Molecular Imaging Of Apelin On Proliferation And Function Of Adipose Tissue Derived Mesenchymal Stem Cells

Background and aimsIschemic vascular diseases are the most dangerous factors to threaten human beings. Vscular regeneration is the main therapeutic method, of which stem cell therapy has becoming a hot spot for the past few years. Nevertheless, a great deal of studies including our departed studies indicated that somatic stem cells, especially mesenchymal stem cells,could hardly survive long-term therapy.Therefore,the difficulty of stem cell therapy is how to promote the survival, integration and proliferation of somatic stem cell in vivo.Apelin, a novel peptide with significant cardioactive properties,which is the endogenic ligand of APJ, has various beneficial effects on vascular protection. Such as vascular dilatation, blood pressure decrease, myocardial protection, myocardial contractility, antagonism to AngiotensinⅡ. Meanwhile, Apelin has a protective effect on vascular endothelial cells and cardiac myocytes. This study was designed to evaluate the contribution of Apelin to the therapeutic efficacy of mesenchymal stem cells under ischemia with molecular imaging method and investigate the mechanism of Apelin's cytoprotective action. MethodsPart one:The isolation and culture of AD-MSCs.Adipose derived mesenchymal stem cells (AD-MSCs) stablely expressing firefly luciferase (Fluc) were isolated from -actin-luc transgenic mice and characterized by flow cytometry and bioluminescence imaging (BLI). Part two: Apelin's effect on the proliferation and apoptosis of AD-MSCs under Hypoxia/Reoxygenation.AD-MSCs were subjected to (1) normal control; (2) Hypoxia 6h+ Reoxygen 2hrs (H/R) (3) H/R+Apelin (10-10¹0-5 mol/L). Cell survival and proliferation were assessed by BLI and MTT assay. Apoptosis was determined by the TUNEL.Part three: Apelin's effect on the survival of AD-MSCs in hindlimb ischemic mice.FVB mice underwent femoral artery ligation and received AD-MSCs(1×10⁶) or AD-MSCs with Apelin intra-quadriceps femoris muscle injection. Cell survival was imaged by BLI.Part four: The mechanism of Apelin's cytoprotective action. Hypoxia(6h)/Reoxygenation(2h)cell model was established.AD-MSCs were subjected to (1) normal control ; (2) 1×10^-6mol/L Apelin (3) Apelin (1×10^-6 mol/L)+LY294002(20μmol/L).The expressions of Akt and pAkt after Apelin administration were analyzed by Western blot. Results:1.AD-MSCs were positive for the CD44 and CD90 by flow cytometry. The Firefly luciferase expression was well correlated with cell numbers which proved by both BLI and luciferase assay.2.Administration of Apelin increased proliferation and decreased apoptosis of AD-MSCs under hypoxia compared with control groups.3. In vivo BLI revealed Apelin increased survival of AD-MSCs in hindlimb ischemic mice.4.Western blot assay revealed the phosphorylation of Akt was higher in Apelin group than that in control groups. (P<0.05)ConclusionThe results suggested that Apelin has beneficial effects on the proliferation and survival of mesenchymal stem cells under hypoxia and might constitute an important therapy target in stem cell therapy for ischemic disease.