Protective Mechanism Of Heat Shock Protein 22 On Endothelial Progenitor Cell Injury Induced By High Fat

Part I(in vitro): HSP22 has a protective effect on EPCs induced by high lipid environment Objective:1.It was confirmed that hyperlipidemia promoted the apoptosis of bone marrow derived EPCs.2.It was confirmed that HSP22 had a protective effect on EPCs induced by high fat environment,and the protective mechanism was verified as follows: under high fat environment,HSP22 activated PI3K/Akt signaling pathway through the CXCL12/CXCR4 axis to mediate EPCs proliferation and angiogenesis.Methods:1.Three hyperlipidemia models of mice with different genotypes were established: HSP22+/+,HSP22-/-and WT mice aged 7-8 weeks were fed with high fat for 6 weeks(i.e.,the high-fat feeding group),and HSP22+/+,HSP22-/-and WT mice aged 7-8 weeks were fed with ordinary feeding for 6 weeks(i.e.,the ordinary feeding group).2.Extraction and primary culture of EPCs: Mice were sacrificed after cervical vertebra removal,Mononuclear cells(MNC)were extracted from bone marrow of mice by differential centrifugation method,and primary culture was performed.3.Flow cytometry was used to detect the proportion of EPCs: bone marrow mononuclear cell populations(high-fat feeding group and ordinary feeding group)of the three mice with different genotypes were extracted,and the surface markers of cells in the endothelial group were detected by flow cytometry: Vascular Growth Factor Receptor 2 and Stem cell surface antigen(SCAL-1 +)were both positive cells,namely EPCs,and the proportion of bone marrow-derived EPCs in high fat environment was compared(%).4.Mononuclear cells from bone marrow of three kinds of mice with different genotypes that have been constructed with high fat model were extracted for primary culture.They were divided into HSP22+/+ control group,PI3 K inhibition group of HSP22+/+,HSP22-/-control group,PI3 K inhibition group of HSP22-/-,WT control group and PI3 K inhibition group of WT by stimulation with or without PI3 K inhibitor(Trade name:LY294002).Flow cytometry was used to compare the apoptosis rate of EPCs in each group,Tube formation ability of EPCs in each group,and Western blot was used to compare the expression of Akt,p-Akt,SDF-1 and CXCR-4 in each group.Results:1.High lipid induces a decrease in the number of bone marrow-derived endothelial progenitor cells(EPCs).2.HSP22 promotes the formation of new tubules of EPCs in high lipid environment.3.HSP22 protects against EPCs induced by high lipid environment through activation of PI3K/Akt signaling pathway.4.HSP22 increased the expression of p-Akt,SDF-1,and CXCR-4 proteins of EPCs under high lipid environment,but the expression of Akt was not different.Conclusion:1.Hyperlipidemia can promote the apoptosis of bone marrow derived EPCs.2.In high lipid environment,HSP22 activates PI3K/Akt signaling pathway through the CXCL12/CXCR4 axis to mediate EPCs proliferation and angiogenesis.Part 2(in vivo): HSP22 increases survival of transplanted EPCs in a high-fat environmentObjective:1.Hsp22-/-knockout mice and Hsp22 +/+ overexpressed mice were constructed to clarify the therapeutic effect of transplantation of overexpressed Hsp22 EPCs in myocardial infarction.2.It was demonstrated that HSP22+/+ EPCs cell transplantation improved cardiac function in mice with myocardial infarction.3.It was confirmed that Hsp22 +/+ EPCs cell transplantation reduced the apoptosis of myocardial cells in mice with myocardial infarction.4.It was demonstrated that Hsp22 +/+ EPCs cell transplantation increased neovascularization density in myocardial infarction mice.Methods:1.Establishment of high fat mouse model: WT mice aged 7-8 weeks were fed with high fat for 6 weeks to make a high fat model.2.Establishment of mouse myocardial infarction model: The anterior descending artery of WT mice was ligated,and the ECG showed clear ST-segment elevation,that is,the mouse myocardial infarction model was successfully made..3.Extraction of EPCs: Three genotypes of mice were sacrificed after cervical vertebra removal,Mononuclear cells(MNC)were extracted from bone marrow of mice by differential centrifugation,and resuspended with culture medium.4.Magnetic bead-sorting mouse bone-marrow derived SCAL-1 + cells: marrow derived mononuclear cells of the three extracted mice with different genotypes were sorted by the magnetic bead-sorting system,and then resuspended with MEC buffer solution to make cell suspension with a concentration of 5×105/ m L.100 ul EPC suspension was injected into WT mice with myocardial infarction through the tail vein.5.After surgery,mice were fed normally for 4 weeks.WT mice transplanted with EPC suspension from three different genotypes of mice were examined by echocardiography,including left ventricular end-systolic diameter(LVESD),left ventricular end-diastolic diameter(LVEDD),left ventricular ejection fraction(EF),and left ventricular short-axis shorting rate(FS%).6.After the echocardiography was completed,the mice were killed,and the hearts of the mice were taken and made into paraffin sections.The myocardial infarction area of the mice was detected by Masson staining,cell apoptosis was detected by Tunnel staining,and the density of new blood vessels was detected by IB4 staining.Results:1.HSP22 improved cardiac function in high-fat WT mice after myocardial infarction.2.HSP22 reduced MI area in post-myocardial infarction WT mice with high fat.3.HSP22 reduced myocardial apoptosis in high-fat WT mice after myocardial infarction.4.HSP22 can induce angiogenesis in high-fat WT mice after myocardial infarction.Conclusion: 1.HSP22 improved cardiac function in high-fat WT mice after myocardial infarction.2.The overexpression of HSP22 endothelial progenitor cells improved cardiac function,reduced myocardial apoptosis,decreased myocardial infarction area and increased neovascularization density in mice with myocardial infarction.