Hydrogen sulfide supplementation as a potential treatment for primary mitochondrial diseases.

Primary mitochondrial diseases (PMD) are amongst the most common inborn errors of metabolism causing fatal outcomes within the first decade of life. With marked heterogeneity in both inheritance patterns and physiological manifestations, these conditions present distinct challenges for targeted drug therapy, where effective therapeutic countermeasures remain elusive within the clinic. Hydrogen sulfide (H2S)-based therapeutics may offer a new option for patient treatment, having been proposed as a conserved mitochondrial substrate and post-translational regulator across species, displaying therapeutic effects in age-related mitochondrial dysfunction and neurodegenerative models of mitochondrial disease. H2S can stimulate mitochondrial respiration at sites downstream of common PMD-defective subunits, augmenting energy production, mitochondrial function and reducing cell death. Here, we highlight the primary signalling mechanisms of H2S in mitochondria relevant for PMD and outline key cytoprotective proteins/pathways amenable to post-translational restoration via H2S-mediated persulfidation. The mechanisms proposed here, combined with the advent of potent mitochondria-targeted sulfide delivery molecules, could provide a framework for H2S as a countermeasure for PMD disease progression.

The Hydrogen Sulfide Donor AP39 Reduces Glutamate-mediated Excitotoxicity in a Rat Model of Brain Ischemia.

The vast majority of stroke cases are classified as ischemic stroke, but effective pharmacotherapy strategies to treat brain infarction are still limited. Glutamate, which is a primary mediator of excitotoxicity, contributes to neuronal damage in numerous pathologies, including ischemia. The aim of this study was to investigate the effect of the hydrogen sulfide donor AP39 on excitotoxicity. AP39 was administered as a single dose of 100 nmol/kg b.w. i.v. 10 min after the restoration of blood flow and 100 min after middle cerebral artery occlusion (MCAO) in male Sprague-Dawley rats. Neurological deficits by Phillips's score, and infarct volume by TTC staining were evaluated (n = 8). LC-MS was used to determine the extracellular glutamate concentration in microdialysates collected intrasurgically and from freely moving animals 24 h and 3 days after reperfusion (n = 6). The expression of proteins involved in the regulation of glutamatergic transmission was investigated 24 h after reperfusion by Western-blot analysis (n = 6). The results were verified by double-immunostaining of brain cryosections (n = 6). The results showed a significant longitudinal decrease in extracellular glutamate concentrations in the motor cortex and hippocampus in MCAO + AP39 rats compared to MCAO rats. Moreover, the administration of AP39 increased the content of the GLT-1 transporter and reduced the content of VGLUT1 in the ischemic core. Upregulation of the GLT-1 transporter responsible for glutamate reuptake from the synaptic cleft, and downregulation of VGLUT1, which regulates glutamate transport to synaptic vesicles, indicate that these are important mechanisms by which AP39 reduces extracellular glutamate concentrations and, consequently, excitotoxicity after ischemia.

Static Cold Storage with Mitochondria-Targeted Hydrogen Sulfide Donor Improves Renal Graft Function in an Ex Vivo Porcine Model of Controlled Donation-after-Cardiac-Death Kidney Transplantation.

The global donor kidney shortage crisis has necessitated the use of suboptimal kidneys from donors-after-cardiac-death (DCD). Using an ex vivo porcine model of DCD kidney transplantation, the present study investigates whether the addition of hydrogen sulfide donor, AP39, to University of Wisconsin (UW) solution improves graft quality. Renal pedicles of male pigs were clamped in situ for 30 min and the ureters and arteries were cannulated to mimic DCD. Next, both donor kidneys were nephrectomized and preserved by static cold storage in UW solution with or without AP39 (200 nM) at 4 °C for 4 h followed by reperfusion with stressed autologous blood for 4 h at 37 °C using ex vivo pulsatile perfusion apparatus. Urine and arterial blood samples were collected hourly during reperfusion. After 4 h of reperfusion, kidneys were collected for histopathological analysis. Compared to the UW-only group, UW+AP39 group showed significantly higher pO2 (p < 0.01) and tissue oxygenation (p < 0.05). Also, there were significant increases in urine production and blood flow rate, and reduced levels of urine protein, serum creatinine, blood urea nitrogen, plasma Na+ and K+, as well as reduced intrarenal resistance in the UW+AP39 group compared to the UW-only group. Histologically, AP39 preserved renal structure by reducing the apoptosis of renal tubular cells and immune cell infiltration. Our finding could lay the foundation for improved graft preservation and reduce the increasingly poor outcomes associated with DCD kidney transplantation.

The mitochondria-targeted sulfide delivery molecule attenuates drugs-induced gastropathy. Involvement of heme oxygenase pathway.

Hydrogen sulfide (H2S) signaling and H2S-prodrugs maintain redox balance in gastrointestinal (GI) tract. Predominant effect of any H2S-donor is mitochondrial. Non-targeted H2S-moieties were shown to decrease the non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrotoxicity but in high doses. However, direct, controlled delivery of H2S to gastric mucosal mitochondria as a molecular target improving NSAIDs-pharmacology remains overlooked. Thus, we treated Wistar rats, i.g. with vehicle, mitochondria-targeted H2S-releasing AP39 (0.004-0.5 mg/kg), AP219 (0.02 mg/kg) as structural control without H2S-releasing ability, or AP39 + SnPP (10 mg/kg) as a heme oxygenase (HMOX) inhibitor. Next, animals were administered i.g. with acetylsalicylic acid (ASA, 125 mg/kg) as NSAIDs representative or comparatively with 75% ethanol to induce translational hemorrhagic or necrotic gastric lesions, that were assessed micro-/macroscopically. Activity of mitochondrial complex IV/V, and DNA oxidation were assessed biochemically. Gastric mucosal/serum content of IL-1ß, IL-10, TNF-α, TGF-ß1/2, ARG1, GST-α, or phosphorylation of mTOR, NF-κB, ERK, Akt, JNK, STAT3/5 were evaluated by microbeads-fluorescent xMAP®-assay; gastric mucosal mRNA level of HMOX-1/2, COX-1/2, SOD-1/2 by real-time PCR. AP39 (but not AP219) dose-dependently (0.02 and 0.1 mg/kg) diminished NSAID- (and ethanol)-induced gastric lesions and DNA oxidation, restoring mitochondrial complexes activity, ARG1, GST-α protein levels and increasing HMOX-1 and SOD-2 expression. AP39 decreased proteins levels or phosphorylation of gastric mucosal inflammation/oxidation-sensitive markers and restored mTOR phosphorylation. Pharmacological inhibition of HMOX-1 attenuated AP39-gastroprotection. We showed that mitochondria-targeted H2S released from very low i.g. doses of AP39 improved gastric mucosal capacity to cope with NSAIDs-induced mitochondrial dysfunction and redox imbalance, mechanistically requiring the activity of HMOX-1.

Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans.

Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic.

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway.

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE-/- mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease.

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves. Lower levels of bioavailable H2S and higher levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine ß-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1ß and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1ß and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation. We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells (GCs) are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (Δψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in GCs. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.

Sulfur amino acid supplementation displays therapeutic potential in a C. elegans model of Duchenne muscular dystrophy.

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a common muscle disease that manifests with muscle weakness, wasting, and degeneration. An emerging theme in DMD pathophysiology is an intramuscular deficit in the gasotransmitter hydrogen sulfide (H2S). Here we show that the C. elegans DMD model displays reduced levels of H2S and expression of genes required for sulfur metabolism. These reductions can be offset by increasing bioavailability of sulfur containing amino acids (L-methionine, L-homocysteine, L-cysteine, L-glutathione, and L-taurine), augmenting healthspan primarily via improved calcium regulation, mitochondrial structure and delayed muscle cell death. Additionally, we show distinct differences in preservation mechanisms between sulfur amino acid vs H2S administration, despite similarities in required health-preserving pathways. Our results suggest that the H2S deficit in DMD is likely caused by altered sulfur metabolism and that modulation of this pathway may improve DMD muscle health via multiple evolutionarily conserved mechanisms.

Mitochondria-targeted hydrogen sulfide donors versus acute oxidative gastric mucosal injury.

Hydrogen sulfide (H2S) as a gaseous molecule prevents gastrointestinal (GI)-tract against various injuries. This study aimed to evaluate for the first time the detailed molecular mechanism of mitochondria-targeting H2S-prodrugs, AP39 and RT01 in gastroprotection against ischemia/reperfusion (I/R)-induced lesions. Wistar rats exposed to I/R were pretreated i.g. with vehicle, AP39 (0.004-2 mg/kg), RT01 (0.1 mg/kg), or with AP219 (0.1 mg/kg) as structural control without ability to release H2S. AP39 was also administered with mTOR1 inhibitor, rapamycin (1 mg/kg i.g.). Gastric damage area was assessed micro-/macroscopically, gastric blood flow (GBF) by laser flowmetry, mRNA level of HIF-1α, GPx, SOD1, SOD2, annexin-A1, SOCS3, IL-1RA, IL-1ß, IL-1R1, IL-1R2, TNFR2, iNOS by real-time PCR. Gastric mucosal and/or serum content of IL-1ß, IL-4, IL-5, IL-10, G-CSF, M-CSF, VEGFA, GRO, RANTES, MIP-1α, MCP1, TNF-α, TIMP1, FABP3, GST-α, STAT3/5 and phosphorylation of mTOR, NF-κB, ERK, Akt was evaluated by microbeads-fluorescent assay. Mitochondrial complexes activities were measured biochemically. RNA damage was assessed as 8-OHG by ELISA. AP39 and RT01 reduced micro-/macroscopic gastric I/R-injury increasing GBF. AP39-gastroprotection was accompanied by maintained activity of mitochondrial complexes, prevented RNA oxidation and enhanced mRNA/protein expression of SOCS3, IL-1RA, annexin-A1, GST-α, HIF-1α. Rapamycin reversed AP-39-gastroprotection. AP39-gastroprotection was followed by decreased NF-κB, ERK, IL-1ß and enhanced Akt and mTOR proteins phosphorylation. AP39-prevented gastric mucosal damage caused by I/R-injury, partly by mitochondrial complex activity maintenance. AP39-mediated attenuation of gastric mucosal oxidation, hypoxia and inflammation involved mTOR1 and Akt pathways activity and modulation of HIF-1α, GST-α, SOCS3, IL1RA and TIMP1 molecular interplay.

Vasorelaxant Activity of AP39, a Mitochondria-Targeted H2S Donor, on Mouse Mesenteric Artery Rings In Vitro.

Mitochondria-targeted hydrogen sulfide (H2S) donor compounds, such as compound AP39, supply H2S into the mitochondrial environment and have shown several beneficial in vitro and in vivo effects in cardiovascular conditions such as diabetes and hypertension. However, the study of their direct vascular effects has not been addressed to date. Thus, the objective of the present study was to analyze the effects and describe the mechanisms of action of AP39 on the in vitro vascular reactivity of mouse mesenteric artery. Protein and gene expressions of the H2S-producing enzymes (CBS, CSE, and 3MPST) were respectively analyzed by Western blot and qualitative RT-PCR, as well the in vitro production of H2S by mesenteric artery homogenates. Gene expression of CSE and 3MPST in the vessels has been evidenced by RT-PCR experiments, whereas the protein expression of all the three enzymes was demonstrated by Western blotting experiments. Nonselective inhibition of H2S-producing enzymes by AOAA abolished H2S production, whereas it was partially inhibited by PAG (a CSE selective inhibitor). Vasorelaxation promoted by AP39 and its H2S-releasing moiety (ADT-OH) were significantly reduced after endothelium removal, specifically dependent on NO-cGMP signaling and SKCa channel opening. Endogenous H2S seems to participate in the mechanism of action of AP39, and glibenclamide-induced KATP blockade did not affect the vasorelaxant response. Considering the results of the present study and the previously demonstrated antioxidant and bioenergetic effects of AP39, we conclude that mitochondria-targeted H2S donors may offer a new promising perspective in cardiovascular disease therapeutics.

Effect of hydrogen sulfide on glycolysis-based energy production in mouse erythrocytes.

Hydrogen sulfide (H2 S) is a gasotransmitter that regulates both physiological and pathophysiological processes in mammalian cells. Recent studies have demonstrated that H2 S promotes aerobic energy production in the mitochondria in response to hypoxia, but its effect on anaerobic energy production has yet to be established. Glycolysis is the anaerobic process by which ATP is produced through the metabolism of glucose. Mammalian red blood cells (RBCs) extrude mitochondria and nucleus during erythropoiesis. These cells would serve as a unique model to observe the effect of H2 S on glycolysis-mediated energy production. The purpose of this study was to determine the effect of H2 S on glycolysis-mediated energy production in mitochondria-free mouse RBCs. Western blot analysis showed that the only H2 S-generating enzyme expressed in mouse RBCs is 3-mercaptopyruvate sulfurtransferase (MST). Supplement of the substrate for MST stimulated, but the inhibition of the same suppressed, the endogenous production of H2 S. Both exogenously administered H2 S salt and MST-derived endogenous H2 S stimulated glycolysis-mediated ATP production. The effect of NaHS on ATP levels was not affected by oxygenation status. On the contrary, hypoxia increased intracellular H2 S levels and MST activity in mouse RBCs. The mitochondria-targeted H2 S donor, AP39, did not affect ATP levels of mouse RBCs. NaHS at low concentrations (3-100 µM) increased ATP levels and decreased cell viability after 3 days of incubation in vitro. Higher NaHS concentrations (300-1000 µM) lowered ATP levels, but prolonged cell viability. H2 S may offer a cytoprotective effect in mammalian RBCs to maintain oxygen-independent energy production.

Mitochondria-Targeted Hydrogen Sulfide Delivery Molecules Protect Against UVA-Induced Photoaging in Human Dermal Fibroblasts, and in Mouse Skin In Vivo.

Aims: Oxidative stress and mitochondrial dysfunction play a role in the process of skin photoaging via activation of matrix metalloproteases (MMPs) and the subsequent degradation of collagen. The activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor controlling antioxidant and cytoprotective defense systems, might offer a pharmacological approach to prevent skin photoaging. We therefore investigated a pharmacological approach to prevent skin photoaging, and also investigated a protective effect of the novel mitochondria-targeted hydrogen sulfide (H2S) delivery molecules AP39 and AP123, and nontargeted control molecules, on ultraviolet A light (UVA)-induced photoaging in normal human dermal fibroblasts (NHDFs) in vitro and the skin of BALB/c mice in vivo. Results: In NHDFs, AP39 and AP123 (50-200 nM) but not nontargeted controls suppressed UVA (8 J/cm2)-mediated cytotoxicity and induction of MMP-1 activity, preserved cellular bioenergetics, and increased the expression of collagen and nuclear levels of Nrf2. In in vivo experiments, topical application of AP39 or AP123 (0.3-1 µM/cm2; but not nontargeted control molecules) to mouse skin before UVA (60 J/cm2) irradiation prevented skin thickening, MMP induction, collagen loss of oxidative stress markers 8-hydroxy-2'-deoxyguanosine (8-OHdG), increased Nrf2-dependent signaling, as well as increased manganese superoxide dismutase levels and levels of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Innovation and Conclusion: Targeting H2S delivery to mitochondria may represent a novel approach for the prevention and treatment of skin photoaging, as well as being useful tools for determining the role of mitochondrial H2S in skin disorders and aging. Antioxid. Redox Signal. 36, 1268-1288.

The Hypothermic Effect of Hydrogen Sulfide Is Mediated by the Transient Receptor Potential Ankyrin-1 Channel in Mice.

Hydrogen sulfide (H2S) has been shown in previous studies to cause hypothermia and hypometabolism in mice, and its thermoregulatory effects were subsequently investigated. However, the molecular target through which H2S triggers its effects on deep body temperature has remained unknown. We investigated the thermoregulatory response to fast-(Na2S) and slow-releasing (GYY4137) H2S donors in C57BL/6 mice, and then tested whether their effects depend on the transient receptor potential ankyrin-1 (TRPA1) channel in Trpa1 knockout (Trpa1-/-) and wild-type (Trpa1+/+) mice. Intracerebroventricular administration of Na2S (0.5-1 mg/kg) caused hypothermia in C57BL/6 mice, which was mediated by cutaneous vasodilation and decreased thermogenesis. In contrast, intraperitoneal administration of Na2S (5 mg/kg) did not cause any thermoregulatory effect. Central administration of GYY4137 (3 mg/kg) also caused hypothermia and hypometabolism. The hypothermic response to both H2S donors was significantly (p < 0.001) attenuated in Trpa1-/- mice compared to their Trpa1+/+ littermates. Trpa1 mRNA transcripts could be detected with RNAscope in hypothalamic and other brain neurons within the autonomic thermoeffector pathways. In conclusion, slow- and fast-releasing H2S donors induce hypothermia through hypometabolism and cutaneous vasodilation in mice that is mediated by TRPA1 channels located in the brain, presumably in hypothalamic neurons within the autonomic thermoeffector pathways.

Hydrogen Sulfide Is a Novel Protector of the Retinal Glycocalyx and Endothelial Permeability Barrier.

Significantly reduced levels of the anti-inflammatory gaseous transmitter hydrogen sulfide (H2S) are observed in diabetic patients and correlate with microvascular dysfunction. H2S may protect the microvasculature by preventing loss of the endothelial glycocalyx. We tested the hypothesis that H2S could prevent or treat retinal microvascular endothelial dysfunction in diabetes. Bovine retinal endothelial cells (BRECs) were exposed to normal (NG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) ± the slow-release H2S donor NaGYY4137 in vitro. Glycocalyx coverage (stained with WGA-FITC) and calcein-labeled monocyte adherence were measured. In vivo, fundus fluorescein angiography (FFA) was performed in normal and streptozotocin-induced (STZ) diabetic rats. Animals received intraocular injection of NaGYY4137 (1 µM) or the mitochondrial-targeted H2S donor AP39 (100 nM) simultaneously with STZ (prevention) or on day 6 after STZ (treatment), and the ratio of interstitial to vascular fluorescence was used to estimate apparent permeability. NaGYY4137 prevented HG-induced loss of BREC glycocalyx, increased monocyte binding to BRECs (p ≤ 0.001), and increased overall glycocalyx coverage (p ≤ 0.001). In rats, the STZ-induced increase in apparent retinal vascular permeability (p ≤ 0.01) was significantly prevented by pre-treatment with NaGYY4137 and AP39 (p < 0.05) and stabilized by their post-STZ administration. NaGYY4137 also reduced the number of acellular capillaries (collagen IV + /IB4-) in the diabetic retina in both groups (p ≤ 0.05). We conclude that NaGYY4137 and AP39 protected the retinal glycocalyx and endothelial permeability barrier from diabetes-associated loss of integrity and reduced the progression of diabetic retinopathy (DR). Hydrogen sulfide donors that target the glycocalyx may therefore be a therapeutic candidate for DR.

The Slow-Releasing and Mitochondria-Targeted Hydrogen Sulfide (H2S) Delivery Molecule AP39 Induces Brain Tolerance to Ischemia.

Ischemic stroke is the third leading cause of death in the world, which accounts for almost 12% of the total deaths worldwide. Despite decades of research, the available and effective pharmacotherapy is limited. Some evidence underlines the beneficial properties of hydrogen sulfide (H2S) donors, such as NaSH, in an animal model of brain ischemia and in in vitro research; however, these data are ambiguous. This study was undertaken to verify the neuroprotective activity of AP39, a slow-releasing mitochondria-targeted H2S delivery molecule. We administered AP39 for 7 days prior to ischemia onset, and the potential to induce brain tolerance to ischemia was verified. To do this, we used the rat model of 90-min middle cerebral artery occlusion (MCAO) and used LC-MS/MS, RT-PCR, LuminexTM assays, Western blot and immunofluorescent double-staining to determine the absolute H2S levels, inflammatory markers, neurotrophic factor signaling pathways and apoptosis marker in the ipsilateral frontal cortex, hippocampus and in the dorsal striatum 24 h after ischemia onset. AP39 (50 nmol/kg) reduced the infarct volume, neurological deficit and reduced the microglia marker (Iba1) expression. AP39 also exerted prominent anti-inflammatory activity in reducing the release of Il-1ß, Il-6 and TNFα in brain areas particularly affected by ischemia. Furthermore, AP39 enhanced the pro-survival pathways of neurotrophic factors BDNF-TrkB and NGF-TrkA and reduced the proapoptotic proNGF-p75NTR-sortilin pathway activity. These changes corresponded with reduced levels of cleaved caspase 3. Altogether, AP39 treatment induced adaptative changes within the brain and, by that, developed brain tolerance to ischemia.

Evaluating the Effects of Subnormothermic Perfusion with AP39 in a Novel Blood-Free Model of Ex Vivo Kidney Preservation and Reperfusion.

The use of blood for normothermic and subnormothermic kidney preservation hinders the translation of these approaches and promising therapeutics. This study evaluates whether adding hydrogen sulfide donor AP39 to Hemopure, a blood substitute, during subnormothermic perfusion improves kidney outcomes. After 30 min of renal pedicle clamping, porcine kidneys were treated to 4 h of static cold storage (SCS-4 °C) or subnormothermic perfusion at 21 °C with Hemopure (H-21 °C), Hemopure + 200 nM AP39 (H200nM-21 °C) or Hemopure + 1 µM AP39 (H1µM-21 °C). Then, kidneys were reperfused with Hemopure at 37 °C for 4 h with metabolic support. Perfusate composition, tissue oxygenation, urinalysis and histopathology were analyzed. During preservation, the H200nM-21 °C group exhibited significantly higher urine output than the other groups and significantly higher tissue oxygenation than the H1µM-21 °C group at 1 h and 2h. During reperfusion, the H200nM-21 °C group exhibited significantly higher urine output and lower urine protein than the other groups. Additionally, the H200nM-21 °C group exhibited higher perfusate pO2 levels than the other groups and significantly lower apoptotic injury than the H-21 °C and the H1µM-21 °C groups. Thus, subnormothermic perfusion at 21 °C with Hemopure + 200 nM AP39 improves renal outcomes. Additionally, our novel blood-free model of ex vivo kidney preservation and reperfusion could be useful for studying other therapeutics.

Subnormothermic Perfusion with H2S Donor AP39 Improves DCD Porcine Renal Graft Outcomes in an Ex Vivo Model of Kidney Preservation and Reperfusion.

Cold preservation is the standard of care for renal grafts. However, research on alternatives like perfusion at higher temperatures and supplementing preservation solutions with hydrogen sulfide (H2S) has gained momentum. In this study, we investigated whether adding H2S donor AP39 to porcine blood during subnormothermic perfusion at 21 °C improves renal graft outcomes. Porcine kidneys were nephrectomized after 30 min of clamping the renal pedicles and treated to 4 h of static cold storage (SCS) on ice or ex vivo subnormothermic perfusion at 21 °C with autologous blood alone (SNT) or with AP39 (SNTAP). All kidneys were reperfused ex vivo with autologous blood at 37 °C for 4 h. Urine output, histopathology and RNAseq were used to evaluate the renal graft function, injury and gene expression profiles, respectively. The SNTAP group exhibited significantly higher urine output than other groups during preservation and reperfusion, along with significantly lower apoptotic injury compared to the SCS group. The SNTAP group also exhibited differential pro-survival gene expression patterns compared to the SCS (downregulation of pro-apoptotic genes) and SNT (downregulation of hypoxia response genes) groups. Subnormothermic perfusion at 21 °C with H2S-supplemented blood improves renal graft outcomes. Further research is needed to facilitate the clinical translation of this approach.

GYY4137 and Sodium Hydrogen Sulfide Relaxations Are Inhibited by L-Cysteine and KV7 Channel Blockers in Rat Small Mesenteric Arteries.

Donors of H2S may be beneficial in treating cardiovascular diseases where the plasma levels of H2S are decreased. Therefore, we investigated the mechanisms involved in relaxation of small arteries induced by GYY4137 [(4-methoxyphenyl)-morpholin-4-yl-sulfanylidene-sulfido-λ5-phosphane;morpholin-4-ium], which is considered a slow-releasing H2S donor. Sulfides were measured by use of 5,5'-dithiobis-(2-nitro benzoic acid), and small rat mesenteric arteries with internal diameters of 200-250 µm were mounted in microvascular myographs for isometric tension recordings. GYY4137 produced similar low levels of sulfides in the absence and the presence of arteries. In U46619-contracted small mesenteric arteries, GYY4137 (10-6-10-3 M) induced concentration-dependent relaxations, while a synthetic, sulfur-free, GYY4137 did not change the vascular tone. L-cysteine (10-6-10-3 M) induced only small relaxations reaching 24 ± 6% at 10-3 M. Premixing L-cysteine (10-3 M) with Na2S and GYY4137 decreased Na2S relaxation and abolished GYY4137 relaxation, an effect prevented by an nitric oxide (NO) synthase inhibitor, L-NAME (Nω-nitro-L-arginine methyl ester). In arteries without endothelium or in the presence of L-NAME, relaxation curves for GYY4137 were rightward shifted. High extracellular K+ concentrations decreased Na2S and abolished GYY4137 relaxation suggesting potassium channel-independent mechanisms are also involved Na2S relaxation while potassium channel activation is pivotal for GYY4137 relaxation in small arteries. Blockers of large-conductance calcium-activated (BKCa) and voltage-gated type 7 (KV7) potassium channels also inhibited GYY4137 relaxations. The present findings suggest that L-cysteine by reaction with Na2S and GYY4137 and formation of sulfides, inhibits relaxations by these compounds. The low rate of release of H2S species from GYY4137 is reflected by the different sensitivity of these relaxations towards high K+ concentration and potassium channel blockers compared with Na2S. The perspective is that the rate of release of sulfides plays an important for the effects of H2S salt vs. donors in small arteries, and hence for a beneficial effect of GYY4137 for treatment of cardiovascular disease.