Abstract 418: Preconditioning Promotes Survival and Proliferation of Mesenchymal Stem Cells in the Infarcted Rat Heart Via Activation of NF-κB Downstream of PI3K/Akt Signaling.
Background: We hypothesize that preconditioning of mesenchymal stem cells (MSCs) with diazoxide (DZ) enhances their survival under oxidant stress through activation of pro-survival transcription factor NFκB downstream of PI3K/Akt.
Methods and Results: MSCs were preconditioned (PCMSCs) with 200μmol DZ for 30 minutes at 37oC. PCMSCs showed higher resistance to oxidant stress (100μmol H2O2 for 4h) as compared to non-preconditioned MSCs (non-PCMSCs) as shown by LDH assay, DePsipher staining and Annexin-V positivity by flow cytometry. Immunoblotting on PCMSCs lysate showed higher phosphorylation of PI3K, Akt, GSK3β and NFκB as compared to non-PCMSCs. Enhanced Akt activity in PCMSCs was confirmed by Kinase Assay. Real-time PCR showed up-regulation of Rela (NFκB family). These effects were abolished by pre-treatment of PCMSCs with 30μmol wortaminin. 2-D gel electrophoresis followed by MALDI-TOF Mass spectroscopy and Western blot showed up-regulation of p-Ezrin (a plasma membrane-microfilament cross-linker) in PCMSCs compared to non-PCMSCs for a possible role upstream of PI3K/Akt. PCMSC also showed up-regulation of secretable growth factors including HGF (4-fold), VEGF (7-fold), FGF-2 (5-fold) and PGF (3-fold). For in vivo studies, 50μl DMEM without cells (group-1) or containing 1.5 x106 male non-PCMSCs (group-2), PCMSCs (group-3) and PCMSCs pre-treated with 30μmol wortaminin (group-4) were injected intramyocardially in a female rat model of acute myocardial infarction. Real-time PCR for sry-gene showed higher survival (3 fold) of PCMSCs in the infarcted heart (n = 4/group; p < 0.01 with all groups).Immunostaining for Ki67 showed higher proliferation in group-3. Animals harvested at six weeks after cell transplantation (n = 8 animals/group) showed significantly reduced infarct size in group-3. Confocal imaging after immunostaining for actinin, connexin-43 and vonWillebrand Factor showed enhanced angiomyogenesis in group-3. Echocardiography at 6 weeks showed improved left ventricular function indices in groups-2 and 3 as compared with groups-1 and 4 (p < 0.05).
Conclusions: Preconditioning of MSCs improves their survival via PI3K/Akt/NFkB signaling and promotes their angiomyogenic potential through release of paracrine factors.