Effect And Role Of Cardiac Mesenchymal Cell-Derived Exosomes On Ischemic Cardiovascular Models

Part I Isolation and identification of Sca-1+ cardiac mesenchymal cell-derived ExosomesObjective: To isolate,purify and identify the exosomes from Sca-1+cardiac mesenchymal cells.Methods: Sca-1+ cardiac mesenchymal cells(CMCs)of 2 to 3 months old C57BL/6 mice were isolated by a two-step procedure as previously described.The hematopoietic cells were removed using the mouse hematopoietic lineage cocktail kit by magnetic activated cell sorting(MACS)followed by enriching Sca-1 + cells with Sca-1 labeled magnetic beads.Sca-1+ CMCs were subjected flow cytometry to analyze the cell surface antigens of CD105,CD44,CD140,and c-kit,while GATA4+ cells were detected by immunofluorescent staining.The cells characterized as cardiac mesenchymal cells were next cultured in media without exosomes containing 10% FBS.After 48 hours the medium was collected and precipitated for isolating exosomes.The obtained exosomes were identified by immuno-electron microscopy image and nanoparticle tracking analysis.Meanwhile,the expression of CD63,CD81,and Tsg101 was detected by Western blot.Results: The Sca-1+ CMCs were successfully obtained by the two-step method.86% of CD105+ cells,91% of CD44+ cells,65% of CD140+ cells,and 6.8% of c-kit+ were identified by flow cytometry analysis.Immunofluorescent staining confirmed that prevailing Sca-1+CMCs were GATA4+ and Ki67+.Sca-1+ cardiac mesenchymal cell-derived Exosomes CMC-Exo was obtained by precipitation method.Immuno-electron microscopy imaging and nanoparticle tracking analysis showed that the isolated exosomes were typical microvesicles with a diameter of approximately120nm.Western blot experiments also showed that CMC-Exo contained exosome-specific proteins,such as CD63,CD81,and Tsg101.Conclusion: Sca-1+ CMC-Exo was obtained successfully by the experimental methods above,which was validated by identification experiments.Part II Protective effect of CMC-Exo on acute myocardial infarction mouse modelsObjective: Echocardiographic studies and histology analysis were performed in order to evaluate the protective effect of CMC-Exo on acute myocardial infarction(MI)mouse modelsMethods: C57BL/6 female mice were subjected to acute MI as previously described.Immediately after coronary occlusion,mice were injected intramyocardialy with 30 ?l PBS or CMC-Exo(50 ?g,30 ?l)in the infarct border zone.Echocardiographic studies were performed at baseline,1 day and 1 month after MI.Animals were sacrificed 1 month after MI for tissue harvesting and histological assay.Masson's trichrome staining was performed to quantify the MI size and ventricular wall thickness.The capillary density and cell proliferation were determined by immunofluorescence of CD31,Ki67,EdU,and TnI.Results: The stable myocardial infarction models were established successfully.The echocardiography analysis showed that CMC-Exo significantly improved cardiac function one month post-MI.Masson's Trichrome staining showed that the ventricle wall of the CMC-Exo treated hearts was dramatically thicker and contained more live cardiomyocytes in comparison with PBS treated hearts,suggesting the the beneficial effect of CMC-Exo in preserving ischemic cardiomyocytes after MI.Immunofluorescent staining of CD31 showed a significantly higher density of capillaries in the infarcted hearts treated with CMC-Exo compared with PBS.Besides,EdU and Ki67 staining showed that more proliferating cells were observed in CMC-Exo treated hearts than PBS treated control hearts,most of which were endothelial cells.Moreover,more TnI+ and Ki67+ cardiomyocytes were observed in CMC-Exo treated hearts.Conclusion: CMC-Exo treatment significantly improved left ventricular function after MI.The histological analysis indicated that CMC-Exo promoted the proliferation of endothelial cells and a few cardiomyocytes,as well as increased capillary density in the myocardial infarction border zone compared with PBS-treated hearts,indicating that CMC-Exo promoted angiogenesis.Part III Protective effect of CMC-Exo on acute hind-limb ischemia mouse modelsObjective: Laser Doppler imaging and histology analysis were performed in order to evaluate the protective effect of CMC-Exo on hind-limb ischemia models.Methods: Twelve C57BL/6 female mice were subjected to acute hind-limb ischemia(HLI)by surgical ligation of the femoral artery.The ischemic hind limbs were intramuscularly injected with PBS or CMC-Exo at 4 different locations after the surgery.The laser Doppler imaging measurements were performed prior to induction of hind-limb ischemia(baseline),1 day,15 days,and 28 days post HLI.At each time point,blood flow in ischemic and non-ischemic limbs was measured.Animals were euthanized one month after surgery,muscle specimens were fixed,dehydrated andparaffin sections were made.The signal of lectin was detected by immunofluorescent staining,followed by limb muscle capillary density calculation.Results: The laser Doppler imaging analysis showed that CMC-Exo significantly promoted the recovery of ischemic limb blood perfusion at 2 weeks and 4 weeks post surgery.On the other hand,the results of lectin immunofluorescence showed that the capillary density in CMC-Exo treated limb slightly increased compared with that in the PBS group(535.5 H 133.5/mm2 vs 479.0 H 110.9/mm2,but there was no statistical difference.Conclusion By establishing the mouse model of hind-limb ischemia,it was found that CMC-Exo significantly improved the ischemic limb blood perfusion at 2 weeks and 4 weeks after surgery although immunofluorescent analysis did not show significant differences in muscle capillary density between the two groups.Part IV In vitro Study of CMC-Exo for the protective effect on ischemic cardiovascular modelsObjective: Further in vitro experiments were undertaken to explore the mechanism of CMC-Exo for the protective effect on ischemic cardiovascular modelsMethods: Tube formation assay was performed to assess the effect of CMC-Exo on angiogenesis.HUVECs were seeded in 50 ?l EGM 2-MV medium containing PBS or CMC-Exo and incubated for 20 hours at 37 °C to allow tube formation.The wells were then imaged for capillary-like structures using an EVOS microscope.Quantification of the tubes was performed by image analysis using Image J.Western Blot was performed to detect the expression of PCNA and Erk 24 hours after HUVECs were treated with the different dose of CMC-Exo.To identify the most enriched,abundant miRNAs in CMC-Exo,total RNAs from CMC and CMC-Exowere extracted and subjected to microarray analysis using GeneChip miRNA 4.0 Array.To determine whether local delivery of CMC-Exo can increase the level of the miRNA in treated muscle,mice were injected 50 ?g CMC-Exo into left tibialis anterior(TA)muscle and used right TA as a non-treated control.The muscles were dissected after 24 hours and total RNAs were isolated.Quantitative PCR was next performed with designed primer to determine RNA quantity.To predict the potential molecular function of miR-7116-5p,we use TargetScan,a popular bioinformatics tool to analyze 5795 predicted gene targets of miR-7116-5p,and then use Funrich software for molecular function enrichment analysisResults: Treatment of cells with CMC-Exo significantly enhanced the tube formation in comparison with PBS,suggesting a pro-angiogenetic effect of CMC-Exo.The expression of PCNA and Erk was not enhanced 24 hours after HUVECs were treated with the different dose of CMC-Exo.The microRNA array showed that there were 72 miRNAs enriched in CMC-Exo compared with that in CMCs(Fold change ?2).Among these miRNAs,miR-7116-5p was enriched to the most extent,which increased 32-fold.And the level of miR-7116-5p increased approximately 20 fold by local CMC-Exo delivery in treated muscle compared with non-treated control muscle by qPCR assay,suggesting that miR-7116-5p can be transferred into muscle by CMC-Exo.The molecular function enrichment analysis showed that CMC-Exo might play a role in ubiquitination,or be related to ATP binding and GTP binding proteins.Conclusion: The results of in vitro experiments indicated the proangiogenetic effect of CMC-Exo.The miR-7116-5p is the most enriched,high-abundant miRNA in CMC-Exo compared with which in CMCs and can be transferred into muscle by CMC-Exo.The miR-7116-5p may play an important role in CMC-Exo mediated paracrine effect.