Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution.

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.

The hibernation-derived compound SUL-138 shifts the mitochondrial proteome towards fatty acid metabolism and prevents cognitive decline and amyloid plaque formation in an Alzheimer's disease mouse model.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease worldwide and remains without effective cure. Increasing evidence is supporting the mitochondrial cascade hypothesis, proposing that loss of mitochondrial fitness and subsequent ROS and ATP imbalance are important contributors to AD pathophysiology. METHODS: Here, we tested the effects of SUL-138, a small hibernation-derived molecule that supports mitochondrial bioenergetics via complex I/IV activation, on molecular, physiological, behavioral, and pathological outcomes in APP/PS1 and wildtype mice. RESULTS: SUL-138 treatment rescued long-term potentiation and hippocampal memory impairments and decreased beta-amyloid plaque load in APP/PS1 mice. This was paralleled by a partial rescue of dysregulated protein expression in APP/PS1 mice as assessed by mass spectrometry-based proteomics. In-depth analysis of protein expression revealed a prominent effect of SUL-138 in APP/PS1 mice on mitochondrial protein expression. SUL-138 increased the levels of proteins involved in fatty acid metabolism in both wildtype and APP/PS1 mice. Additionally, in APP/PS1 mice only, SUL-138 increased the levels of proteins involved in glycolysis and amino acid metabolism pathways, indicating that SUL-138 rescues mitochondrial impairments that are typically observed in AD. CONCLUSION: Our study demonstrates a SUL-138-induced shift in metabolic input towards the electron transport chain in synaptic mitochondria, coinciding with increased synaptic plasticity and memory. In conclusion, targeting mitochondrial bioenergetics might provide a promising new way to treat cognitive impairments in AD and reduce disease progression.

The 6-hydroxychromanol derivative SUL-109 ameliorates renal injury after deep hypothermia and rewarming in rats.

Background: Mitochondrial dysfunction plays an important role in kidney damage in various pathologies, including acute and chronic kidney injury and diabetic nephropathy. In addition to the well-studied ischaemia/reperfusion (I/R) injury, hypothermia/rewarming (H/R) also inflicts acute kidney injury. Substituted 6-hydroxychromanols are a novel class of mitochondrial medicines that ameliorate mitochondrial oxidative stress and protect the mitochondrial network. To identify a novel 6-hydroxychromanol that protects mitochondrial structure and function in the kidney during H/R, we screened multiple compounds in vitro and subsequently assessed the efficacy of the 6-hydroxychromanol derivatives SUL-109 and SUL-121 in vivo to protect against kidney injury after H/R in rats. Methods: Human proximal tubule cell viability was assessed following exposure to H/R for 48/4 h in the presence of various 6-hydroxychromanols. Selected compounds (SUL-109, SUL-121) or vehicle were administered to ketamine-anaesthetized male Wistar rats (IV 135 µg/kg/h) undergoing H/R at 15°C for 3 h followed by rewarming and normothermia for 1 h. Metabolic parameters and body temperature were measured throughout. In addition, renal function, renal injury, histopathology and mitochondrial fitness were assessed. Results: H/R injury in vitro lowered cell viability by 94 ± 1%, which was counteracted dose-dependently by multiple 6-hydroxychomanols derivatives. In vivo, H/R in rats showed kidney injury molecule 1 expression in the kidney and tubular dilation, accompanied by double-strand DNA breaks and protein nitrosylation. SUL-109 and SUL-121 ameliorated tubular kidney damage, preserved mitochondrial mass and maintained cortical adenosine 5'-triphosphate (ATP) levels, although SUL-121 did not reduce protein nitrosylation. Conclusions: The substituted 6-hydroxychromanols SUL-109 and SUL-121 ameliorate kidney injury during in vivo H/R by preserving mitochondrial mass, function and ATP levels. In addition, both 6-hydroxychromanols limit DNA damage, but only SUL-109 also prevented protein nitrosylation in tubular cells. Therefore SUL-109 offers a promising therapeutic strategy to preserve kidney mitochondrial function.

The 6-chromanol derivate SUL-109 enables prolonged hypothermic storage of adipose tissue-derived stem cells.

Encouraging advances in cell therapy research with adipose derived stem cells (ASC) require an effective short-term preservation method that provides time for quality control and transport of cells from their manufacturing facility to their clinical destination. Hypothermic storage of cells in their specific growth media offers an alternative and simple preservation method to liquid nitrogen cryopreservation or commercial preservation fluids for short-term storage and transport. However, accumulation of cell damage during hypothermia may result in cell injury and death upon rewarming through the production of excess reactive oxygen species (ROS). Here, the ability of the cell culture medium additive SUL-109, a modified 6-chromanol, to protect ASC from hypothermia and rewarming damage is examined. SUL-109 conveys protective effects against cold-induced damage in ASC as is observed by preservation of cell viability, adhesion properties and growth potential. SUL-109 does not reduce the multilineage differentiation capacity of ASC. SUL-109 conveys its protection against hypothermic damage by the preservation of the mitochondrial membrane potential through the activation of mitochondrial membrane complexes I and IV, and increases maximal oxygen consumption in FCCP uncoupled mitochondria. Consequently, SUL-109 alleviates mitochondrial ROS production and preserves ATP production. In summary, here we describe the generation of a single molecule cell preservation agent that protects ASC from hypothermic damage associated with short-term cell preservation that does not affect the differentiation capacity of ASC.

The novel compound Sul-121 inhibits airway inflammation and hyperresponsiveness in experimental models of chronic obstructive pulmonary disease.

COPD is characterized by persistent airflow limitation, neutrophilia and oxidative stress from endogenous and exogenous insults. Current COPD therapy involving anticholinergics, ß2-adrenoceptor agonists and/or corticosteroids, do not specifically target oxidative stress, nor do they reduce chronic pulmonary inflammation and disease progression in all patients. Here, we explore the effects of Sul-121, a novel compound with anti-oxidative capacity, on hyperresponsiveness (AHR) and inflammation in experimental models of COPD. Using a guinea pig model of lipopolysaccharide (LPS)-induced neutrophilia, we demonstrated that Sul-121 inhalation dose-dependently prevented LPS-induced airway neutrophilia (up to ~60%) and AHR (up to ~90%). Non-cartilaginous airways neutrophilia was inversely correlated with blood H2S, and LPS-induced attenuation of blood H2S (~60%) was prevented by Sul-121. Concomitantly, Sul-121 prevented LPS-induced production of the oxidative stress marker, malondialdehyde by ~80%. In immortalized human airway smooth muscle (ASM) cells, Sul-121 dose-dependently prevented cigarette smoke extract-induced IL-8 release parallel with inhibition of nuclear translocation of the NF-κB subunit, p65 (each ~90%). Sul-121 also diminished cellular reactive oxygen species production in ASM cells, and inhibited nuclear translocation of the anti-oxidative response regulator, Nrf2. Our data show that Sul-121 effectively inhibits airway inflammation and AHR in experimental COPD models, prospectively through inhibition of oxidative stress.

Platelet dynamics during natural and pharmacologically induced torpor and forced hypothermia.

Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster) and non-hibernating (rat and mouse) species without changing platelet function. Pharmacological torpor induced by injection of 5'-AMP in mice did not induce thrombocytopenia, possibly because 5'-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of platelets, dependent on intrinsic platelet functionality, governs clearance of circulating platelets during torpor.