Peroxiredoxin 6 suppresses ferroptosis in lung endothelial cells.

Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation by its phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyltransferase activities. Prdx6 is highly expressed in the lung, and intact lungs and cells null for Prdx6 or with single-point mutations that inactivate either Prdx6-peroxidase or aiPLA2 activity alone exhibit decreased viability, increased lipid peroxidation, and incomplete repair when exposed to paraquat, hyperoxia, or organic peroxides. Ferroptosis is form of cell death driven by the accumulation of phospholipid hydroperoxides. We studied the role of Prdx6 as a ferroptosis suppressor in the lung. We first compared the expression Prdx6 and glutathione peroxidase 4 (GPx4) and visualized Prdx6 and GPx4 within the lung. Lung Prdx6 mRNA levels were five times higher than GPx4 levels. Both Prdx6 and GPx4 localized to epithelial and endothelial cells. Prdx6 knockout or knockdown sensitized lung endothelial cells to erastin-induced ferroptosis. Cells with genetic inactivation of either aiPLA2 or Prdx6-peroxidase were more sensitive to ferroptosis than WT cells, but less sensitive than KO cells. We then conducted RNA-seq analyses in Prdx6-depleted cells to further explore how the loss of Prdx6 sensitizes lung endothelial cells to ferroptosis. Prdx6 KD upregulated transcriptional signatures associated with selenoamino acid metabolism and mitochondrial function. Accordingly, Prdx6 deficiency blunted mitochondrial function and increased GPx4 abundance whereas GPx4 KD had the opposite effect on Prdx6. Moreover, we detected Prdx6 and GPx4 interactions in intact cells, suggesting that both enzymes cooperate to suppress lipid peroxidation. Notably, Prdx6-depleted cells remained sensitive to erastin-induced ferroptosis despite the compensatory increase in GPx4. These results show that Prdx6 suppresses ferroptosis in lung endothelial cells and that both aiPLA2 and Prdx6-peroxidase contribute to this effect. These results also show that Prdx6 supports mitochondrial function and modulates several coordinated cytoprotective pathways in the pulmonary endothelium.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Hypoxia blunts angiogenic signaling and upregulates the antioxidant system in elephant seal endothelial cells.

Elephant seals experience extreme hypoxemia during diving bouts. Similar depletions in oxygen availability characterize pathologies including myocardial infarction and ischemic stroke in humans, but seals manage these repeated episodes without injury. However, the real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture system to assess the molecular response to prolonged hypoxia. Seal and human cells exposed to 1% O 2 for up to 6 h demonstrated differential responses to both acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling at both the transcriptional and cellular level. Rapid upregulation of genes involved in the glutathione (GSH) metabolism pathway supported maintenance of GSH pools and increases in intracellular succinate in seal but not human cells during hypoxia exposure. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurred in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. In sum, our studies show that in contrast to human cells, seal cells adapt to hypoxia exposure by dampening angiogenic signaling, increasing antioxidant protection, and maintaining mitochondrial morphological integrity and function.

The Antarctic Weddell seal genome reveals evidence of selection on cardiovascular phenotype and lipid handling.

The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes were SUMO2 and EP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.

Repeated stimulation of the HPA axis alters white blood cell count without increasing oxidative stress or inflammatory cytokines in fasting elephant seal pups.

The hypothalamic-pituitary-adrenal (HPA) axis controls the release of glucocorticoids, which regulate immune and inflammatory function by modulating cytokines, white blood cells and oxidative stress via glucocorticoid receptor (GR) signaling. Although the response to HPA activation is well characterized in many species, little is known about the impacts of HPA activation during extreme physiological conditions. Hence, we challenged 18 simultaneously fasting and developing elephant seal pups with daily intramuscular injections of adrenocorticotropin (ACTH), a GR antagonist (RU486), or a combination of the two (ACTH+RU486) for 4 days. We collected blood at baseline, 2 h and 4 days after the beginning of treatment. ACTH and ACTH+RU486 elevated serum aldosterone and cortisol at 2 h, with effects diminishing at 4 days. RU486 alone induced a compensatory increase in aldosterone, but not cortisol, at 4 days. ACTH decreased neutrophils at 2 h, while decreasing lymphocytes and increasing the neutrophil:lymphocyte ratio at 4 days. These effects were abolished by RU486. Despite alterations in white blood cells, there was no effect of ACTH or RU486 on transforming growth factor-ß or interleukin-6 levels; however, both cytokines decreased with the 4 day fasting progression. Similarly, ACTH did not impact protein oxidation, lipid peroxidation or antioxidant enzymes, but plasma isoprostanes and catalase activity decreased while glutathione peroxidase increased with fasting progression. These data demonstrate differential acute (2 h) and chronic (4 days) modulatory effects of HPA activation on white blood cells and that the chronic effect is mediated, at least in part, by GR. These results also underscore elephant seals' extraordinary resistance to oxidative stress derived from repeated HPA activation.

Potentiation of Acetylcholine-Induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) Rats: Sex-Specific Responses.

Previous reports suggest that diabetes may differentially affect the vascular beds of females and males. The objectives of this study were to examine whether there were (1) sex differences in aortic function and (2) alterations in the relative contribution of endothelium-derived relaxing factors in modulating aortic reactivity in UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats. Endothelium-dependent vasorelaxation (EDV) in response to acetylcholine (ACh) was measured in aortic rings before and after exposure to pharmacological inhibitors. Relaxation responses to sodium nitroprusside were assessed in endothelium-denuded rings. Moreover, contractile responses to phenylephrine (PE) were measured before and after incubation of aortic rings with a nitric oxide synthase (NOS) inhibitor in the presence of indomethacin. Metabolic parameters and expression of molecules associated with vascular and insulin signaling as well as reactive oxygen species generation were determined. Diabetes slightly but significantly impaired EDV in response to ACh in aortas from females but potentiated the relaxation response in males. The potentiation of EDV in diabetic male aortas was accompanied by a traces of nitric oxide (NO)- and prostanoid-independent relaxation and elevated aortic expression of small- and intermediate conductance Ca2+-activated K+ channels in this group. The smooth muscle sensitivity to NO was not altered, whereas the responsiveness to PE was significantly enhanced in aortas of diabetic groups in both sexes. Endothelium-derived NO during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was reduced in aortas of diabetic rats regardless of sex. Accordingly, decreases in pAkt and peNOS were observed in aortas from diabetic rats in both sexes compared with controls. Our data suggest that a decrease in insulin sensitivity via pAkt-peNOS-dependent signaling and an increase in oxidative stress may contribute to the elevated contractile responses observed in diabetic aortas in both sexes. This study demonstrates that aortic function in UCD-T2DM rats is altered in both sexes. Here, we provide the first evidence of sexual dimorphism in aortic relaxation in UCD-T2DM rats.

Fasting ameliorates oxidative stress: A review of physiological strategies across life history events in wild vertebrates.

Fasting is a component of many species' life history due to environmental factors or behavioral patterns that limit access to food. Despite metabolic and physiological challenges associated with these life history stages, fasting-adapted wild vertebrates exhibit few if any signs of oxidative stress, suggesting that fasting promotes redox homeostasis. Here we review mammalian, avian, reptilian, amphibian, and piscine examples of animals undergoing fasting during prolonged metabolic suppression (e.g. hibernation and estivation) or energetically demanding processes (e.g. migration and breeding) to better understand the mechanisms underlying fasting tolerance in wild vertebrates. These studies largely show beneficial effects of fasting on redox balance via limited oxidative damage. Though some species exhibit signs of oxidative stress due to energetically or metabolically extreme processes, fasting wild vertebrates largely buffer themselves from the negative consequences of oxidative damage through specific strategies such as elevating antioxidants, selectively maintaining redox balance in critical tissues, or modifying behavioral patterns. We conclude with suggestions for future research to better elucidate the protective effects of fasting on oxidative stress as well as disentangle the impacts from other life history stages. Further research in these areas will facilitate our understanding of the mechanisms wild vertebrates use to mitigate the negative impacts associated with metabolically-extreme life history stages as well as potential translation into therapeutic interventions in non-fasting-adapted species including humans.

Functional Studies with Primary Cells Provide a System for Genome-to-Phenome Investigations in Marine Mammals.

Marine mammals exhibit some of the most dramatic physiological adaptations in their clade and offer unparalleled insights into the mechanisms driving convergent evolution on relatively short time scales. Some of these adaptations, such as extreme tolerance to hypoxia and prolonged food deprivation, are uncommon among most terrestrial mammals and challenge established metabolic principles of supply and demand balance. Non-targeted omics studies are starting to uncover the genetic foundations of such adaptations, but tools for testing functional significance in these animals are currently lacking. Cellular modeling with primary cells represents a powerful approach for elucidating the molecular etiology of physiological adaptation, a critical step in accelerating genome-to-phenome studies in organisms in which transgenesis is impossible (e.g., large-bodied, long-lived, fully aquatic, federally protected species). Gene perturbation studies in primary cells can directly evaluate whether specific mutations, gene loss, or duplication confer functional advantages such as hypoxia or stress tolerance in marine mammals. Here, we summarize how genetic and pharmacological manipulation approaches in primary cells have advanced mechanistic investigations in other non-traditional mammalian species, and highlight the need for such investigations in marine mammals. We also provide key considerations for isolating, culturing, and conducting experiments with marine mammal cells under conditions that mimic in vivo states. We propose that primary cell culture is a critical tool for conducting functional mechanistic studies (e.g., gene knockdown, over-expression, or editing) that can provide the missing link between genome- and organismal-level understanding of physiological adaptations in marine mammals.

Natural Tolerance to Ischemia and Hypoxemia in Diving Mammals: A Review.

Reperfusion injury follows ischemia/reperfusion events occurring during myocardial infarction, stroke, embolism, and other peripheral vascular diseases. Decreased blood flow and reduced oxygen tension during ischemic episodes activate cellular pathways that upregulate pro-inflammatory signaling and promote oxidant generation. Reperfusion after ischemia recruits inflammatory cells to the vascular wall, further exacerbating oxidant production and ultimately resulting in cell death, tissue injury, and organ dysfunction. Diving mammals tolerate repetitive episodes of peripheral ischemia/reperfusion as part of the cardiovascular adjustments supporting long duration dives. These adjustments allow marine mammals to optimize the use of their body oxygen stores while diving but can result in selectively reduced perfusion to peripheral tissues. Remarkably, diving mammals show no apparent detrimental effects associated with these ischemia/reperfusion events. Here, we review the current knowledge regarding the strategies marine mammals use to suppress inflammation and cope with oxidant generation potentially derived from diving-induced ischemia/reperfusion.

Muscular apoptosis but not oxidative stress increases with old age in a long-lived diver, the Weddell seal.

Seals experience repeated bouts of ischemia-reperfusion while diving, potentially exposing their tissues to increased oxidant generation and thus oxidative damage and accelerated aging. We contrasted markers of oxidative damage with antioxidant profiles across age and sex for propulsive (longissismus dorsi) and maneuvering (pectoralis) muscles of Weddell seals to determine whether previously observed morphological senescence is associated with oxidative stress. In longissismus dorsi, old (age 17-26 years) seals exhibited a nearly 2-fold increase in apoptosis over young (age 9-16 years) seals. There was no evidence of age-associated changes in lipid peroxidation or enzymatic antioxidant profiles. In pectoralis, 4-hydroxynonenal-Lys (4-HNE-Lys) levels increased 1.5-fold in old versus young seals, but lipid hydroperoxide levels and apoptotic index did not vary with age. Glutathione peroxidase activity was 1.5-fold higher in pectoralis of old versus young animals, but no other antioxidants changed with age in this muscle. With respect to sex, no differences in lipid hydroperoxides or apoptosis were observed in either muscle. Males had higher HSP70 expression (1.4-fold) and glutathione peroxidase activity (1.3-fold) than females in longissismus dorsi, although glutathione reductase activity was 1.4-fold higher in females. No antioxidants varied with sex in pectoralis. These results show that apoptosis is not associated with oxidative stress in aged Weddell seal muscles. Additionally, the data suggest that adult seals utilize sex-specific antioxidant strategies in longissismus dorsi but not pectoralis to protect skeletal muscles from oxidative damage.

Low guanylyl cyclase activity in Weddell seals: implications for peripheral vasoconstriction and perfusion of the brain during diving.

Nitric oxide (NO) is a potent vasodilator, which improves perfusion and oxygen delivery during tissue hypoxia in terrestrial animals. The vertebrate dive response involves vasoconstriction in select tissues, which persists despite profound hypoxia. Using tissues collected from Weddell seals at necropsy, we investigated whether vasoconstriction is aided by downregulation of local hypoxia signaling mechanisms. We focused on NO-soluble guanylyl cyclase (GC)-cGMP signaling, a well-known vasodilatory transduction pathway. Seals have a lower GC protein abundance, activity, and capacity to respond to NO stimulation than do terrestrial mammals. In seal lung homogenates, GC produced less cGMP (20.1 ± 3.7 pmol·mg protein-1·min-1) than the lungs of dogs (-80 ± 144 pmol·mg protein-1·min-1 less than seals), sheep (-472 ± 96), rats (-664 ± 104) or mice (-1,160 ± 104, P < 0.0001). Amino acid sequences of the GC enzyme α-subunits differed between seals and terrestrial mammals, potentially affecting their structure and function. Vasoconstriction in diving Weddell seals is not consistent across tissues; perfusion is maintained in the brain and heart but decreased in other organs such as the kidney. A NO donor increased median GC activity 49.5-fold in the seal brain but only 27.4-fold in the kidney, consistent with the priority of cerebral perfusion during diving. Nos3 expression was high in the seal brain, which could improve NO production and vasodilatory potential. Conversely, Pde5a expression was high in the seal renal artery, which may increase cGMP breakdown and vasoconstriction in the kidney. Taken together, the results of this study suggest that alterations in the NO-cGMP pathway facilitate the diving response.

Intrinsic anti-inflammatory properties in the serum of two species of deep-diving seal.

Weddell and elephant seals are deep-diving mammals, which rely on lung collapse to limit nitrogen absorption and prevent decompression injury. Repeated collapse and re-expansion exposes the lungs to multiple stressors, including ischemia-reperfusion, alveolar shear stress and inflammation. There is no evidence, however, that diving damages pulmonary function in these species. To investigate potential protective strategies in deep-diving seals, we examined the inflammatory response of seal whole blood exposed to lipopolysaccharide (LPS), a potent endotoxin. Interleukin-6 (IL6) cytokine production elicited by LPS exposure was 50 to 500 times lower in blood of healthy northern elephant seals and Weddell seals compared with that of healthy human blood. In contrast to the ∼6× increased production of IL6 protein from LPS-exposed Weddell seal whole blood, isolated Weddell seal peripheral blood mononuclear cells, under standard cell culture conditions using medium supplemented with fetal bovine serum (FBS), produced a robust LPS response (∼300×). Induction of Il6 mRNA expression as well as production of IL6, IL8, IL10, KC-like and TNFα were reduced by substituting FBS with an equivalent amount of autologous seal serum. Weddell seal serum also attenuated the inflammatory response of RAW 267.4 mouse macrophage cells exposed to LPS. Cortisol level and the addition of serum lipids did not impact the cytokine response in cultured cells. These data suggest that seal serum possesses anti-inflammatory properties, which may protect deep divers from naturally occurring inflammatory challenges such as dive-induced hypoxia-reoxygenation and lung collapse.

The Ability of Nitric Oxide to Lower Intraocular Pressure Is Dependent on Guanylyl Cyclase.

Purpose: While nitric oxide (NO) donors are emerging as treatments for glaucoma, the mechanism by which NO lowers intraocular pressure (IOP) is unclear. NO activates the enzyme guanylyl cyclase (GC) to produce cyclic guanosine monophosphate. We studied the ocular effects of inhaled and topically applied NO gas in mice and lambs, respectively. Methods: IOP and aqueous humor (AqH) outflow were measured in WT and GC-1α subunit null (GC-1-/-) mice. Mice breathed 40 parts per million (ppm) NO in O2 or control gas (N2/O2). We also studied the effect of ocular NO gas exposure (80, 250, 500, and 1000 ppm) on IOP in anesthetized lambs. NO metabolites were measured in AqH and plasma. Results: In awake WT mice, breathing NO for 40 minutes lowered IOP from 14.4 ± 1.9 mm Hg to 10.9 ± 1.0 mm Hg (n = 11, P < 0.001). Comparable results were obtained in anesthetized WT mice (n = 10, P < 0.001). In awake or anesthetized GC-1-/- mice, IOP did not change under similar experimental conditions (P ≥ 0.08, n = 20). Breathing NO increased in vivo outflow facility in WT but not GC-1-/- mice (+13.7 ± 14.6% vs. -12.1 ± 9.4%, n = 4 each, P < 0.05). In lambs, ocular exposure to NO lowered IOP in a dose-dependent manner (-0.43 mm Hg/ppm NO; n = 5 with 40 total measurements; P = 0.04) without producing corneal pathology or altering pulmonary and systemic hemodynamics. After ocular NO exposure, NO metabolites were increased in AqH (n = 8, P < 0.001) but not in plasma. Conclusions: Breathing NO reduced IOP and increased outflow facility in a GC-dependent manner in mice. Exposure of ovine eyes to NO lowers IOP.