Xiangying Jiao

Adiponectin Cardioprotection After Myocardial Ischemia/Reperfusion Involves the Reduction of Oxidative/Nitrative Stress

Background— Several clinical studies have demonstrated that levels of adiponectin are significantly reduced in patients with type 2 diabetes and that adiponectin levels are inversely related to the risk of myocardial ischemia. The present study was designed to determine the mechanism by which adiponectin exerts its protective effects against myocardial ischemia/reperfusion. Methods and Results— Adiponectin−/− or wild-type mice were subjected to 30 minutes of myocardial ischemia followed by 3 hours or 24 hours (infarct size and cardiac function) of reperfusion. Myocardial infarct size and apoptosis, production of peroxynitrite, nitric oxide (NO) and superoxide, and inducible NO synthase (iNOS) and gp91phox protein expression were compared. Myocardial apoptosis and infarct size were markedly enhanced in adiponectin−/− mice (P<0.01). Formation of NO, superoxide, and their cytotoxic reaction product, peroxynitrite, were all significantly higher in cardiac tissue obtained from adiponectin−/− than from wild-type mice (P<0.01). Moreover, myocardial ischemia/reperfusion–induced iNOS and gp91phox protein expression was further enhanced, but endothelial NOS phosphorylation was reduced in cardiac tissue from adiponectin−/− mice. Administration of the globular domain of adiponectin 10 minutes before reperfusion reduced myocardial ischemia/reperfusion–induced iNOS/gp91phox protein expression, decreased NO/superoxide production, blocked peroxynitrite formation, and reversed proapoptotic and infarct-enlargement effects observed in adiponectin−/− mice. Conclusion— The present study demonstrates that adiponectin is a natural molecule that protects hearts from ischemia/reperfusion injury by inhibition of iNOS and nicotinamide adenine dinucleotide phosphate-oxidase protein expression and resultant oxidative/nitrative stress.

Endothelial dysfunction in adiponectin deficiency and its mechanisms involved

Endothelial dysfunction is the earliest pathologic alteration in diabetic vascular injury and plays a critical role in the development of atherosclerosis. Plasma levels of adiponectin (APN), a novel vasculoprotective adipocytokine, are significantly reduced in diabetic patients, but its relationship with endothelial dysfunction remains unclear. The present study aims to determine whether APN deficiency may cause endothelial dysfunction and to investigate the involved mechanisms. Vascular rings were made from the aortic vessels of wild type (WT) or APN knockout (APN(-/-)) mice. Endothelial function, total NO production, eNOS expression/phosphorylation, superoxide production, and peroxynitrite formation were determined. ACh and acidified NaNO2 (endothelial dependent and independent vasodilators, respectively) caused similar concentration-dependent vasorelaxation in WT vascular rings. APN(-/-) rings had a normal response to acidified NaNO2, but a markedly reduced response to ACh (>50% reduction vs. WT, P<0.01). Both superoxide and peroxynitrite production were increased in APN(-/-) vessels (P<0.01 vs. WT). Pretreatment with superoxide scavenger Tiron significantly, but incompletely restored vascular vasodilatory response to ACh. In APN(-/-) vessels, eNOS expression was normal, but NO production and eNOS phosphorylation was significantly reduced (P<0.01 vs. WT). Treatment of APN(-/-) mice in vivo with the globular domain of adiponectin reduced aortic superoxide production, increased eNOS phosphorylation, and normalized vasodilatory response to ACh. Increased NO inactivation combined with decreased basal NO production contributes to endothelial dysfunction development when there is a paucity of APN production. Interventions directed towards increasing plasma APN levels may improve endothelial function, and reduce cardiovascular complications suffered by diabetic patients.

Nitrative Inactivation of Thioredoxin-1 and Its Role in Postischemic Myocardial Apoptosis

Background— Intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased tissues but not in normal tissues; definitive evidence to support a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is limited, however. The aims of the present study were to determine whether thioredoxin (Trx), a novel antioxidant and antiapoptotic molecule, is susceptible to nitrative inactivation and to establish a causative link between Trx nitration and postischemic myocardial apoptosis. Methods and Results— In vitro exposure of human Trx-1 to 3-morpholinosydnonimine resulted in significant Trx-1 nitration and almost abolished Trx-1 activity. 3-morpholinosydnonimine–induced nitrative Trx-1 inactivation was completely blocked by MnTE-2-PyP5+ (a superoxide dismutase mimetic) and markedly attenuated by PTIO (a nitric oxide scavenger). Administration of either reduced or oxidized Trx-1 in vivo attenuated myocardial ischemia/reperfusion injury (>50% reduction in apoptosis and infarct size, P<0.01). However, administration of nitrated Trx-1 failed to exert a cardioprotective effect. In cardiac tissues obtained from ischemic/reperfused heart, significant Trx-1 nitration was detected, Trx activity was markedly inhibited, Trx-1/ASK1 (apoptosis signal-regulating kinase-1) complex formation was abolished, and apoptosis signal-regulating kinase-1 activity was increased. Treatment with either FP15 (a peroxynitrite decomposition catalyst) or MnTE-2-PyP5+ 10 minutes before reperfusion blocked nitrative Trx inactivation, attenuated apoptosis signal-regulating kinase-1 activation, and reduced postischemic myocardial apoptosis. Conclusions— These results strongly suggest that nitrative inactivation of Trx plays a proapoptotic role under those pathological conditions in which production of reactive nitrogen species is increased and that antinitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/reperfusion, in which pathological apoptosis is increased.

Chronic β-Adrenergic Receptor Stimulation Induces Cardiac Apoptosis and Aggravates Myocardial Ischemia/Reperfusion Injury by Provoking Inducible Nitric-Oxide Synthase-Mediated Nitrative Stress

The present study provides evidence that inducible nitric-oxide synthase (iNOS)-mediated nitrative stress plays a pivotal role in chronic β-adrenergic receptor (AR) stimulation-induced cardiac damage. In mice, 14 days of isoproterenol (ISO) stimulation via an osmotic minipump induced an up-regulation of iNOS as evidenced by increases in mRNA, protein expression, and immunochemical staining of myocardial iNOS. Serum level of C-reactive protein, an inflammatory mediator, was also markedly increased. Under chronic ISO stimulation, the up-regulated iNOS produced a significantly increased amount of nitric oxide (NO) and its byproduct, peroxynitrite, in the circulation and heart and subsequently resulted in an accelerated myocardial apoptosis. Forty-minute myocardial ischemia (MI) and 24-h reperfusion (R) further increased NO production and peroxynitrite formation and resulted in an enlarged infarct size in mice receiving chronic ISO stimulation. However, the treatment with a selective iNOS inhibitor [N-(3-(aminomethyl) benzyl)acetamidine] (1400W) or the use of a genetic modified animal (iNOS-knockout mice) markedly reduced iNOS-mediated production of NO and formation of peroxynitrite and consequently significantly decreased myocardial apoptosis and infarct size, showing a crucial link between iNOS-mediated nitrative stress and myocardial injury. In conclusion, chronic β-AR stimulation up-regulates iNOS expression and increases NO production in the heart, which subsequently markedly enhances formation of reactive nitrogen species/peroxynitrite in the heart, thereby eliciting myocardial apoptosis and potentiating MI/R injury.

INO-4885 [5,10,15,20-Tetra[N-(benzyl-4′-carboxylate)-2-pyridinium]-21H,23H-porphine Iron(III) Chloride], a Peroxynitrite Decomposition Catalyst, Protects the Heart against Reperfusion Injury in Mice

Oxidative/nitrative stress caused by peroxynitrite, the reaction product of superoxide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}) and nitric oxide (NO), is the primary cause of myocardial ischemia/reperfusion injury. The present study determined whether INO-4885 [5,10,15,20-tetra[N-(benzyl-4′-carboxylate)-2-pyridinium]-21H,23H-porphine iron(III) chloride], a new peroxynitrite decomposition catalyst, may provide cellular protection and protect heart from myocardial ischemia/reperfusion injury. Adult male mice were subjected to 30 min of ischemia and 3 or 24 h of reperfusion. Mice were randomized to receive vehicle, INO-4885 without catalytic moiety, or INO-4885 (3-300 μg/kg i.p.) 10 min before reperfusion. Infarct size, apoptosis, nitrotyrosine content, NO/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} production, and inducible nitric-oxide synthase (iNOS)/NADPH oxidase expression were determined. INO-4885 treatment reduced ischemia/reperfusion-induced protein nitration and caspase 3 activation in a dose-dependent fashion in the range of 3 to 100 μg/kg. However, doses exceeding 100 μg/kg produced nonspecific effects and attenuated its protective ability. At the optimal dose (30 μg/kg), INO-4885 significantly reduced infarct size (p < 0.01), decreased apoptosis (p < 0.01), and reduced tissue nitrotyrosine content (p < 0.01). As expected, INO-4885 had no effect on ischemia/reperfusion-induced iNOS expression and NO overproduction. To our surprise, this compound significantly reduced superoxide production and partially blocked NADPH oxidase overexpression in the ischemic/reperfused cardiac tissue. Additional experiments demonstrated that INO-4885 provided better cardioprotection than N-(3-(aminomethyl)benzyl)acetamidine (1400W, a selective iNOS inhibitor), apocynin (an NADPH oxidase inhibitor), or Tiron (a cell-permeable superoxide scavenger). Taken together, our data demonstrated that INO-4885 is a cardioprotective molecule that attenuates myocardial reperfusion injury by facilitating peroxynitrite decomposition and inhibiting NADPH oxidase-derived \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} production.

Tonic beta-adrenergic drive provokes proinflammatory and proapoptotic changes in aging mouse heart.

Tonic activation of adrenergic drive has been found to be associated with aging, and its further activation is also seen in aging patients with major surgery or congestive heart failure. Nevertheless, its potential effect on the aging heart remains enigmatic. In the present study, at baseline, significant inflammatory and apoptotic changes were found in the aging mouse (20 months old), as evidenced by increases in inducible nitric oxide synthase (iNOS) expression, myocardial apoptosis in the heart, and C-reactive protein (CRP) release in the circulation. These phenotypic changes in aging animals can be induced in young animals (3 months old) by chronic beta-adrenergic receptor (AR) stimulation with isoproterenol (ISO), and they can be markedly reduced in aging animals by chronic beta-blockade with propranolol. Compared with young animals, chronic beta-AR stimulation with ISO in aging animals induced larger increases in iNOS expression, nitrotyrosine formation in the heart, and nitric oxide (NO) production and CRP release in the circulation; it also accelerated myocardial apoptosis and resulted in an enlarged infarct size when animals were subjected to myocardial ischemia and reperfusion (MI/R). However, the pretreatment of 1400W (N-(3-(aminomethyl) benzyl)acetamidine)-a specific iNOS inhibitor-significantly reduced iNOS-mediated nitrative stress associated with a marked decrease in myocardial apoptosis and infarct size in aging mice. These results demonstrate that tonic activation of the beta-adrenergic system associated with aging induces proinflammatory and proapoptotic changes in the heart and that additional beta-AR stimulation results in an exaggerated nitrative stress, mediated by iNOS, that is associated with more severe myocardial injury in aging mice.

Thioredoxin glycation: A novel posttranslational modification that inhibits its antioxidant and organ protective actions.

Thioredoxin (Trx) is an antioxidant and antiapoptotic molecule, and its activity is regulated by posttranslational modifications. Trx-1 has recently been reported to exert potent protective action against endotoxic liver injury. However, whether Trx-1 activity is affected by endotoxin has never been previously investigated. The aim of the present study was to determine endotoxic regulation of Trx-1, and the potential mechanism involved. In vitro coincubation of Trx-1 with lipopolysaccharide (LPS) inhibited Trx-1 activity in a dose- and time-dependent fashion. The core (polysaccharide containing) region of LPS had a greater inhibitory effect on Trx-1 activity than its Lipid A fragment, suggesting the involvement of sugar groups. Periodic acid-Schiff staining and fructosamine assay demonstrated that Trx-1 was rapidly glycated by LPS. Aminoguanidine, a competitive glycation-inhibitor, completely blocked the inhibitory effect of LPS on Trx-1. Moreover, Trx-1 activity was also significantly inhibited by in vitro ribose incubation. Finally, in vivo administration of Trx-1, but not glycated Trx-1, reduced LPS-induced hepatic injury. Taken together, these results demonstrated for the first time that Trx-1 is susceptible to glycative inactivation. This novel posttranslational Trx-1 modification contributes to LPS cytotoxicity, suggesting that blockading protein glycation might be a new therapeutic strategy against endotoxic organ injury.