pancreatin + type II collagenase combined digestion method,combined with differential adherence method,and adding 5-bromodeoxyuridine(5-BrdU)to the medium to inhibit other cells,so as to purify the cultured neonatal rat cardiomyocytes in vitro,and immunofluorescence(IF)was used to identify the cardiomyocytes we cultured,and the specific marker molecule cardiac troponin T(cTnT)expression.2.Subculture,cryopreservation,resuscitation and identification of CMECs: The myocardial microvascular endothelial cell line used in this experiment was purchased from Shanghai Sixin Biotechnology Co.,Ltd.,and was subcultured,and its specific marker molecule CD31 expression was identified by IF.3.Myocardial cells treated with AngⅡ: Myocardial cells were treated with different concentrations of angiotensin II for 48 hours,and Western Blot was used to detect the expression of central necrosis Protein: ANP,BNP,myh6,myh7 and so on.The cytoskeleton was labeled with phalloidin,and the cell sizes of the Nor group and the AngⅡ treated group were observed under a inverted fluorescence microscope.4.Extraction and identification of exosome: collect normal cardiomyocytes and AngⅡ-pretreated cardiomyocytes cell culture fluids,labeled,and then Ultracentrifugation(UC)was used to extract normal myocardial cells exosome(Normal exosome(Nor-exo))and AngⅡ treated myocardial cells exosome(AngiotensinII exosome,AngⅡ-exo).Western Blot was used to detect the expression of exosome-sPecific surface marker Protein molecules CD9,CD63,Alix,and HSP70.Nanoparticle tracking analysis(NTA)is used for analysis of the population characteristics of the extracted exosome.Transmission electron microscope(TEM)was used to observe the morphology of our extracted exosome.5.Validation of Exosome internalization: Exosome(300 μg / mL)were labeled with red fluorescent dye Dil and co-cultured with myocardial microvascular endothelial cells for 8 hours,and the uptake of exosome by endothelial cells was observed by IF.6.Exosome’ effect on endothelial cell Proliferation,migration and angiogenesis: To verify the effects of Nor-exo and Ang Ⅱ-exo on the Proliferation,migration and angiogenesis of CMECs,the exPeriment was divided into 3 groups:(1)Normal(Nor)group;(2)Nor-exo group;(3)AngⅡ-exo group;Nor group endothelial cells only added comPlete culture medium,Nor-exo group added 300 μ g / mL normal myocardial exosome to endothelial cell culture medium,and AngⅡ-exo group added 300 μg / mL of endothelial cell culture medium Cardiac cell-derived exosome treated with AngⅡ.FCM was used to detect the effect of AngⅡ-exo on the cell cycle of endothelial cells,and EdU staining was used to detect its effect on the proliferation of endothelial cells;Western Blot was used to detect its Proliferation-related Proteins: PCNA,VE-cadherin,ZO-1,etc.Transwell cell migration assay was used to detect its effect on the migration ability of CMECs in vitro;basement membrane assay was used to detect its effect on the ability of endothelial cells to form tubes.7.Relative expression of miR-29 a in Nor-exo and AngⅡ-exo: qRT-PCR was used to detect the expression of miR-29 a in each group.8.Effect verification of miR-29 a on CMECs: Transfect miR-29 a mimics,inhibitors and their negative controls with endobo cells using riboFECT ? CP transfection reagent,and the effects of miR-29 a on the Proliferation,migration and angiogenesis of CMECs were observed.The Experimental groups were:(1)(1)Nor group;(2)miR-29 a mimics group;(3)MNC(mimics negative control)group;(2)(1)Nor group;(2)inhibitors group;(3)INC(inhibitor negative control)group.FCM was used to detect the effect of miR-29 a on the cell cycle of endothelial cells,and Western Blot was used to detect its effect on the expression of Proliferation-related Proteins: PCNA,VE-cadherin,ZO-1,etc.;Transwell cell migration assay was used to detect its in vitro migration to CMECs.EdU staining was used to detect its effect on the proliferation of endothelial cells.Basement membrane experiments were used to test its effect on endothelial cell tube forming ability.9.Validate that CMs-exos mainly inhibits the Proliferation,migration and angiogenesis of myocardial microvascular endothelial cells by transmitting miR-29 a.Firstly we transfect miR-29 a and its negative control to CMs with riboFECT ? CP transfection reagent and collect and extract the culture fluid and extract the exosome for subsequent experiments.The negative control of miR-29 a to CMECs was divided into 4 groups:(1)Nor group;(2)AngⅡ-exo group;(3)AngⅡ-exo + miR29 a inhibitor group;(4)AngⅡ-exo + INC(inhibitor negative control)group;qRT-PCR was used to detect miR-29 a expression in CMECs of different treatment groups.FCM was used to detect the cell cycle in each group,and EdU staining was used to detect the DNA replication activity and Proliferation ability of each group of cells.Western Blot was used to detect the expression of Proliferation-related Proteins PCNA,ZO-1,and VE in each group.Transwell cell migration assay was used to detect the migration caPacity of CMECs in each group in vitro;the basement membrane test was used to detect the angiogenesis ability of CMECs in each group.Result:1.The Primary neonatal rat cardiomyocytes are small and round.After 24 hours of culture,some cells have been observed to adhere to the wall.After 48 hours of culture,the cardiomyocytes grow in triangles,Polygons,and irregular shapes,and there is voluntary Pulsation.Immunofluorescence identified the Positive expression of cTnT in our cultured cardiomyocytes.2.The myocardial microvascular endothelial cell lines we Purchased were exchanged and Passaged.The cells in normal logarithmic Phase were irregular,sPindle-shaPed,etc.,and arranged in Paving stones.Immunofluorescence identified it and found that its sPecific surface molecule CD31 was Positively expressed.3.After treating cardiomyocytes with different concentrations of AngⅡ for 48 hours,the expression levels of central senescent Proteins: ANP,BNP,myh7,etc.in each group increased comPared with the control group,and increased along with the increasing of AngⅡ concentration,the expression of myh6 decreased.At the same time,there is no significant difference between 10-6mmol / L and 10-5mmol / L,so the subsequent experiments used Ang Ⅱ concentration of 10-6mmol / L,that is,1umol / L,as the Processing conditions.The cytoskeleton was labeled with Phalloidin,and the surface area of cells in the AngⅡ-treated group was significantly increased comPared with the Nor group under a fluorescent microscoPe(P <0.05).4.Nor-exo and AngⅡ-exo were extracted by ultracentrifugation resPectively.Western Blot results showed that the surface marker Proteins CD9,Alix,CD63,and HSP70 of our extracted exosome were Positively expressed;they were observed to have different sizes under TEM,which is saucer typed,oval or biconcave disk-shaped vesicle bodies covered with liPid membranes;NTA results showed that the Peak diameters and average values of the two samples were in the range of 30-150 nm,and the Particle sizes were uniform.It was 109.4 nm,indicating that the vesicle-like substance extracted from us is exosomes.5.After co-culturing Dil-labeled exosome(300 μg / mL)with CMECs for 8 hours,the fluorescence microscoPe observation revealed that most of the exosome were taken uP by them.6.ComPared with Nor group and Nor-exo group,the Proliferation,migration and angiogenesis of endothelial cells in AngⅡ-exo group were reduced(P <0.05).7.As detected by qRT-PCR,comPared with Nor and Nor-exo,the expression of miR-29 a in the AngⅡ-exo group was significantly increased(P <0.05).8.Combining the results of Previous research by our research group,when transfecting myocardial cells with lentivirus,the transfection efficiency of MOI = 100 is the highest.When transfecting myocardial microvascular endothelial cells,the transfection efficiency is the highest when MOI = 50.Therefore,in this exPeriment,MOI = 50 was selected as the best MOI value for transfection of myocardial microvascular endothelial cells,and MOI = 100 was used as the best MOI value for transfection of myocardial cells.ComPared with the control group and MNC,endothelial cell Proliferation,migration and angiogenesis was reduced in miR-29 a mimics group(P <0.05),while inhibitors group showed the oPPosite trend(P <0.05).9.The expression of miR-29 a in each group was detected by qRT-PCR.The results showed that,comPared with the Nor group,the expression of miR-29 a increased in the AngⅡ-exo group and the AngⅡ-exo + INC group(P <0.05).The expression level was the lowest in the AngⅡ-exo + inhibitors group(P <0.05).ComPared with other groups,the AngⅡ-exo group inhibited the Proliferation,migration and angiogenesis of endothelial cells(P <0.05),while the AngⅡ-exo + inhibitors group reversed the treatment of Ang Ⅱ-exo Reduced Proliferation,migration and angiogenesis of endothelial cells(P <0.05).Summary:1.Ang Ⅱ-Pretreated cardiomyocyte-derived exosome can inhibit the Proliferation,migration and angiogenesis of CMECs.2.MiR-29 a was highly expressed in Ang Ⅱ-Pretreated cardiomyocyte-derived exosome.3.miR-29 a can inhibit the Proliferation,migration and angiogenesis of CMECs.Part II AngⅡ-exosome inhibits VEGFA,regulates theProliferation,migration and angiogenesis of CMECs bytransmitting miR-29 a Objective: To investigate the effects of AngⅡ-exosome miR-29 a on the Proliferation,migration and angiogenesis of CMECs is achieved by regulating VEGFA.Method:1.Validation of the correlation between miR-29 a and VEGFA: First,use luciferase report experiments to verify whether miR-29 a can be combined with VEGFA;then use riboFECT ? CP transfection reagent to transfect miR-29 a mimics,inhibitors and its negative control to CMECs.Experimental grouping:(1)Nor group;(2)miR-29 a mimics group;(3)miR-29 a inhibitor group.Western Blot and qRT-PCR were used to detect the expression of VEGFA in each group.2.Verification of VEGFA transfection: Lentiviral transfection was used to overexpress or silence VEGFA in CMECs.The Experimental groups were:(1)(1)Normal(Nor)group;(2)Lentiviral vector(LV)group;(3)LV-VEGFA group;(2)(1)Normal(Nor)group;(2)Lentiviral vector(LV)group;(3)LV-VEGFA group.The fluorescence intensity after transfection was observed under a fluorescence microscope.QRT-PCR and Western Blot were used to detect the expression of VEGFA Protein and mRNA in CMECs of different treatment groups.3.Verification of MiR-29 a and VEGFA interaction: Experimental grouping:(1)Nor group;(2)miR-29 a mimics group;(3)miR-29 a mimics + LV-VEGFA group;Western Blot and qRT-PCR were used to detect differences in VEGFA Protein and mRNA expression levels;Transwell cell migration experiments were used to detect differences in CMECs in vitro migration ability,FCM was used to detect cell cycle differences in each group,and basement membrane experiments were used to detect CMECs angiogenesis ability in each group.4.AngII-treated cardiomyocyte-derived exosomes inhibit VEGFA expression by transmitting miR-29 a and the inhibitory effect on endothelial cell Proliferation,migration,and angiogenesis were verified.The Experimental groups were:(1)Nor group;(2)AngⅡ-exo group;exo + LV-VEGFA group;(4)AngⅡ-exo + LV-VEGFA + miR-29 a mimics group,using Western Blot,qRT-PCR to detect differences in VEGFA Protein and mRNA expression levels in each group;basement membrane test to detect angiogenesis caPacity of CMECs in each group.The transwell cell migration exPeriment was used to detect the differences in the migration ability of CMECs in each group,and the FCM was used to detect the cell cycle differences in each group.Result:1.The results of the luciferase rePort exPeriment showed that miR-29 a has a binding site with VEGFA.Western Blot and qRT-PCR results showed that when miR-29 a expression was increased,VEGFA expression was inhibited,while(P <0.05),and when miR-29 a expression was decreased,it showed the oPPosite trend(P <0.05).2.Western Blot and qRT-PCR results showed that comPared with the blank control group and the emPty vector group,the expression of VEGFA in the LV-VEGFA group increased(P <0.05),while the VEGFA-inhibited group showed the oPPosite trend,which Proved transfection success.3.ComPared with the blank control group,the expression of VEGFA in the AngⅡ-exo and miR-29 a mimics groups was significantly reduced(P <0.05),and the Proliferation and migration and angiogenesis of endothelial cells were inhibited(P <0.05);In the case of VEGFA,miR-29 a mimics also showed an inhibitory effect(P <0.05).Summary:AngⅡ treated cardiomyocyte-derived exosome inhibited the expression of VEGFA by transmitting miR-29 a,thereby inhibiting the Proliferation,migration and angiogenesis of CMECs.Conclusion:1.Angiotensin II treated cardiomyocytes can release cardiac hypertrophyexosome,in additional,exosomes can inhibit the proliferation,migration and angiogenesis of myocardial microvascular endothelial cells in vitro.2.The underlying mechanism of angiotensin II-treated cardiomyocyte-derived exosomes in inhibiting the proliferation,migration,and angiogenesis of myocardial microvascular endothelial cells may play a role by inhibiting the expression of VEGFA through the delivery of miR-29 a. |
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