Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis - a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Here, by using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology we discovered that execution of ferroptosis involves a highly organized oxygenation center, whereby only one class of phospholipids, phosphatidylethanolamines (PE), undergoes oxidation in the ER-associated compartments with the specificity towards two fatty acyls – arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 acts as a specific anti-ferroptotic rescue pathway. Lipoxygenases (LOX) generate doubly- and triply-oxygenated (15-hydroperoxy)-di-acylated PE species which act as death signals while tocopherols and tocotrienols suppress LOX and protect against ferroptosis suggesting an unforeseen homeostatic physiological role of vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.

Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid.

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δmice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1−/Δmice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-a (SIRP-a) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-a in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-a on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-a. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-a and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47 phox (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-a. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxidemediated vasodilation in a SIRP-a-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-a signaling axis and was associated with increased superoxide production and cell death. A SIRP-a antibody that blocks thrombospondin-1 activation of SIRP-a mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-a activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-a on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.