P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway

Abstract

Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine γ-lyase (CSE), cystathionine β- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na 2 S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe. SPY imaging was performed to determine the collateral formation in different time points. Capillary myofiber ratio was determined by NOVA Red staining with CD31 antibody. Mature vessel density and proliferation index were determined by the ratio of smooth muscle actin (SMA) to DAPI and Ki67 to DAPI positive areas respectively. TUNEL assay was measured to investigate the apoptosis. Nitric oxide (NO) was measured by NO analyzer (NOA). CPTIO (1 mg/kg/day) was injected intra-peritoneally to determine the effect of NO in sulfide induced ischemic angiogenesis. VEGF aptamer (50 mg/kg/day) was injected locally to the ischemic muscle to determine the role of VEGF in sulfide induced ischemic angiogenesis. Lastly, VEGF expression was determined by ELISA. Blood perfusion, blush rate by SPY imaging, angiogenic index, proliferation index and mature vessel density were significantly decreased in CSEKO mice compared WT mice indicating the role of sulfide on ischemic angiogeneis and arteriogenesis. Na 2 S dose dependently increases the angiogenesis index, proliferation index, mature vessel density and restores the blood flow in WT mice. Hydrogen sulfide also rescues the impaired blood flow in CSEKO mice by increasing angiogenic index, proliferation index and mature vessel density. Blood tissue perfusion, proliferation index, capillary to myofiber ratio and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limbs of aged diabetic mice treated with Na 2 S compared to PBS control. There was no effect of sulfide on blood glucose, body weight and lipid profiles of diabetic mice.Total NO production was increased in sulfide treated group compared to PBS which was inhibited by CPTIO treatment. Lastly, VEGF164 aptamer blocked sulfide induced augmentation of blood flow and reduced the expression of VEGF164 in WT and diabetic mice hind limb ischemia indicating VEGF164 as the sulfide induced angiogenic stimulator. Sodium sulfide therapy restores tissue perfusion of critical limb ischemia by increasing production of NO, increasing expression of VEGF and preventing apoptosis in diabetic mouse chronic hind limb ischemia which may be beneficial for the diabetic patient with peripheral vascular disease.