Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Endogenous hydrogen sulfide production is essential for dietary restriction benefits.

Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK:

In vivo magnetic resonance 31 P-Spectral Analysis With Neural Networks: 31P-SPAWNN.

PURPOSE: We have introduced an artificial intelligence framework, 31P-SPAWNN, in order to fully analyze phosphorus-31 ( 31 $$ {}^{31} $$ P) magnetic resonance spectra. The flexibility and speed of the technique rival traditional least-square fitting methods, with the performance of the two approaches, are compared in this work. THEORY AND METHODS: Convolutional neural network architectures have been proposed for the analysis and quantification of 31 $$ {}^{31} $$ P-spectroscopy. The generation of training and test data using a fully parameterized model is presented herein. In vivo unlocalized free induction decay and three-dimensional 31 $$ {}^{31} $$ P-magnetic resonance spectroscopy imaging data were acquired from healthy volunteers before being quantified using either 31P-SPAWNN or traditional least-square fitting techniques. RESULTS: The presented experiment has demonstrated both the reliability and accuracy of 31P-SPAWNN for estimating metabolite concentrations and spectral parameters. Simulated test data showed improved quantification using 31P-SPAWNN compared with LCModel. In vivo data analysis revealed higher accuracy at low signal-to-noise ratio using 31P-SPAWNN, yet with equivalent precision. Processing time using 31P-SPAWNN can be further shortened up to two orders of magnitude. CONCLUSION: The accuracy, reliability, and computational speed of the method open new perspectives for integrating these applications in a clinical setting.

Hydrogen Sulphide Release via the Angiotensin Converting Enzyme Inhibitor Zofenopril Prevents Intimal Hyperplasia in Human Vein Segments and in a Mouse Model of Carotid Artery Stenosis.

OBJECTIVE: Hypertension is a major risk factor for intimal hyperplasia (IH) and re-stenosis following vascular and endovascular interventions. Preclinical studies suggest that hydrogen sulphide (H2S), an endogenous gasotransmitter, limits re-stenosis. While there is no clinically available pure H2S releasing compound, the sulfhydryl containing angiotensin converting enzyme inhibitor zofenopril is a source of H2S. Here, it was hypothesised that zofenopril, due to H2S release, would be superior to other non-sulfhydryl containing angiotensin converting enzyme inhibitors (ACEi) in reducing intimal hyperplasia. METHODS: Spontaneously hypertensive male Cx40 deleted mice (Cx40-/-) or wild type (WT) littermates were randomly treated with enalapril 20 mg or zofenopril 30 mg. Discarded human vein segments and primary human smooth muscle cells (SMCs) were treated with the active compound enalaprilat or zofenoprilat. IH was evaluated in mice 28 days after focal carotid artery stenosis surgery and in human vein segments cultured for seven days ex vivo. Human primary smooth muscle cell (SMC) proliferation and migration were studied in vitro. RESULTS: Compared with control animals (intima/media thickness 2.3 ± 0.33 µm), enalapril reduced IH in Cx40-/- hypertensive mice by 30% (1.7 ± 0.35 µm; p = .037), while zofenopril abrogated IH (0.4 ± 0.16 µm; p < .002 vs. control and p > .99 vs. sham operated Cx40-/- mice). In WT normotensive mice, enalapril had no effect (0.9665 ± 0.2 µm in control vs. 1.140 ± 0.27 µm; p > .99), while zofenopril also abrogated IH (0.1623 ± 0.07 µm; p < .008 vs. control and p > .99 vs. sham operated WT mice). Zofenoprilat, but not enalaprilat, also prevented IH in human vein segments ex vivo. The effect of zofenopril on carotid and SMCs correlated with reduced SMC proliferation and migration. Zofenoprilat inhibited the mitogen activated protein kinase and mammalian target of rapamycin pathways in SMCs and human vein segments. CONCLUSION: Zofenopril provides extra beneficial effects compared with non-sulfhydryl ACEi in reducing SMC proliferation and re-stenosis, even in normotensive animals. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases and hypertension.

Predictive Factors of Surgical Complications in the First Year Following Kidney Transplantation.

BACKGROUND: In the recent years, an increased use of marginal donors and grafts and a growing prevalence of peripheral arterial disease in the recipients have been observed. Meanwhile, the open surgical technique for kidney transplantation has not changed. The aim of this study is to analyze all surgical complications occurring in the first year after kidney transplant and to determine potential predictive risk factors. METHODS: Data of the 399 patients who underwent kidney transplant in our University Hospital between January 2006 and December 2015 were retrospectively reviewed. The primary endpoint was the overall rate of vascular, parietal and urological complications at 1 year following kidney transplantation. The secondary outcomes were graft and patient' survival rates, and the identification of predictive factors of the surgical complications. RESULTS: 24% of patients developed 134 complications. Vascular complication represented 39% of all complications and resulted in 9 graft losses. Parietal and urological complications represented 46-15% of all complications, respectively, No parietal or urological complications were associated with graft loss. 5 patients died during the 1st year, none of these cases was associated with graft loss. The graft survival rate reached 96% at 1 year, including patients still alive. The occurrence of surgical complication was associated with reduced graft survival at 1 year. Using a multivariate analysis, 4 predictive factors were identified: age, deceased donor, operative time and dyslipidemia. CONCLUSION: Surgical complications after kidney transplantation remained frequent and age, deceased kidney donors, and operative time were identified as risk factors. As vascular complications were a major cause of early graft loss, efforts should aim to reduce their occurrence to increase graft survival.

Differential role of nicotinamide adenine dinucleotide deficiency in acute and chronic kidney disease.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous coenzyme involved in electron transport and a co-substrate for sirtuin function. NAD+ deficiency has been demonstrated in the context of acute kidney injury (AKI). METHODS: We studied the expression of key NAD+ biosynthesis enzymes in kidney biopsies from human allograft patients and patients with chronic kidney disease (CKD) at different stages. We used ischaemia-reperfusion injury (IRI) and cisplatin injection to model AKI, urinary tract obstruction [unilateral ureteral obstruction (UUO)] and tubulointerstitial fibrosis induced by proteinuria to investigate CKD in mice. We assessed the effect of nicotinamide riboside (NR) supplementation on AKI and CKD in animal models. RESULTS: RNA sequencing analysis of human kidney allograft biopsies during the reperfusion phase showed that the NAD+de novo synthesis is impaired in the immediate post-transplantation period, whereas the salvage pathway is stimulated. This decrease in de novo NAD+ synthesis was confirmed in two mouse models of IRI where NR supplementation prevented plasma urea and creatinine elevation and tubular injury. In human biopsies from CKD patients, the NAD+de novo synthesis pathway was impaired according to CKD stage, with better preservation of the salvage pathway. Similar alterations in gene expression were observed in mice with UUO or chronic proteinuric glomerular disease. NR supplementation did not prevent CKD progression, in contrast to its efficacy in AKI. CONCLUSION: Impairment of NAD+ synthesis is a hallmark of AKI and CKD. NR supplementation is beneficial in ischaemic AKI but not in CKD models.

Recent progress and remaining hurdles toward clinical xenotransplantation.

BACKGROUND: Xenotransplantation has made tremendous progress over the last decade. METHODS: We discuss kidney and heart xenotransplantation, which are nearing initial clinical trials. RESULTS: Life sustaining genetically modified kidney xenografts can now last for approximately 500 days and orthotopic heart xenografts for 200 days in non-human primates. Anti-swine specific antibody screening, preemptive desensitization protocols, complement inhibition and targeted immunosuppression are currently being adapted to xenotransplantation with the hope to achieve better control of antibody-mediated rejection (AMR) and improve xenograft longevity. These newest advances could probably facilitate future clinical trials, a significant step for the medical community, given that dialysis remains difficult for many patients and can have prohibitive costs. Performing a successful pig-to-human clinical kidney xenograft, that could last for more than a year after transplant, seems feasible but it still has significant potential hurdles to overcome. The risk/benefit balance is progressively reaching an acceptable equilibrium for future human recipients, e.g. those with a life expectancy inferior to two years. The ultimate question at this stage would be to determine if a "proof of concept" in humans is desirable, or whether further experimental/pre-clinical advances are still needed to demonstrate longer xenograft survival in non-human primates. CONCLUSION: In this review, we discuss the most recent advances in kidney and heart xenotransplantation, with a focus on the prevention and treatment of AMR and on the recipient's selection, two aspects that will likely be the major points of discussion in the first pig organ xenotransplantation clinical trials.

Proximal deep vein thrombosis and pulmonary embolism in COVID-19 patients: a systematic review and meta-analysis.

BACKGROUND: COVID-19 appears to be associated with a high risk of venous thromboembolism (VTE). We aimed to systematically review and meta-analyze the risk of clinically relevant VTE in patients hospitalized for COVID-19. METHODS: This meta-analysis included original articles in English published from January 1st, 2020 to June 15th, 2020 in Pubmed/MEDLINE, Embase, Web of science, and Cochrane. Outcomes were major VTE, defined as any objectively diagnosed pulmonary embolism (PE) and/or proximal deep vein thrombosis (DVT). Primary analysis estimated the risk of VTE, stratified by acutely and critically ill inpatients. Secondary analyses explored the separate risk of proximal DVT and of PE; the risk of major VTE stratified by screening and by type of anticoagulation. RESULTS: In 33 studies (n = 4009 inpatients) with heterogeneous thrombotic risk factors, VTE incidence was 9% (95%CI 5-13%, I2 = 92.5) overall, and 21% (95%CI 14-28%, I2 = 87.6%) for patients hospitalized in the ICU. Proximal lower limb DVT incidence was 3% (95%CI 1-5%, I2 = 87.0%) and 8% (95%CI 3-14%, I2 = 87.6%), respectively. PE incidence was 8% (95%CI 4-13%, I2 = 92.1%) and 17% (95%CI 11-25%, I2 = 89.3%), respectively. Screening and absence of anticoagulation were associated with a higher VTE incidence. When restricting to medically ill inpatients, the VTE incidence was 2% (95%CI 0-6%). CONCLUSIONS: The risk of major VTE among COVID-19 inpatients is high but varies greatly with severity of the disease. These findings reinforce the need for the use of thromboprophylaxis in all COVID-19 inpatients and for clinical trials testing different thromboprophylaxis regimens in subgroups of COVID-19 inpatients. TRIAL REGISTRATION: The review protocol was registered in PROSPERO International Prospective Register of Systematic Reviews ( CRD42020193369 ).

Scoring systems as outcomes assessment of the treatments for haemorrhoidal disease: a systematic review of the literature.

PURPOSE: The comparison between haemorrhoidal treatments is still unclear. Attempts have been made to adopt a unifying postoperative scoring system and thus ensure adequate comparison between clinical trials. We aimed to systematically review the available outcome scores of haemorrhoidal treatment. METHODS: MEDLINE/Pubmed, Web of science, Embase and Cochrane were searched from database implementation until the December 6th 2019. All studies describing or referencing a score to assess haemorrhoidal disease treatment were included. Likert scale alone, incontinence score alone, general assessment of quality of life or scores developed for other proctologic disorders were excluded. The main outcome measures were validation of the scores and correlation of the score items to the core outcome set for haemorrhoidal disease developed by the European Society of Coloproctology. RESULTS: From the 633 records initially screened, 22 studies were included: 8 original articles describing a scoring system and 14 referencing a previously described scoring system. Only 1 score was validated by an external prospective cohort. All the scores evaluated the symptoms of haemorrhoidal disease. No score integrated the disease recurrences or patient's satisfaction. Scores values tended to decrease postoperatively. CONCLUSIONS: The scores described by Gerjy et al. and by Shanmugan et al. are available questionnaires, which have been validated and used in various studies. These scores might help researchers for comparative studies between treatment modalities and optimize haemorrhoids treatment.

Preoperative Protein or Methionine Restriction Preserves Wound Healing and Reduces Hyperglycemia.

BACKGROUND: Dietary restriction (DR), defined as reduced nutrient intake without malnutrition, is associated with longevity extension, improved glucose metabolism, and increased stress resistance, but also poor wound healing. Short-term preoperative DR followed by a return to normal feeding after surgery results in improved surgical outcomes in preclinical models. However, the effect of preoperative DR on wound healing and perioperative glucose homeostasis is currently unknown. Here, we tested the effects of two different preoperative DR regimens-protein restriction (PR) and methionine restriction (MR)-on wound healing and perioperative glucose homeostasis using an established murine model of wound healing in both nondiabetic and diabetic mice. MATERIALS AND METHODS: Surgical outcomes were tested using the McFarlane flap in nondiabetic and streptozotocin-induced diabetic mice. Short-term dietary preconditioning included 1 wk of PR or MR diet (1-2 wk) versus an isocaloric complete diet before surgery; all mice were returned to a complete diet postoperatively. Outcome measures of flap wound recovery included skin viability and laser Doppler imaging of flap perfusion and assessment of CD45+ cell infiltration. Glucose homeostasis was assessed by glucose tolerance testing and by perioperative glucose levels in the diabetic cohort. RESULTS: No significant differences were observed in percentage of viable skin, perfusion, or immune cell infiltration at 7-10 d after surgery in PR or MR mice compared with controls in healthy or diabetic mice. Preoperative glucose tolerance and postoperative glucose levels were however significantly improved by both PR and MR in diabetic mice. CONCLUSIONS: Short-term dietary preconditioning with PR or MR did not impair wound healing in nondiabetic or diabetic mice. However, both regimens reduced preoperative hyperglycemia in diabetic mice. Thus, brief preoperative dietary manipulations stand as strategies to potentially improve perioperative hyperglycemia with no deleterious effects on wound healing in mice.

Ten Year Experience of Using Cryopreserved Arterial Allografts for Distal Bypass in Critical Limb Ischaemia.

OBJECTIVE/BACKGROUND: In critical limb ischaemia (CLI), current guidelines recommend revascularisation whenever possible, preferentially through endovascular means. However, in the case of long occlusions or failed endovascular attempts, distal bypasses still have a place. Single segment great saphenous vein (GSV), which provides the best conduit, is often not available and currently there is no consensus about the best alternative graft. METHODS: From January 2006 to December 2015, 42 cryopreserved arterial allografts were used for a distal bypass. Autologous GSVs or alternative autologous conduits were unavailable for all patients. The patients were observed for survival, limb salvage, and allograft patency. The results were analysed with Kaplan-Meier graphs. RESULTS: Estimates of secondary patency at one, two and five years were 81%, 73%, and 57%, respectively. Estimates of primary patency rates at one, two and five years were 60%, 56%, and 26%, respectively. Estimates of limb salvage rates at one, two and five years were 89%, 89%, and 82%, respectively. Estimates of survival rates at one, two and five years were 92%, 76% and 34%, respectively. At 30 days, major amputations and major adverse cardiac events were one and zero, respectively. Six major amputations occurred during the long-term follow up. CONCLUSION: Despite a low primary patency rate at two years, the secondary patency of arterial allografts is acceptable for distal bypasses. This suggests that cryopreserved arterial allografts are a suitable alternative for limb saving distal bypasses in the absence of venous conduits, improving limb salvage rates and, possibly, quality of life.

Is Overnight Fasting before Surgery Too Much or Not Enough? How Basic Aging Research Can Guide Preoperative Nutritional Recommendations to Improve Surgical Outcomes: A Mini-Review.

Dietary restriction (DR) is best known for extending lifespan in experimental model organisms, but also increases resistance to a variety of clinically relevant stressors, including those associated with surgery. Extended periods of DR, lasting months to years, are required for optimal longevity benefits in rodents, but short-term dietary preconditioning (less than 1 week) remarkably protects from acute injury. Here, we discuss recent advances in our understanding of the mechanistic basis of short-term DR and fasting in the context of surgical stress resistance, including upstream amino acid sensing by the GCN2 and mTORC1 pathways, and downstream effector mechanisms including increased insulin-dependent prosurvival signaling and elevated endogenous hydrogen sulfide production. We also review the current trend in preoperative nutrition away from preoperative fasting and towards carbohydrate loading. Finally, we discuss the rationale for the nonmutually exclusive use of brief DR or pharmacological DR mimetics to precondition against the stress and potential complications of surgery.

Adipose phenotype predicts early human autogenous arteriovenous hemodialysis remodeling.

OBJECTIVE: Substantial proportions of autogenous arteriovenous fistulas (AVFs) for hemodialysis access fail to mature for unclear reasons. AVFs develop in a large mass of surrounding adipose tissue that is increasingly recognized as an active participant in the vascular response to injury via paracrine and endocrine mechanisms. We thus hypothesized that baseline phenotypic characteristics of the adipose tissue juxtaposed to the developing AVF associate with subsequent inward or outward vein wall remodeling. METHODS: Clinical data and subcutaneous adipose tissue were collected from 22 consented patients undergoing AVF creation. Tissue was assayed (protein levels) for interleukin (IL)-6, IL-8, leptin, tumor necrosis factor-α, monocyte chemoattractant protein-1 (MCP-1), resistin, and adiponectin. Vein dimensions were acquired by duplex ultrasound imaging, preoperatively and at 4 to 6 weeks postoperatively, 1 cm cephalad to the arteriovenous anastomosis, which is the most common location of AVF stenosis). RESULTS: The vein at the assayed location outwardly remodeled 55.7% on average (median before, 3.7 mm; median after, 4.7 mm; P = .005). The preoperative vein diameter failed to correlate with postoperative size at the point of assay (R = 0.31; P = .155) unless two outliers were excluded (R = 0.64; P = .002). After removal of the same outliers, the correlation coefficient between venous diameter change (preoperative vs postoperative) and IL-8, tumor necrosis factor-α, MCP-1, resistin, and adiponectin was -0.49, -0.79, -0.66, -0.64, and -0.69, respectively (P < .05). Postoperative AVF flow volume correlated with MCP-1 (R = -0.53; P < .05) and adiponectin (R = -0.47; P < .05). CONCLUSIONS: These data reveal a novel relationship between local adipose phenotype and the eventual venous wall response to hemodynamic perturbation in humans. The predictive value of these mediators generally equaled or exceeded that of preoperative vein size. Beyond providing mechanistic insights into vascular wall adaptations due to flow perturbations, this discovery suggests that strategies focused on altering adipose tissue biology may improve AVF maturation.

Preoperative dietary restriction reduces intimal hyperplasia and protects from ischemia-reperfusion injury.

OBJECTIVE: Whereas chronic overnutrition is a risk factor for surgical complications, long-term dietary restriction (reduced food intake without malnutrition) protects in preclinical models of surgical stress. Building on the emerging concept that acute preoperative dietary perturbations can affect the body's response to surgical stress, we hypothesized that short-term high-fat diet (HFD) feeding before surgery is detrimental, whereas short-term nutrient/energy restriction before surgery can reverse negative outcomes. We tested this hypothesis in two distinct murine models of vascular surgical injury, ischemia-reperfusion (IR) and intimal hyperplasia (IH). METHODS: Short-term overnutrition was achieved by feeding mice a HFD consisting of 60% calories from fat for 2 weeks. Short-term dietary restriction consisted of either 1 week of restricted access to a protein-free diet (protein/energy restriction) or 3 days of water-only fasting immediately before surgery; after surgery, all mice were given ad libitum access to a complete diet. To assess the impact of preoperative nutrition on surgical outcome, mice were challenged in one of two fundamentally distinct surgical injury models: IR injury to either kidney or liver, or a carotid focal stenosis model of IH. RESULTS: Three days of fasting or 1 week of preoperative protein/energy restriction attenuated IH development measured 28 days after focal carotid stenosis. One week of preoperative protein/energy restriction also reduced plasma urea, creatinine, and damage to the corticomedullary junction after renal IR and decreased aspartate transaminase, alanine transaminase, and hemorrhagic necrosis after hepatic IR. However, exposure to a HFD for 2 weeks before surgery had no significant impact on kidney or hepatic function after IR or IH after focal carotid stenosis. CONCLUSIONS: Short-term dietary restriction immediately before surgery significantly attenuated the vascular wall hyperplastic response and improved IR outcome. The findings suggest plasticity in the body's response to these vascular surgical injuries that can be manipulated by novel yet practical preoperative dietary interventions.

Perivascular adipose adiponectin correlates with symptom status of patients undergoing carotid endarterectomy.

BACKGROUND AND PURPOSE: Recent symptoms stand as a major determinant of stroke risk in patients with carotid stenosis, likely reflective of atherosclerotic plaque destabilization. In view of emerging links between vascular and adipose biology, we hypothesized that human perivascular adipose characteristics associate with carotid disease symptom status. METHODS: Clinical history, carotid plaques, blood, and subcutaneous and perivascular adipose tissues were prospectively collected from patients undergoing carotid endarterectomy. Nine adipose-associated biological mediators were assayed and compared in patients with symptomatic (n=15) versus asymptomatic (n=19) disease. Bonferroni correction was performed for multiple testing (α/9=0.006). RESULTS: Symptomatic patients had 1.9-fold higher perivascular adiponectin levels (P=0.005). Other circulating, subcutaneous, and perivascular biomarkers, as well as microscopic plaque characteristics, did not differ between symptomatic and asymptomatic patients. CONCLUSIONS: Symptomatic and asymptomatic carotid endarterectomy patients display a tissue-specific difference in perivascular adipose adiponectin. This difference, which was not seen in plasma or subcutaneous compartments, supports a potential local paracrine relationship with vascular disease processes that may be related to stroke mechanisms.

UW Supplementation with AP39 Improves Liver Viability Following Static Cold Storage.

Static cold storage of donor livers at 4°C incompletely arrests metabolism, ultimately leading to decreases in ATP levels, oxidative stress, cell death, and organ failure. Hydrogen Sulfide (H2S) is an endogenously produced gas, previously demonstrated to reduce oxidative stress, reduce ATP depletion, and protect from ischemia and reperfusion injury. H2S is difficult to administer due to its rapid release curve, resulting in cellular death at high concentrations. AP39, a mitochondrially targeted, slow-release H2S donor, has been shown to reduce ischemia-reperfusion injury in hearts and kidneys. Thus, we investigated whether the addition of AP39 during 3-day static cold storage can improve liver graft viability. At the end of storage, livers underwent six hours of acellular normothermic machine perfusion, a model of transplantation. During simulated transplantation, livers stored with AP39 showed reduced resistance, reduced cellular damage (ALT and AST), and reduced apoptosis. Additionally, bile production and glucose, as well as energy charge were improved by the addition of AP39. These results indicate that AP39 supplementation improves liver viability during static cold storage.