Mechanism Of Apelin On Enhancing Functional Survival Of Adipose Derived Mesenchymal Stem Cells By Regulation Of Autophagy

Background and aims:Ischemic cardiovascular diseases are the most serious diseases to threaten humanbeings because of the high morbidity and mortality. The main therapeutic method isangiogenesis. Stem cell therapy has becoming a hot spot in a growing number of diseasesto promote angiogenesis in recent years. However, a number of studies found that stemcells could hardly survive because of ischemia and rejection. Therefore, the mostimportant problem of stem cell therapy is how to promote cells’ survival aftertransplantation in a long term.Apelin, a adipocytokine extracted from bovine, which is the endogenic ligand of APJ,presents in various organs of human body and therefore has multiple biological effects.Such as myocardial contractility, vascular dilatation and blood pressure decrease. Meanwhile, Apelin has a protective effect on vascular endothelial cells, cardiomyocytesand stem cells.This study was designed to assess Apelin’s promotional effect on survival ofadipose-derived mesenchymal stem cells, and to investigate the mechanism of Apelin’scytoprotective action with molecular imaging method.Methods:Part one: Apelin’s effect on the functional survival of ADMSCs in hindlimb ischemicmice. Hindlimb ischemia model was established. ADMSCs and Apelin were injected to (1)Sham+ADMSCs (2) Sham+ADMSCs+Apelin (3) ADMSCs (4) ADMSCs+Apelin. Bloodflow was evaluated by laser-Doppler. Angiogenesis was determined byimmunohistochemistry. Functional rehabilitation was evaluated by autoamputationpercentage, scores of ischemic damage and ambulatory impairment. Cell survival wasimaged by BLI and immunofluorescence.Part two: Apelin’s effect on survival of ADMSCs under hypoxia/reoxygenation byregulation of autophagy. Hypoxia(4h)/reoxygenation(12h) cell model was established.Cell proliferation was assessed by BLI and MTT assay. Apoptosis was determined by theTUNEL. Autophagy was evaluated by immunofluorescence and transmission electronmicroscope. FVB mice underwent femoral artery ligation and received ADMSCs withApelin or ADMSCs with Apelin and LY294002or ADMSCs with Apelin and CompoundC intra-quadriceps femoris muscle injection. Cell survival was imaged by BLI. Blood flowwas evaluated by laser-Doppler. Angiogenesis was determined by immunohistochemistry.Functional rehabilitation was evaluated by autoamputation percentage, scores of ischemicdamage and ambulatory impairment. Part three: The mechanism of Apelin’s cytoprotective action. Hypoxia （4h）/Reoxygenation（12h）cell model was established. ADMSCs were subjected to (1) normalcontrol;(2)1×10-6mol/L Apelin (3) Apelin＋LY294002(4) Apelin＋compound C. Theexpressions of proteins in autophagy pathways after Apelin administration were analyzedby Western blot.Results:1. In vivo BLI revealed Apelin increased survival of ADMSCs in hindlimb ischemicmice. ADMSCs administrated with Apelin promoted revascularization, blood reflow andfunctional rehabilitation in hindlimb ischemic mice.2. Administration of Apelin increased proliferation and decreased apoptosis ofADMSCs under hypoxia compared with control groups. Apelin promoted autophagyduring ischemia and inhibited autophagy during reoxygenation. Apelin increased survivalof ADMSCs in hindlimb ischemic mice promoted revascularization, blood reflow andfunctional rehabilitation by inhibition of autophagy during reperfusion.3. Western blot assay revealed Apelin promoted autophagy throughAMPK-mTOR-ULK1pathway during ischemia and inhibited autophagy throughAkt-Bcl2-Beclin1pathway during reoxygenation.Conclusion:Apelin has beneficial effects on the therapeutic efficacy and survival maintenance ofmesenchymal stem cells in hindlimb mice possibly through a differential regulation ofautophagy through AMPK-mTOR and Akt-beclin1signal pathways.