Abstract 703: HIF-1α Dependent Expression of iNOS in Bone Marrow Stem Cells Mediates Protection of Cardiomyocytes against Ischemia
We hypothesized that inducible nitric oxide synthase (iNOS) upregulated in bone marrow stem cells (BMSCs) protect ischemic cardiomyocytes via hypoxia inducible factor-1α (HIF-1α) pathway.
Methods: Isolated BMSCs were exposed to hypoxia for 24 hours. The level of HIF-1α protein and its activated form were measured by ELISA. The expression of iNOS and HIF-1α were analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry. Cardiomyocytes in co-culture with BMSCs were subjected to hypoxia and H2O2 (200 μmol). LDH release, DNA fragmentation and annexin-V positive cells were used as injury markers.
Results: HIF-1α protein and its activated form were markedly increased (Fig. A, B⇓) and translocated to the nucleus or peri-nuclear area of BMSCs subjected to hypoxia, in parallel with increased expression of HIF-1α target gene iNOS, which was blocked by pretreating cells with neutralizing HIF-1α antibody (Fig. C⇓). Co-culture of cardiomyocytes with BMSCs not only prevented and reduced cardiomyocyte apoptosis induced by hypoxia and H2O2 but also significantly reduced LDH release from cardiomyocytes (Fig. D–G⇓). The cardiac protection by BMSC was abolished by neutralizing HIF-1α antibodies, and by the selective iNOS inhibitor, 1400W (10 mg/L) as well as a potent competitive inhibitor of NOS, L-NAME (200 μmol/L). However, no effects of neutralizing HIF-1α antibody, L-NAME or 1400W on cardiomyocytes alone were seen. SNAP (300 μmol/L), a NO donor had no synergistic effect on the cardioprotective effect of BMSC.
Conclusion: iNOS upregulated in bone marrow stem cells by hypoxia protects cardiomyocytes against ischemic injury via activating HIF-1α.