Matthew Whiteman

Background: We have evidence that hydrogen sulfide (H₂S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H₂S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H₂S-releasing compounds against hydrogen peroxide (H₂O₂)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H₂S biosynthesis called L-cysteine, with the fast H₂S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H₂S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H₂O₂ (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H₂S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H₂S-releasing compounds. Results: Exposure of the isolated retinas to H₂O₂ produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H₂O₂-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H₂O₂ damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H₂O₂ alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H₂O₂-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H₂S-releasing compounds and the substrate, L-cysteine, can prevent H₂O₂-induced lipid peroxidation in an isolated bovine retina.

During COVID-19 there was an increase in antibiotic utilization across all settings. West Virginia has the highest outpatient antibiotic prescribing rate in the country. As part of our outpatient antibiotic stewardship initiative, we developed and utilized a dashboard to track and report prescribing data in two pediatric clinics in a large health system. We reviewed prescribing data from Oct 2020- Mar 2022 in two outpatient pediatric clinics using a dashboard within the electronic medical record. Both clinics are affiliated with a large health system but clinic A was academic and clinic B was non-academic clinic. We also collected data on total number of visits and visits that had antibiotics prescribed. We reviewed prescription data on visits that were diagnosed with upper respiratory tract illness (URI) like acute otitis media, acute sinusitis, acute pharyngitis etc. We compared URI antibiotic prescriptions in Jan 2021-Mar 2021 with Jan 2022- Mar 2022. There was an increase in overall prescriptions for antibiotics and prescriptions for URI in both clinic sites. This was most likely due to the prolonged viral season following cessation of COVID-19 mitigation measures. There were significant differences noted in prescribing practices, like cefdinir overuse for treatment of otitis media, which will guide our antimicrobial stewardship interventions in future. All Authors: No reported disclosures

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.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable disease requiring novel therapeutics. High baseline monocyte count associates with increased risk of progression and mortality in IPF. Perturbations in monocyte phenotype and mitochondrial function, IL6 production, ACOD1 gene expression and metabolism have been observed in IPF patients. IPF patients also have reduced biosynthetic capacity for the immunomodulatory gas hydrogen sulfide (H2S) - an endogenous regulator of mitochondrial function and inflammation. However, the impact of H2S modulation in monocytes is unknown. Aims: Investigate the anti-fibrotic and anti-inflammatory effects of mitochondrial-targeted H2S donor compounds (mtH2SD) in human monocytes. Methods: Longitudinal monocyte counts were investigated in a retrospective IPF cohort (n=131). Fresh monocytes were isolated from healthy donors (n=11), stimulated with IFNγ/TNFα (20ng/ml) +/- mtH2SD (100nM) for 24 hours prior to multispectral flow cytometry, Seahorse metabolic assays, ELISA, qPCR analysis. Results: Rising monocyte count (>0.01 K/µL/month) was associated with worse survival (p=0.04). In vitro mtH2SD treatment of stimulated monocytes resulted in reduced pro-fibrotic marker levels (CD206 and CD163; p<0.05), more energetic and glycolytic metabolism, augmentation of anti-fibrotic ACOD1 expression, decreased IL-6 production and gene expression (p<0.01). Conclusions: Monocytic functionality is implicated in IPF pathogenesis. Modulation of phenotype and metabolism, pro-fibrotic and pro-inflammatory gene expression by mtH2SD may have therapeutic potential. Future work will recapitulate these data in IPF-derived monocytes.

Living longer without simultaneously extending years spent in good health (“health span”) is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H ₂ S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H ₂ S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH ₂ S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H ₂ S (NaGYY4137, 100 µM and 100 nM) and mtH ₂ S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H ₂ S 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 mtH ₂ S donor-mediated health span. Developmentally administered mtH ₂ S (100 nM) improved life/health span vs. equivalent untargeted H ₂ S doses. mtH ₂ S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H ₂ S metabolism enzymes and FoxO/ daf-16 prevented the positive health span effects of mtH ₂ S, whereas DCAF11/ wdr-23 – Nrf2/ skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH ₂ S treatments. Adult mtH ₂ S 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. H ₂ S 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 mtH ₂ S doses required for health span extension, combined with efficacy in adult animals, suggest mtH ₂ S is a potential healthy aging therapeutic.

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 (H ₂ S) 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 n M ) but not nontargeted controls suppressed UVA (8 J/cm ² )-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 μ M /cm ² ; but not nontargeted control molecules) to mouse skin before UVA (60 J/cm ² ) 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 H ₂ S 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 H ₂ S in skin disorders and aging. Antioxid. Redox Signal . 36, 1268–1288.

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 SK_Ca channel opening. Endogenous H₂S seems to participate in the mechanism of action of AP39, and glibenclamide-induced K_ATP 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.