Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic animals through an eNOS/HIF‐1/VEGF dependent pathway

Abstract

Objective To determine the effect and molecular mechanism of Na2S induced neovascularization in critical limb ischemia (CLI) during diabetes. Methods Permanent unilateral hind limb ischemia was induced in 42 wk old Db/Db diabetic or eNOSKO mice (n=8, each group). PBS or 0.5 mg/kg Na2S was administered twice daily by retro-orbital injection. Body weight, blood glucose, lipid panels, hind limb perfusion, angiogenic index, capillary to myofiber ratio, proliferation index and TUNEL assay were measured. Endothelial cell proliferation was also performed under hypoxic condition. Lastly, HIF -1 activity was measured using a HIF -1 reporter assay and 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 limb of aged diabetic mice treated with Na2S compared to PBS control. Na2S therapy did not completely restore eNOS KO ischemic hind limb blood flow to pre-ligation levels and cPTIO treatment significantly prevented Na2S mediated hypoxic endothelial cell proliferation activity. Conclusion Sodium sulfide therapy restores tissue perfusion of critical limb ischemia in aged diabetic mice by increasing HIF-1 activation and expression of VEGF that involves NO synthesis and bioavailability.