cPTIO Treatment Research Articles

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

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.

Targeting of Pollen Tubes to Ovules Is Dependent on Nitric Oxide (NO) Signaling

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for pollen tube growth in lily (Lilium longiflorum). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen-pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen-pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases (nr1 and nr2), which collectively are thought to trigger a putative NO signaling pathway. Semi-vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca(2+) signaling. A role of NO in modulating Ca(2+) signaling was further substantiated by direct imaging the cytosolic free Ca(2+) concentration during NO-induced re-orientation, where two peaks of Ca(2+) occur-one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen-pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule's micropyle by modulating the action of its diffusible factors.