Exosomal CircHIPK3 Released From Cardiomyocytes Pretreated With Hypoxia Regulates Apoptosis And Proliferation Of Cardiac Microvascular Endothelial Cells Under Oxidative Stress

Background:Cardiac microvascular endothelial cells?CMECs?play an obligatory role in regulating and maintaining cardiac function by connecting to each other and constituting continuous endothelium between the circulation and cardiomyocytes?CMs?.Under local oxidative stress after myocardial infarction,cardiac microvascular endothelial cells undergo apoptosis or necrosis,and their proliferation and migration are limited,which severely damages the integrity of microvessels.This leads to insufficient horizontal perfusion of cardiomyocytes after coronary artery recanalization,and promotes the apoptosis or necrosis of cardiomyocytes,which originally survive.Cardiac structure and function appear changes under various pathophysiological effects.This changes eventually lead to cardiac dysfunction and cardiogenic death.In recent years,the paracrine effect plays a pivotal role in improving the local microenvironment of myocardial infarction and repairing infarcted myocardium.Exosomes are the main substances that play a role in the paracrine pathway and have become a key regulator of angiogenesis and cardiac repair.Exosomes are rich in non-coding RNAs,proteins,cytokine nucleic acids and other biologically active substances,which can mediate the exchange of information between cells and cells.The exosomal circRANs has attracted much attention due to its better conservation and stability.Studies have shown that cardiomyocytes can regulate the biological activity of target cells by releasing exosomes?CMs-exosomes?to surrounding contact cells?fibroblasts,endothelial cells,etc.?,but the specific mechanism is unknown.Our previous studies found that exosomes released from cardiomyocytes pretreated with hypoxia can reduce the area of myocardial infarction in mice and increase the density of neovascularization in the infarct border zone by transmitting high expression of circHIPK3.However,the mechanism of hypoxia preconditioning cardiomyocyte-derived exosomal circHIPK3 regulating the apoptosis,proliferation and migration of CMECs under oxidative stress has not been reported.Therefore,based on the previous studies,this study intends to further establish the oxidative stress microenvironment of acute myocardial infarction?AMI?in vitro,and to investigate the regulation and mechanism of hypoxia preconditioning cardiomyocyte-derived exosomal circHIPK3 on apoptosis,proliferation and migration of CMECs under oxidative stress.Part I Effects of exosomes derived from hypoxic pretreatedCMs on oxidative damage of CMECsObjective:To observe the effects of exosomes of cardiomyocytes derived from normoxia and hypoxia on oxidative damage of CMECs.Methods:1.Culture of cardiomyocyte:Mouse CMs were isolated and cultured in vitro by double enzyme digestion combined with differential adherence method and bromodeoxyuridine?BrdU?purification method.Immunofluorescence?IF?was used to identify the expression of cardiac troponin?cTnT?in CMs.2.Establishment of hypoxic preconditioning time of cardiomyocytes:Cell Counting Kit-8?CCK-8?was used to detect the effect of HPC on the activity of CMs at different time?0 h,6 h,12 h,18 h,24 h?.3.Culture of myocardial microvascular endothelial cells:The CMECs required for the experiments were cultured by enzyme digestion combined with differential adherence method.IF was used to identify the cell markers vWF and CD31 of CMECs.4.Extraction and identification of Exosomes:Normal exosomes?Nor-exos?and hypoxic exosomes?HPC-exos?were extracted from normoxic cardiomyocytes and hypoxic pretreated cardiomyocytes by ultracentrifugation?UC?.The expression of CD9,HSP70and Alix on the surface markers of exosomes were detected by Western Blot.The morphology of exosomes was observed by transmission electron microscope?TEM?.Nanoparticle tracking analysis?NTA?was used to analyze the exosomal population characteristics.5.Establishment of oxidative stress model of CMECs in vitro:The optimal concentration of H₂O₂ simulated oxidative stress microenvironment was explored by treating CMECs with different concentrations of H₂O₂?50?M,100?M,200?M,300?M?for 3 h.CMECs were treated with 200?M H₂O₂ for different time?1 h,2 h,3 h,4 h?.CMECs in normoxic state were used as control group.Fluorescence-activated cell sorting?FCM?was used to detect the effects of different concentrations of H₂O₂ and different time on the apoptosis of CMECs.To explore the optimal concentration and time for establishing oxidative stress microenvironment in vitro.6.Internalization verification of Exosomes:Exosomes were labeled with Dil red fluorescent dye and co-cultured with CMECs for different time?4 h,8 h,12 h?.The intake of exosomes of CMECs was observed by IF.Determining the optimal co-culture time as a condition for subsequent exosomes to treat CMECs.FCM was used to detect the effects of exosomes?0,100,200,300 and 400 ug/mL?on the proliferation of CMECs in oxidative stress microenvironment after 8 hours of co-culture with CMECs.7.Antioxidative stress effect of Exosomes:To verify the effects of Nor-exos and HPC-exos on on apoptosis,proliferation and migration of CMECs under oxidative stress,the experiment was divided into five groups:?1?Nor group;?2?H₂O₂ group;?3?exos-depleted group;?4?Nor-exos group;?5?HPC-exos group.Annexin V-FITC/PI double staining was used to detect the externalization of phosphatidylserine?PS?of the inner cell membrane of apoptotic cells.2',7'-dichlorofluorescein diacetate?DCFH-DA?fluorescent probe was used to detect the release of reactive oxygen species?ROS?in cells.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax and Caspase3.The DNA fragmentation was detected by Tunel staining.The cell cycle was detected by FCM,the DNA replication activity and proliferation ability were detected by EdU staining,and the expressions of proliferation-related proteins PCNA,CyclinD1 and P21 were detected by Western Blot.Transwell cell migration assay was used to detect the migration ability of CMECs in vitro.Results:1.The primary CMs were small and round.One day later,some cells were observed to adhere to the wall by microscopy.Two days later,the cells were triangular,spindle,polygon and irregular,and there was spontaneous pulsation of cells.IF identified cTnT as a positive expression.2.CCK-8 results showed that the cell viability of CMs under hypoxic preconditioning for 12 h was significantly up-regulated?P<0.05?.3.CMECs cultured by enzymatic digestion combined with differential adherence method were adhered to the wall for two days.After three-four days,the growth rate was faster and the cytoplasm was stretched.After six days,the cells were irregular,fusiform,with paving stone-like arrangement.IF observed cell surface molecules vWF and CD31were positive.4.The results of Western Blot showed that Nor-exos and HPC-exos surface marker protein CD9,Alix,HSP70 were all positive.TEM showed that the vesicles were round or double concave discs with lipid membranes.NTA results showed that the particle sizes of Nor-exos and HPC-exos were 151 nm.5.The results of FCM showed that the early apoptotic rate of CMECs treated with200?M H₂O₂ for 3 h was significantly up-regulated?P<0.05?,and the necrosis rate had no significant change?P>0.05?.Therefore,200?M H₂O₂ 3 h group was selected as the optimal treatment condition for inducing oxidative stress microenvironment of CMECs.6.After Dil-labeled exosomes and CMECs were co-cultured for 8 h,most of the exosomes were taken up by CMECs by fluorescence microscopy.FCM results showed that the proliferation rate of CMECs treated with 300?g/mL exosomes for 8 h was the highest?P<0.05?compared with 0?g/mL,100?g/mL and 200?g/m L groups.There was no significant difference in cell proliferation rate between the 400?g/ml group and the 300?g/mL group?P>0.05?.Therefore,we adopted 300 ug/mL exosomes and CMECs were co-cultured for 8 hours as the following experimental treatment conditions.7.Compared with the Normal group,H₂O₂ could significantly induced apoptosis of CMECs and inhibited proliferation and migration of CMECs?P<0.05?.Compared with the H₂O₂ group,in the Nor-exos group and HPC-exos group,apoptosis decreased,cell proliferation and migration increased?P<0.05?.Moreover,HPC-exos significantly improve CMECs viability when subjected to oxidative stress,compared to cells pretreated with Nor-exos.Summary:Under oxidative stress,Nor-exos and HPC-exos can inhibit the apoptosis of CMECs,promote the proliferation and migration of CMECs,and play a protective role of CMECs,but the effect of HPC-exos is more significant.Part II HPC-exosomal circHIPK3 regulates apoptosis,proliferation and migration of CMECs under oxidative stressvia miR-29aObjective:To investigate the effects of HPC-exosomal circHIPK3 on the apoptosis,proliferation and migration of CMECs under oxidative stress microenvironment.Methods:1.Relative expression of circHIPK3 in HPC-exosomes:qRT-PCR was used to detect the expression of circHIPK3 in Nor-exos and HPC-exos.Whether the expression of circHIPK3 in cells was changed after treatment of CMECs by Nor-exos and HPC-exos,the experiment was divided into four groups:?1?Nor group;?2?H₂O₂ group;?3?Nor-exos group;?4?HPC-exos group.qRT-PCR was used to detect the expression level of circHIPK3in each group.2.Determination of circHIPK3 multiplicity of infection:Overexpression or silencing of circHIPK3 in CMECs or CMs using lentiviral transfection methods.Experimental groups:CMECs:MOI=10?MOI=50?MOI=100;CMs:MOI=50?MOI=100?MOI=200,qRT-PCR was used to detect the transfection efficiency.3.To verify that HPC-exo exerts anti-cellular oxidative stress effects primarily by delivering circHIPK3,the experiment is divided into eight groups:?1?H₂O₂ group;?2?Lentiviral vector?LV?group;?3?LV-circHIPK3 group;?4?LV-si-circHIPK3 group;?5?HPC-exos group;?6?LV-exos group;?7?LV-si-HIPK3 mRNA-exos group;?8?LV-si-circHIPK3-exos group.qRT-PCR was used to detect the expression of circHIPK3 in CMECs of different treatment groups.Apoptosis was detected by Annexin V-FITC/PI double staining.DCFH-DA fluorescent probe was used to detect the release of intracellular ROS.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax and Caspase3.Tunel staining was used to detect cellular DNA fragmentation.Cell cycle was detected by FCM,DNA replication activity and proliferation ability were detected by EdU staining,and expressions of proliferation-related proteins PCNA,CyclinD1 and P21 were detected by Western Blot.Transwell cell migration assay was used to detect the migration ability of CMECs in vitro.4.Verification of the correlation between circHIPK3 and miR-29a:qRT-PCR was used to detect the expression of circHIPK3 and miR-29a in the H₂O₂ group,LV group,LV-circHIPK3 group and LV-si-circHIPK3 group.The binding of circHIPK3 to miR-29a was verified by luciferase reporter assay and Ago2 immunoprecipitation assay.RNA FISH assay was used to detect the location of circHIPK3 and miR-29a.5.To detect the relative expression of miR-29a in CMECs under oxidative stress,the experiment was divided into two groups:Normal CMECs group?control group?and H₂O₂treated CMECs group?H₂O₂ group?.qRT-PCR was used to detect the expression of miR-29a in CMECs under oxidative stress.6.Verification of the role of miR-29a in oxidative stress microenvironment CMECs:miR-29a and its negative control were transfected with riboFECTTM CP transfection reagent to CMECs under oxidative stress,and the effect of miR-29a on apoptosis of CMECs under oxidative stress was observed.The experiment was divided into five groups:?1?H₂O₂ group;?2?miR-29a mimics group;?3?MNC?mimics negative control?group;?4?miR-29a Inhibitors group;?5?INC?inhibitor negative control?group.Apoptosis was detected by Annexin V-FITC/PI double staining.DCFH-DA fluorescent probe was used to detect the release of intracellular ROS.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax and Caspase3.Tunel staining was used to detect cellular DNA fragmentation.Cell cycle was detected by FCM,DNA replication activity and proliferation ability were detected by EdU staining,and expressions of proliferation-related proteins PCNA,CyclinD1 and P21 were detected by Western Blot.Transwell cell migration assay was used to detect the migration ability of CMECs in vitro.7.HPC-exos mediates the role of miR-29a in regulating apoptosis,proliferation and migration of CMECs.The experiment was divided into six groups:?1?H₂O₂ group;?2?HPC-exos group;?3?HPC-exos+miR-29a mimics group;?4?HPC-exos+MNC group;?5?HPC-exos+miR-29a Inhibitors group;?6?HPC-exos+INC group.Apoptosis was detected by Annexin V-FITC/PI double staining.DCFH-DA fluorescent probe was used to detect the release of intracellular ROS.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax and Caspase3.Tunel staining was used to detect cellular DNA fragmentation.Cell cycle was detected by FCM,DNA replication activity and proliferation ability were detected by EdU staining,and expressions of proliferation-related proteins PCNA,CyclinD1 and P21 were detected by Western Blot.Transwell cell migration assay was used to detect the migration ability of CMECs in vitro.Results:1.The results of qRT-PCR showed that the expression of cricHIPK3 in Hypoxic preconditioning CMs-exosomes was significantly higher than that in normoxic CMs-exosomes?P<0.05?.On the basis of oxidative stress,the expression of circHIPK3 in cells treated with Nor-exos and HPC-exos was also up-regulated?P<0.05?,and the effect of HPC-exos was more obvious?P<0.05?.2.The results of qRT-PCR showed that the transfection efficiency of MOI=50 was the highest when lentivirus was transfected into CMECs.the transfection efficiency of MOI=100 was the highest when lentivirus was transfected into CMs.Therefore,MOI=50was used as the best MOI for transfecting CMECs,and MOI=100 was used as the best MOI for transfecting CMs.3.Compared with the H₂O₂ group,the expression of circHIPK3 in the LV-circHIPK3group and the HPC-exos group was significantly up-regulated?P<0.05?,and the expression of circHIPK3 in the LV-si-circHIPK3 group was significantly down-regulated?P<0.05?.Compared with the HPC-exos group,the expression of circHIPK3 in the LV-si-HIPK3mRNA-exos group had no significant difference?P>0.05?,and the expression of circHIPK3 in the LV-si-circHIPK3-exos group was significantly down-regulated?P<0.05?.Compared with the H₂O₂ group,in LV-circHIPK3 group and HPC-exos group,the externalization of PS was decreased?P<0.05?.The release of intracellular ROS was reduced?P<0.05?.The expression of Bax and Cleaved caspase3 were decreased,and the expression of Bcl-2 was increased?P<0.05?.DNA fragmentation was reduced?P<0.05?.In LV-si-circHIPK3 group,the externalization of PS was increased?P<0.05?,the release of intracellular ROS was increased?P<0.05?,the expression of Bax and Cleaved caspase3were increased,the expression of Bcl-2 was decreased?P<0.05?,and the DNA fragmentation of cells was increased?P<0.05?.Compared with the HPC-exos group,the externalization of PS,the release of intracellular ROS,the expression of Bcl-2,Bax and Cleaved caspase3,and DNA fragmentation were not significantly different in the LV-si-HIPK3 mRNA-exos group,the externalization of PS was decreased?P<0.05?,the release of intracellular ROS was decreased?P<0.05?,the expression of Bax and Cleaved caspase3 were decreased,the expression of Bcl-2 was increased?P<0.05?,and the DNA fragmentation of cells was decreased?P<0.05?.Compared with the H₂O₂ group,in the LV-circHIPK3 group and HPC-exos group,the ratio of G0/G1 phase decreased and the ratio of S+G2/M phase increased?P<0.05?,the cell proliferation ability increased significantly?P<0.05?,the expression of PCNA and Cyclin D1 increased significantly and the expression of P21 decreased significantly?P<0.05?,the number of cell migration increased significantly?P<0.05?.In the LV-si-circHIPK3 group,the ratio of G0/G1 phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and CyclinD1 decreased significantly and the expression of P21 increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.Compared with the HPC-exos group,the ratio of G0/G1 phase and S+G2/M phase,the cell proliferation ability,the expression of PCNA,CyclinD1 and P21,the number of cell migration were not significantly different in the LV-si-HIPK3 mRNA-exos group?P>0.05?.In the LV-si-circHIPK3-exos group,the ratio of G0/G1 phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and CyclinD1 decreased significantly and the expression of P21increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.4.Compared with the H₂O₂ group,in LV-circHIPK3 group,the relative expression of circHIPK3 was significantly up-regulated?P<0.05?,in LV-si-circHIPK3,the relative expression of circHIPK3 was significantly down-regulated?P<0.05?.However,the relative expression of miR-29a did not change significantly?P>0.05?.The luciferase reporter assay showed that circHIPK3 binds to miR-29a.The results of RNA FISH showed that circHIPK3 and miR-29a co-localize to the cytoplasm of cells.Ago2 co-precipitation assay showed that circHIPK3,miR-29a and Ago2 can form a ternary complex.5.The results of qRT-PCR showed that the relative expression of miR-29a in H₂O₂group was significantly higher than that in Control group?P<0.05?.6.Compared with the H₂O₂ group,in miR-29a mimics group,the externalization of PS was increased?P<0.05?,the release of intracellular ROS was increased?P<0.05?,the expression of Bax and Cleaved caspase3 were increased,the expression of Bcl-2 was decreased?P<0.05?,and the DNA fragmentation of cells was increased?P<0.05?.In the miR-29a Inhibitors group,the externalization of PS was decreased?P<0.05?,the release of intracellular ROS was decreased?P<0.05?,the expression of Bax and Cleaved caspase3were decreased,the expression of Bcl-2 was increased?P<0.05?,and the DNA fragmentation of cells was decreased?P<0.05?.Compared with the H₂O₂ group,in the miR-29a mimics group,the ratio of G0/G1phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and CyclinD1 decreased significantly and the expression of P21 increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.In the miR-29a Inhibitors group,the ratio of G0/G1 phase decreased and the ratio of S+G2/M phase increased?P<0.05?,the cell proliferation ability increased significantly?P<0.05?,the expression of PCNA and CyclinD1 increased significantly and the expression of P21 decreased significantly?P<0.05?,the number of cell migration increased significantly?P<0.05?.7.Compared with the H₂O₂ group,in the HPC-exos group,the externalization of PS was decreased?P<0.05?,the release of intracellular ROS was decreased?P<0.05?,the expression of Bax and Cleaved caspase3 were decreased,the expression of Bcl-2 was increased?P<0.05?,and the DNA fragmentation of cells was decreased?P<0.05?.Compared with the HPC-exos group,in HPC-exos+mimics group,the externalization of PS was increased?P<0.05?,the release of intracellular ROS was increased?P<0.05?,the expression of Bax and Cleaved caspase3 were increased,the expression of Bcl-2 was decreased?P<0.05?,and the DNA fragmentation of cells was increased?P<0.05?.In the HPC-exos+Inhibitors group,the externalization of PS was decreased?P<0.05?,the release of intracellular ROS was decreased?P<0.05?,the expression of Bax and Cleaved caspase3were decreased,the expression of Bcl-2 was increased?P<0.05?,and the DNA fragmentation of cells was decreased?P<0.05?.Compared with the H₂O₂ group,in the HPC-exos group,the ratio of G0/G1 phase decreased and the ratio of S+G2/M phase increased?P<0.05?,the cell proliferation ability increased significantly?P<0.05?,the expression of PCNA,CyclinD1 increased significantly and the expression of P21 decreased significantly?P<0.05?,the number of cell migration increased significantly?P<0.05?.Compared with the HPC-exos group,in HPC-exos+mimics group,the ratio of G0/G1 phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and CyclinD1 decreased significantly and the expression of P21increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.In the HPC-exos+Inhibitors group,the ratio of G0/G1 phase decreased and the ratio of S+G2/M phase increased?P<0.05?,the cell proliferation ability increased significantly?P<0.05?,the expression of PCNA,CyclinD1 increased significantly and the expression of P21 decreased significantly?P<0.05?,the number of cell migration increased significantly?P<0.05?.Summary:HPC-exosomal circHIPK3 can inhibit the apoptosis of oxidative damaged CMECs and promote their proliferation and migration by regulating the role of microRNA-29a.Part III HPC-exosomal circHIPK3 regulates IGF-1 and VEGFA through miR-29a to regulate apoptosis?proliferation and migration of CMECs under oxidative stressObjective:To investigate the targeting of HPC-exosomal circHIPK3 to inhibit miR-29a in CMECs,promote the expression of downstream IGF-1 and VEGFA,and regulate the apoptosis,proliferation and migration of CMECs under oxidative stress microenvironment.Methods:1.Verification of the correlation between miR-29a and IGF-1:Luciferase reporter assay was used to verify the binding of miR-29a to IGF-1.Transfected miR-29a and its negative control with riboFECT^TM CP transfection reagent to CMECs under oxidative stress.The experiment is divided into five groups:?1?H₂O₂ group;?2?miR-29a mimics group;?3?MNC group;?4?miR-29a Inhibitors group;?5?INC group.qRT-PCR was used to detect the relative expression of miR-29a and IGF-1 in each group.Western Blot was used to detect the expression level of IGF-1.2.Verification of the correlation between miR-29a and VEGFA:Luciferase reporter assay was used to verify the binding of miR-29a to VEGFA.Transfected miR-29a and its negative control with riboFECT^TM CP transfection reagent to CMECs under oxidative stress.The experiment is divided into five groups:?1?H₂O₂ group;?2?miR-29a mimics group;?3?MNC group;?4?miR-29a Inhibitors group;?5?INC group.qRT-PCR was used to detect the relative expression of VEGFA in each group.Western Blot was used to detect the protein expression level of VEGFA in each group.3.HPC-exos regulates the effects of miR-29a on apoptosis,proliferation and migration of CMECs under oxidative stress by transferring circHIPK3.The experiment is divided into six groups:?1?HPC-exos group;?2?si-circ-exos group;?3?si-circ-exos+mimics group;?4?si-circ-exos+MNC group;?5?si-circ-exos+Inhibitors group;?6?si-circ-exos+INC group.Apoptosis was detected by Annexin V-FITC/PI double staining.DCFH-DA fluorescent probe was used to detect the release of intracellular ROS.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax,Caspase3 and IGF-1.Tunel staining was used to detect cellular DNA fragmentation.Cell cycle was measured by FCM.EdU staining was used to detect cellular DNA replication activity and proliferation ability.Western Blot was used to detect the expression of proliferation-related proteins PCNA,CyclinD1,P21 and VEGFA.Transwell cell migration assay was used to detect the migration ability of CMECs in vitro.Results:1.The results of Luciferase Report assay showed that there were binding sites between miR-29a and IGF-1.Compared with the H₂O₂ group,the expression levels of miR-29a and IGF-1 in the MNC group and the INC group did not change significantly.In the miR-29a mimics group,the expression of miR-29a was increased significantly and the expression of IGF-1 was decreased significantly?P<0.05?.In the miR-29a Inhibitors group,the expression of miR-29a was decreased significantly and the expression of IGF-1was increased significantly?P<0.05?.Compared with the H₂O₂ group,the expression of IGF-1 was significantly down-regulated in miR-29a mimics group?P<0.05?,the expression of IGF-1 was significantly up-regulated in the miR-29a Inhibitors group?P<0.05?.2.The results of Luciferase Report assay showed that there were binding sites between miR-29a and VEGFA.Compared with the H₂O₂ group,the expression levels of VEGFA in the MNC group and the INC group did not change significantly.In the miR-29a mimics group,the expression of VEGFA was decreased significantly?P<0.05?.In the miR-29a Inhibitors group,the expression of VEGFA was increased significantly?P<0.05?.Compared with the H₂O₂ group,the expression of VEGFA was significantly down-regulated in miR-29a mimics group?P<0.05?,the expression of VEGFA was significantly up-regulated in the miR-29a Inhibitors group?P<0.05?.3.Compared with the HPC-exos group,in the si-circ-exos group,the externalization of PS was increased?P<0.05?,the release of intracellular ROS was increased?P<0.05?,the expression of Bax and Cleaved caspase3 were increased,the expression of Bcl-2 and IGF-1 were decreased?P<0.05?,and the DNA fragmentation of cells was increased?P<0.05?.Compared with the si-circ-exos group,in si-circ-exos+mimics group,the externalization of PS was increased?P<0.05?,the release of intracellular ROS was increased?P<0.05?,the expression of Bax and Cleaved caspase3 were increased,the expression of Bcl-2 and IGF-1 were decreased?P<0.05?,and the DNA fragmentation of cells was increased?P<0.05?.In the si-circ-exos+Inhibitors group,the externalization of PS was decreased?P<0.05?,the release of intracellular ROS was decreased?P<0.05?,the expression of Bax and Cleaved caspase3 were decreased,the expression of Bcl-2 and IGF-1 were increased?P<0.05?,and the DNA fragmentation of cells was decreased?P<0.05?.Compared with the HPC-exos group,in the si-circ-exos group,the ratio of G0/G1phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and Cyclin D1 decreased significantly and the expression of P21 increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.Compared with the si-circ-exos group,in si-circ-exos+mimics group,the ratio of G0/G1 phase increased and the ratio of S+G2/M phase decreased?P<0.05?,the cell proliferation ability decreased significantly?P<0.05?,the expression of PCNA and Cyclin D1 decreased significantly and the expression of P21increased significantly?P<0.05?,the number of cell migration decreased significantly?P<0.05?.In the si-circ-exos+Inhibitors group,the ratio of G0/G1 phase decreased and the ratio of S+G2/M phase increased?P<0.05?,the cell proliferation ability increased significantly?P<0.05?,the expression of PCNA,Cyclin D1 and VEGFA increased significantly and the expression of P21 decreased significantly?P<0.05?,the number of cell migration increased significantly?P<0.05?.Summary:In the oxidative stress microenvironment,HPC-exosomal circHIPK3 targeting inhibits miR-29a to regulate the expression of IGF-1 and VEGFA,thereby inhibiting the apoptosis of CMECs and promoting the proliferation and migration of CMECs.Conclusion:1.Hypoxic preconditioning of cardiomyocytes can release protective exosomes to inhibit apoptosis and promote proliferation and migration of myocardial microvascular endothelial cells after oxidative damage.2.The potential mechanism of hypoxic preconditioning of cardiomyocyte-derived exosomes may play a protective role by transmitting circHIPK3,competitively binding to miR-29a,and regulating the expression of IGF-1 and VEGFA.