The Novel Mitochondria-Targeted Hydrogen Sulfide
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Pharmacological Research 113 (2016) 186–198 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs Invited perspective The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro a,∗ a,b b a Domokos Gero˝ , Roberta Torregrossa , Alexis Perry , Alicia Waters , c a b a,∗∗ Sophie Le-Trionnaire , Jacqueline L. Whatmore , Mark Wood , Matthew Whiteman a University of Exeter Medical School, Exeter, UK b Biosciences, College of Life and Environmental Sciences, University of Exeter, UK c IRSET-UMR INSERM U1085, Equipe 3-Stress, Membrane et Signalisation, Rennes Cedex, France a r t i c l e i n f o a b s t r a c t Article history: The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive Received 30 June 2016 oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in Received in revised form 10 August 2016 the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochon- Accepted 14 August 2016 drial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial Available online 23 August 2016 oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel Keywords: mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 Hydrogen sulfide respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvas- Oxidative stress cular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric Electron transport Superoxide acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was Hyperglycemia increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 Endothelial cells (30–300 nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited Bioenergetics the mitochondrial oxidant production. Both H2S donors (30–300 nM) increased the electron transport at Complex II respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained SQR the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications. © 2016 Published by Elsevier Ltd. 1. Introduction the next 20 years [1]. Diabetes diagnostic criteria were established based on the increased risk of microvascular and cardiovascular Diabetic complications are responsible for the majority of complications in patients with increased plasma glucose level [2] expenses associated with diabetes treatment and the costs of dia- and glycemic control represent the foundation of diabetes ther- betes that currently accounts for 10% of total healthcare costs, is apy, still it provides little protection against cardiovascular disease projected to increase to 17% of health resource expenditure over (CVD) [3]. Since glucose control is ineffective against cardiovascu- lar events in diabetic patients [3–5], it is important to find novel therapies that reduce the progression of cardiovascular disease in diabetes. Hyperglycemia induces oxidant production in the ves- ∗ Corresponding author at: University of Exeter Medical School, St Luke’s Campus, sels and oxidative stress is considered as a major contributor to Heavitree Road, Exeter, Devon, EX1 2LU, UK. ∗∗ vascular damage [6]. Oxidative stress induced by hyperglycemia Corresponding author at: University of Exeter Medical School, St. Luke’s Campus, persists in the cells long after glucose levels are normalised and Magdalen Road, Exeter, EX1 2LU, UK. E-mail addresses: [email protected] (D. Gero),˝ this phenomenon is known as “glucose memory” [7]. The limited [email protected] (M. Whiteman). http://dx.doi.org/10.1016/j.phrs.2016.08.019 1043-6618/© 2016 Published by Elsevier Ltd. D. Gero˝ et al. / Pharmacological Research 113 (2016) 186–198 187 CVD risk reduction in diabetes may be explained by the persistence nephropathy, vasculopathy and cardiomyopathy [29–31] and its of deleterious downstream effects that occur after intermittent active constituents were found to be diallyldisulfide (DADS) and hyperglycemic episodes, despite lower glycated hemoglobin lev- diallyltrisulfide (DATS) [32], which are slower H2S donors than els. Na2S [33]. Since the protective effect of H2S is mostly mitochon- Hyperglycemia-induced mitochondrial superoxide generation drial in the hyperglycemic endothelium, we tested the efficacy is an upstream player in the development of endothelial dysfunc- of novel mitochondria-targeted H2S donors against the glucose- tion and it is responsible for the activation of other sources of induced oxidant production. AP39 is a slow-release H2S donor oxidants in the cells [8]. In endothelial cells, high glucose supply that was shown to accumulate in the mitochondria [36,37] and results in increased glucose oxidation: more electron donors are protect against oxidative stress-induced mitochondrial DNA and pushed into the electron transport chain and the voltage gradi- protein damage in endothelial cells [34]. We compared the effi- ent across the mitochondrial membrane increases. The increased cacy of AP39 and AP123, a newer mitochondrial H2S donor, against transmembrane voltage induces electron leakage between com- glucose-induced endothelial dysfunction and we found that mito- plexes II and III and the inappropriate transfer of electrons to chondrial slow-release H2S donors are >1000-fold more potent molecular oxygen generates superoxide [9]. If the mitochondrial than Na2S against hyperglycemia-induced oxidant production and potential is normalised in the cells by uncoupling protein-1 (UCP-1) also have beneficial effect on cellular bioenergetics in endothelial overexpression or the mitochondrial respiratory chain is inacti- cells. vated by mitochondrial DNA depletion hyperglycemia does not generate superoxide [8]. Blockage of mitochondrial superoxide 2. Methods generation by the above methods or neutralisation by manganese superoxide dismutase (MnSOD) inhibits other sources of oxidants 2.1. Synthesis of mitochondria-targeted H S donor AP123 (10-(4- in endothelial cells: the activation of protein kinase C (PKC) and 2 carbamothioylphenoxy)-10-oxodecyl)triphenylphosphonium the polyol pathway, the formation of advanced glycation end bromide) product (AGE) and the hexosamine pathway [8]. Similarly, we found that mitochondrial superoxide scavenging using paroxetine AP39 was synthesised as previously described by us [35], [10] or induction of uncoupling protein-2 (UCP-2) also blocked −1 −1 extinction coefficient in DMSO ( = 6162 M cm ; the glucose-induced oxidant production in endothelial cells [11]. 400nm −1 −1 ( = 12000 M cm ). AP123 was synthesised using While these methods all reduce the mitochondrial ROS produc- 327nm 3 the following procedure: acetonitrile (8 cm ) was added to tion and the associated cellular damage, neither ROS scavenging 10-bromodecanoic acid (400 mg, 1.59 mmol) and triphenylphos- nor mitochondrial uncoupling fully restore the mitochondrial phine (418 mg, 1.59 mmol) and the resulting mixture was energy production. If electrons are used for superoxide genera- stirred and heated under reflux for 48 h [36]. The acetoni- tion and the protons are released through uncoupling proteins, trile was evaporated in vacuo and the colourless, oily residue there will be a drop in ATP production via oxidative phosphory- 3 lation. was triturated with toluene (3 × 10 cm ) before thorough dry- ing on a rotary evaporator and dissolution in dichloromethane H2S is an endogenously produced ‘gasotransmitter’ that plays 3 (15 cm ). At room temperature, 4-hydroxythiobenzamide (244 mg, key roles in regulating vascular tone, inflammation, cell death 1.59 mmol) was added to the stirred solution, followed by a solu- and proliferation as well as vascular protection [12–15]. Lower tion of N,N-dicyclohexylcarbodiimide (330 mg, 1.60 mmol) in H2S bioavailability has been reported in the diabetic vascula- 3 dichloromethane (8 cm ) and 4-dimethylaminopyridine (10 mg, ture in humans and correlates to poorer microcirculatory blood 0.08 mmol). After stirring for 22 h, the reaction mixture was fil- flow [16]. Impaired vascular H2S synthesis and/or bioavailability tered through a cotton wool plug and after removal of the solvent is also observed in the vasculature of several animal models of in vacuo, the crude product was applied as a dichloromethane diabetes induced either pharmacologically- (e.g. streptozotocin- 3 solution onto a silica gel flash chromatography column ca 120 cm induced [17]) or genetically-induced (e.g. Akita [18], db/db [19] and silica gel, 3 cm diameter column). After flushing the silica gel with NOD mice [20]). The ‘loss’ of vasculoprotective H2S is thought to 3 ethyl acetate (200 cm ), the product was eluted with methanol contribute to vascular endothelial dysfunction and