Phillip Chumley

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Binding of Xanthine Oxidase to Vascular Endothelium KINETIC CHARACTERIZATION AND OXIDATIVE IMPAIRMENT OF NITRIC OXIDE-DEPENDENT SIGNALING

Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 μg/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (K d = 6 nm) at 4 °C to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by ∼50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome creduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both protease-reversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.

Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation

Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

Nitric oxide regulation of superoxide-dependent lung injury: Oxidant-protective actions of endogenously produced and exogenously administered nitric oxide

The influence of endogenous cell .NO production and .NO derived from exogenous sources on oxidant injury to cultured fetal rat lung alveolar epithelium and an animal model of pulmonary oxidant injury was examined. Confluent fetal rat alveolar epithelial cell monolayers were stimulated to produce .NO after treatment with a combination of cytokines (IL-1 beta, TNF-alpha, IFN-gamma), LPS and zymosan-activated serum (CZ). Cell injury, assessed by 14C-adenine release, was significantly increased compared to basal and CZ-induced cells after inhibition of .NO synthesis by L-NMMA. Cell monolayer macromolecule barrier function was determined by the rate of diffusion of 125I-albumin from the apical to basolateral side of monolayers. Following exposure to CZ and/or O2.- generated by xanthine oxidase + lumazine (XO), endogenous cell .NO production and exogenously administered .NO (from .NO donors S-nitrosyl-glutathione and S-nitroso-N-acetylpenicillamine) significantly inhibited the increased monolayer permeability induced by exposure to reactive oxygen species. Furthermore, inhalation of 5-10 ppm of .NO significantly reduced the toxicity of > 95% oxygen to adult rats. We conclude that when cultured pulmonary epithelial cells and lung tissue in vivo are subjected to inflammatory mediators or acute oxidative stress, .NO can play a protective role by inhibiting O2.(-)-dependent toxicity.

Nitrolinoleate, a nitric oxide-derived mediator of cell function: Synthesis, characterization, and vasomotor activity

Nitric oxide (•NO) and •NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO2). Nitroselenylation of linoleic acid yielded LNO2 that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, 15N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO2 metabolism, using L[15N]O2 as an internal standard, revealed that LNO2 is rapidly metabolized by rat aortic smooth muscle (RASM) monolayers and rat thoracic aorta, resulting in nitrite production and up to 3-fold increases in cGMP (ED50 = 30 μM for RASM, 50 μM for aorta). LNO2 induced endothelium-independent relaxation of preconstricted rat aortic rings, which was unaffected by LG-nitro-l-arginine methyl ester addition and inhibited by the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one and the •NO scavenger HbO2. These results reveal that synthetic LNO2, identical to lipid derivatives produced biologically by the reaction of •NO and •NO-derived species with oxidizing unsaturated fatty acids (e.g., linoleate), can transduce vascular signaling actions of •NO.

Nitration of unsaturated fatty acids by nitric oxide-derived reactive species

Reactions of linoleate (and presumably other unsaturated fatty acids) with reactive nitrogen species that form in biological systems from secondary reactions of .NO yield two main nitration product groups, LNO2 (formed by ONOO-, .NO2, or NO2+ reaction with linoleate), and LONO2 (formed by HONO reaction with 13(S)-HPODE, or .NO termination with LOO.). Comparison of HPLC retention times and m/z for lipid nitration products indicate that the mechanisms of nitrated product formation converge at several points: (i) The initial product of HONO attack on LOOH will be LOONO, which is identical to the initial termination product of LOO. reaction with .NO. (ii) Dissociation of LOONO to give LO. and .NO2 via caged radicals, which recombine to give LONO2 (m/z 340) will occur, regardless of how LOONO is formed (Fig. 7). (iii) In some experiments, the reaction of O2- (where oxidation is initiated by xanthine oxidase-derived O2- production and metal-dependent decomposition of H2O2) with .NO will result in generation of ONOO-. Nitration of unsaturated lipid by this species will yield a species demonstrated herein to be LNO2. Lipid oxidation leads to formation of bioactive products, including hydroxides, hydroperoxides, and isoprostanes. In vivo, nitrated lipids (LNO2, LONO2) may also possess bioactivity, for example through eicosanoid receptor binding activity, or by acting as antagonists/competitive inhibitors of eicosanoid receptor-ligand interactions. In addition, nitrated lipids could mediate signal transduction via direct .NO donation, transnitrosation, or following reductive metabolism. Similar bioactive products are formed following ONOO- reaction with glucose, glycerol, and other biomolecules.

Manganese-porphyrin reactions with lipids and lipoproteins

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.

Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 μg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (∼ 50%), NADPH oxidase 4 (NOX4; ∼100%), and transforming growth factor-β expression (∼200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.