Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer

Nitrite (NO2−) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II–IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet

A Novel Hybrid Compound LLP2A-Ale Both Prevented and Rescued the Osteoporotic Phenotype in a Mouse Model of Glucocorticoid-Induced Osteoporosis.

Prolonged glucocorticoid (GC) administration causes secondary osteoporosis (GIOP) and non-traumatic osteonecrosis. LLP2A-Ale is a novel bone-seeking compound that recruits mesenchymal stem cells to the bone surface, stimulates bone formation, and increases bone mass. The purpose of this study was to determine if treatment with LLP2A-Ale alone or in combination with parathyroid hormone (PTH) could prevent or treat GIOP in a mouse model. Four-month-old male Swiss-Webster mice were randomized to a prevention study with placebo, GC (day 1-28), and GC + LLP2A-Ale (IV, day 1) or a treatment study with placebo, GC (days 1-56), GC + LLP2A-Ale (IV, day 28), GC + PTH, and GC + LLP2A-Ale + PTH (days 28-56). Mice were killed on day 28 (prevention study) or on day 56 (treatment study). The study endpoints included bone mass, bone strength, serum markers of bone turnover (P1NP and CTX-I) and angiogenesis (VEGF-A), surface-based bone turnover, and blood vessel density. LLP2A-Ale prevented GC-induced bone loss and increased mechanical strength in the vertebral body (days 28 and 56) and femur (day 56). LLP2A-Ale, PTH, and LLP2A-Ale + PTH treatment significantly increased the mineralizing surface, bone formation rate, mineral apposition rate, double-labeled surface, and serum P1NP level on day 56. LLP2A-Ale and PTH treatment increased femoral blood vessel density and LLP2A-Ale increased serum VEGF-A on day 28. Therefore, LLP2A-Ale monotherapy could be a potential option to both prevent and treat GC-induced osteoporosis and bone fragility

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer — the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40–60% saturated with oxygen (near the Hb P(50)), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control

IRAK-1 Contributes to Lipopolysaccharide-induced Reactive Oxygen Species Generation in Macrophages by Inducing NOX-1 Transcription and Rac1 Activation and Suppressing the Expression of Antioxidative Enzymes*

Inflammatory stimulants such as bacterial endotoxin (lipopolysaccharide (LPS)) are known to induce tissue damage and injury partly through the induction of reactive oxygen species (ROS). Although it is recognized that the induction of ROS in macrophages by LPS depends upon the expression and activation of NADPH oxidase, as well as the suppression of antioxidative enzymes involved in ROS clearance, the underlying molecular mechanisms are poorly defined. In this study, we examined the contribution of the interleukin-1 receptor-associated kinase 1 (IRAK-1) to LPS-induced generation of ROS. We observed that LPS induced significantly less ROS in IRAK-1−/− macrophages, indicating that IRAK-1 is critically involved in the induction of ROS. Mechanistically, we observed that IRAK-1 is required for LPS-induced expression of NOX-1, a key component of NADPH oxidase, via multiple transcription factors, including p65/RelA, C/EBPβ, and C/EBPδ. On the other hand, we demonstrated that IRAK-1 associated with and activated small GTPase Rac1, a known activator of NOX-1 oxidase enzymatic activity. IRAK-1 forms a close complex with Rac1 via a novel LWPPPP motif within the variable region of IRAK-1. On the other hand, we also observed that IRAK-1 is required for LPS-mediated suppression of peroxisome proliferator-activated receptor α and PGC-1α, nuclear factors essential for the expression of antioxidative enzymes such as GPX3 and catalase. Consequently, injection of LPS causes significantly less plasma lipid peroxidation in IRAK-1−/− mice compared with wild type mice. Taken together, our study reveals IRAK-1 as a novel component involved in the generation of ROS induced by LPS