Abstract 3819: The Canonical Activation of NF-kB Is Responsible for Secretion of Cardioprotective Paracrine Factors from Preconditioned Stem Cells
Background: We investigated that canonical activation of NF-kB in preconditioned mesenchymal stem cells (PCMSCs) is responsible for sustained cytoprotection under oxidant stress and for release of paracrine factors to induce angiomyogenesis in the infarcted heart.
Methods and Results: Bone marrow derived MSCs were preconditioned with 200 μM diazoxide for 30 min and subsequently treated with 100μM H2O2 for 4-h either immediately or 24-h after preconditioning. PCMSCs were more resistant to oxidant stress over a period of 24-h than untreated MSCs (non-PCMSCs) (p<0.001) as shown by lactate dehydrogenase assay. Higher Akt activity (p<0.001) and increased phospho-NF-κB p65 in the nuclear protein fraction were observed in PCMSCs (p<0.001 vs non-PCMSCs) and was abrogated by wortmannin (30μM) pretreatment. Pretreatment of PCMSCs with IKKα/IKKβ inhibitor (NEMO; 10μM) abrogated increase in nuclear phospho-NF-κB p65 which suggested the activation of canonical pathway. This was quantified by p65 NF- kB TransAM assay. The use of NF- kB specific decoy and NEMO prior to preconditioning resulted in loss of cytoprotection at 24-h after preconditioning. Real-time PCR showed upregulation of COX-2 (3-fold), SFRP-2 (5-fold), and NPY (3.6-fold) in PCMSCs which were significantly reduced upon use of NF- kB decoy. Real-time PCR for sry-gene on day-4 after sex-mismatched engraftment of cells in female rat model of acute coronary artery ligation (n=6/group) showed higher survival of male PCMSCs than Non-PCMSCs (p<0.01). Wortmannin pretreatment of PCMSCs reduced their survival (p<0.01 vs PCMSCs). Immunohistology for cardiac actinin and von Willibrand factor revealed increased myogenesis and higher blood vessel density (42±9 blood vessels per 0.155mm2 in the infarct zone ( p<0.05 vs control) in PCMSCs transplanted hearts. Echocardiography at 6 weeks (n=8/group) revealed improved ejection fraction (49.5±5.9) and fractional shortening (29.1±4.2) in PCMSCs transplanted animals (p<0.05 vs controls). Infarct size attenuation was consistent with echocardiography findings.
Conclusion: Canonical NF-kB activation is responsible for sustained cytoprotection of donor cells and cardiac repair by secretion of paracrine factors with angiomyogenic potential.