Abstract 12865: Hydrogen Sulfide Therapy Rescues Critical Limb Ischemia in Aged Diabetic Animals Through an NOS Independent NO/HIF-1/VEGF Dependent Pathway
Introduction: Hydrogen sulfide is reported to have vasodilatory, anti-inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. However, the mechanisms of sulfide-mediated protection during chronic tissue ischemia remain unclear and also unknown with regard to NO mediated effects during chronic ischemia.
Objective: To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia.
Methods: Hind limb ischemia was induced in 10 week old wild type, 42 wk old Db/Db diabetic, or eNOSKO mice (n=8, each group). PBS, 0.1, 0.5 and 1mg/kg Na2S was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe. Angiogenic index was determined by the ratio of CD31 to DAPI positive staining. Cell proliferation index was measured as the ratio of Ki67 to DAPI positive areas and TUNEL assay was measured to investigate the apoptosis. Endothelial cell proliferation was also performed under hypoxic condition. HIF -1 activity was measured using a HIF -1 reporter assay in vitro and expression of HIF -1 in vivo was measured by western blot assay. Lastly, VEGF expression was determined by ELISA.
Result: Blood tissue perfusion, angiogenic index, proliferation index, capillary to myofiber ratio, HIF-1 activity and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limbs of aged diabetic mice treated with Na2S compared to PBS control. Na2S therapy partially restored eNOS KO ischemic hind limb blood flow to pre-ligation levels which was inhibited by cPTIO. cPTIO also significantly prevented Na2S mediated hypoxic endothelial cell proliferation activity suggesting the involvement of non-enzymatic induction of NO by sulfide during revascularization. Lastly, VEGF164 aptamer blocked sulfide induced augmentation of blood flow and reduced the expression VEGF164 in WT and diabetic mice hind limb ischemia indicating VEGF164 as the sulfide induced angiogenic stimulator.
Conclusion: Sodium sulfide therapy restores tissue perfusion of critical limb ischemia by increasing HIF-1 activation and expression of VEGF that involves non-enzymatic NO synthesis and bioavailability.
- © 2011 by American Heart Association, Inc.