Hydrogen sulfide (H2S) has been proposed as a novel endogenous regulator of vascular tone and inflammation. Novel slow release H2S donors (SRHOs) (e.g. GYY4137) have been shown to regulate blood pressure in experimental/genetically induced hypertension, and to inhibit tissue damage, oedema and inflammatory signalling in sepsis and arthritis. All these conditions involve perturbed mitochondrial function, oxidative stress and eventually apoptotic cell death. In the literature, the majority of studies pertaining to the vascular effects of H2S have been limited to the use of simple sulfide salts, NaSH and Na2S, which release H2S far too quickly to be physiologically relevant. Indeed, a study comparing NaSH and GYY 4137 showed that the level of H2S released from NaSH was rapidly high (400I-lM in the first 20min) and declined dramatically until disappearance. In contrast, H2S release from GYY4137 was low and continuous in time (-40I-lM up to 7 days). Therefore, to circumvent this problem, our team has designed several novel SRHOs, such as GYY4137, AP39, AP123, AP67 and AP72. These compounds offer the advantage over sulfide salts (i) to be not toxic (ii) to release H2S in a slow and sustained manner, (iii) to exert their effects at considerably lower H2S concentrations, and (iiii) to selectively target the mitochondria (in the case of AP39 and AP123). The latter combine a mitochondria-targeting moiety (triphenylphosphonium), a C2-C18 linker and H2S releasing moieties (dithiolethione or 4-hydroxythiobenzamide respectively). Preliminary experiments on these mitochondria-targeting SRHO showed a C1Q linker to be optimal; therefore AP39 and AP123 were chosen. Human cerebral microvascular endothelial cells (hCMEC/03) were exposed to oxidative stress agents, mitochondrial toxins and apoptosis inducing drugs in the presence/absence of the SRHOs. Cell toxicity was assessed using alamarBlue and flow cytometry, whereas cytosolic/mitochondrial reactive oxygen species (ROS) generation was estimated using H20CFOA, MitoSOX Red and Oihydroethidium fluorescence (spectrophotometry, flow cytometry). Mitochondrial membrane potential (~4Jm) was assessed using TMRM. Western immunoblotting and fluorescence activity assays were used to determine caspase3/7 activation/activity. Quantification of cytochrome c release from the 3 mitochondria was also attempted. Intracellular levels of ATP were assessed by the luciferase activity assay. Some antioxidant assays were finally used to determine whether the potential antioxidant effects of the SRHDs would be due to any scavenging effect. Cell viability was significantly preserved by all SRHDs. Besides, the potency of cytoprotection was substantially increased by the targeting mitochondria SRHD (AP39/AP123) from the j..IM to the nM range. The collapse of L14Jm normally observed in toxic conditions was inhibited by SRHDs. Overall "ROS" generation was markedly decreased after treatment with SRHDs. AP67 and AP72 inhibited caspase 3/7 activity when apoptosis was induced by staurosporine/etoposide. Cytochrome c release was positively modulated with our donors, as well as the ATP synthesis. These data suggest that SRHDs can inhibit/reverse oxidative stressmediated cellular injury, and highlight the increased potency of the mitochondria- targeting H2S donors AP39 and AP123 compared to GVY4137, AP67 and AP72. The measurement of the antioxidant capacity in cell-free system of the SRHDs showed (i) no scavenging effect of AP39 and AP123, confirming their release of H2S only in the mitochondria (ii) a scavenging effect of GVY4137, AP67 and AP72 only for high concentrations (>250j..lM), confirming that the antioxidant effects of these SRHDs in our cell system was unlikely due to any scavenging effect. Strategies increasing H2S bioavailability, in particular targeting mitochondria, may represent a new therapeutic opportunity to limit mitochondrial dysfunction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:657595 |
| Date | January 2014 |
| Creators | Le Trionnaire, Sophie |
| Publisher | Exeter and Plymouth Peninsula Medical School |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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