Yuexing Yuan

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.

C1q/TNF-Related Proteins, A Family of Novel Adipokines, Induce Vascular Relaxation Through the Adiponectin Receptor-1/AMPK/eNOS/Nitric Oxide Signaling Pathway

Objective—Reduced plasma adiponectin (APN) in diabetic patients is associated with endothelial dysfunction. However, APN knockout animals manifest modest systemic dysfunction unless metabolically challenged. The protein family CTRPs (C1q/TNF-related proteins) has recently been identified as APN paralogs and some CTRP members share APN′s metabolic regulatory function. However, the vasoactive properties of CTRPs remain completely unknown. Methods and Results—The vasoactivity of currently identified murine CTRP members was assessed in aortic vascular rings and underlying molecular mechanisms was elucidated in human umbilical vein endothelial cells. Of 8 CTRPs, CTRPs 3, 5, and 9 caused significant vasorelaxation. The vasoactive potency of CTRP9 exceeded that of APN (3-fold) and is endothelium-dependent and nitric oxide (NO)-mediated. Mechanistically, CTRP9 increased AMPK/Akt/eNOS phosphorylation and increased NO production. AMPK knockdown completely blocked CTRP9-induced Akt/eNOS phosphorylation and NO production. Akt knockdown had no significant effect on CTRP9-induced AMPK phosphorylation, but blocked eNOS phosphorylation and NO production. Adiponectin receptor 1, but not receptor 2, knockdown blocked CTRP9-induced AMPK/Akt/eNOS phosphorylation and NO production. Finally, preincubating vascular rings with an AMPK-inhibitor abolished CTRP9-induced vasorelaxative effects. Conclusion—We have provided the first evidence that CTRP9 is a novel vasorelaxative adipocytokine that may exert vasculoprotective effects via the adiponectin receptor 1/AMPK/eNOS dependent/NO mediated signaling pathway.

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.

Cardiomyocyte-derived adiponectin is biologically active in protecting against myocardial ischemia-reperfusion injury

Adiponectin (APN) has traditionally been viewed as an adipocyte-specific endocrine molecule with cardioprotective effects. Recent studies suggest that APN is also expressed in cardiomyocytes. However, biological significances of this locally produced APN remain completely unknown. The aim of this study was to investigate the pathological and pharmacological significance of cardiac-derived APN in cardiomyocyte pathology. Adult cardiomyocytes from wild-type littermates (WT) or gene-deficient mice were pretreated with vehicle (V) or rosiglitazone (RSG) for 6 h followed by simulated ischemia-reperfusion (SI/R, 3 h/12 h). Compared with WT cardiomyocytes, myocytes from APN knockout (APN-KO) mice sustained greater SI/R injury, evidenced by greater oxidative/nitrative stress, caspase-3 activity, and lactate dehydrogenase (LDH) release (P < 0.05). Myocytes from adiponectin receptor 1 knockdown (AdipoR1-KD) or AdipoR1-KD/AdipoR2-KO mice had slightly increased SI/R injury, but the difference was not statistically significant. RSG significantly (P < 0.01) increased APN mRNA and protein expression, upregulated AdipoR1/AdipoR2 expression, reduced SI/R-induced apoptosis, and decreased LDH release in WT cardiomyocytes. However, the anti-oxidative/anti-nitrative and cell protective effects of RSG were completely lost in APN-KO cardiomyocytes (P > 0.05 vs. vehicle group), although a comparable degree of AdipoR1/AdipoR2 upregulation was observed. The upregulatory effect of RSG on APN mRNA and protein expression was significantly potentiated in AdipoR1-KD/AdipoR2-KO cardiomyocytes. However, the cellular protective effects of RSG were significantly blunted, although not completely lost, in these cells. These results demonstrated that cardiomyocyte APN is biologically active in protecting cells against SI/R injury. Moreover, this locally produced APN achieves its protective effect primarily through paracrine/autocrine activation of APN receptors.

C1q/Tumor Necrosis Factor-Related Protein-3, a Newly Identified Adipokine, Is a Novel Antiapoptotic, Proangiogenic, and Cardioprotective Molecule in the Ischemic Mouse Heart

Background— Obesity and diabetes mellitus adversely affect postischemic heart remodeling via incompletely understood mechanisms. C1q/tumor necrosis factor–related protein-3 (CTRP3) is a newly identified adipokine exerting beneficial metabolic regulation, similar to adiponectin. The aim of the present study was to determine whether CTRP3 may regulate postischemic cardiac remodeling and cardiac dysfunction, and, if so, to elucidate the underlying mechanisms. Methods and Results— Male adult mice were subjected to myocardial infarction (MI) via left anterior descending coronary artery occlusion. Both the effect of MI on endogenous CTRP3 expression/production and the effect of exogenous CTRP3 (adenovirus or recombinant CTRP3) replenishment on MI injury were investigated. MI significantly inhibited adipocyte CTRP3 expression and reduced the plasma CTRP3 level, reaching a nadir 3 days after MI. CTRP3 replenishment improved survival rate (P<0.05), restored cardiac function, attenuated cardiomyocyte apoptosis, increased revascularization, and dramatically reduced interstitial fibrosis (all P<0.01). CTRP3 replenishment had no significant effect on cardiac AMP-activated protein kinase phosphorylation but significantly increased Akt phosphorylation and expression of hypoxia inducing factor-1&agr; and vascular endothelial growth factor. Surprisingly, treatment of human umbilical vascular endothelial cells with CTRP3 did not directly affect nitric oxide production or tube formation. However, preconditioned medium from CTRP3-treated cardiomyocytes significantly enhanced human umbilical vascular endothelial cell tube formation, an effect blocked by either pretreatment of cardiomyocytes with a PI3K inhibitor or pretreatment of human umbilical vascular endothelial cells with a vascular endothelial growth factor inhibitor. Finally, the protective effect of adipocyte-conditioned medium against hypoxia-induced cardiomyocyte injury is significantly blunted when CTRP3 is knocked down. Conclusion— CTRP3 is a novel antiapoptotic, proangiogenic, and cardioprotective adipokine, the expression of which is significantly inhibited after MI.

Adiponectin: An Indispensable Molecule in Rosiglitazone Cardioprotection Following Myocardial Infarction

Rationale: Patients treated with peroxisome proliferator-activated receptor (PPAR)-&ggr; agonist manifest favorable metabolic profiles associated with increased plasma adiponectin (APN). However, whether increased APN production as a result of PPAR-&ggr; agonist treatment is an epiphenomenon or is causatively related to the cardioprotective actions of PPAR-&ggr; remains unknown. Objective: To determine the role of APN in rosiglitazone (RSG) cardioprotection against ischemic heart injury. Methods and Results: Adult male wild-type (WT) and APN knockdown/knockout (APN+/− and APN−/−) mice were treated with vehicle or RSG (20 mg/kg per day), and subjected to coronary artery ligation 3 days after beginning treatment. In WT mice, RSG (7 days) significantly increased adipocyte APN expression, elevated plasma APN levels (2.6-fold), reduced infarct size (17% reduction), decreased apoptosis (0.23±0.02% versus 0.47±0.04% TUNEL-positive in remote nonischemic area), attenuated oxidative stress (48.5% reduction), and improved cardiac function (P<0.01). RSG-induced APN production and cardioprotection were significantly blunted (P<0.05 versus WT) in APN+/−, and completely lost in APN−/− (P>0.05 versus vehicle-treated APN−/− mice). Moreover, treatment with RSG for up to 14 days significantly improved the postischemic survival rate of WT mice (P<0.05 versus vehicle group) but not APN knockdown/knockout mice. Conclusions: The cardioprotective effects of PPAR-&ggr; agonists are critically dependent on its APN stimulatory action, suggesting that under pathological conditions where APN expression is impaired (such as advanced type 2 diabetes), the harmful cardiovascular effects of PPAR-&ggr; agonists may outweigh its cardioprotective benefits.

C1q/Tumor Necrosis Factor–Related Protein-9, a Novel Adipocyte-Derived Cytokine, Attenuates Adverse Remodeling in the Ischemic Mouse Heart via Protein Kinase A Activation

Background— C1q/tumor necrosis factor–related protein-9 (CTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. However, the role of CTRP9 in postmyocardial infarction remodeling remains completely unknown. This study determined whether CTRP9 may regulate cardiac remodeling after acute myocardial infarction (AMI) and elucidated the underlying mechanisms. Methods and Results— Male adult mice were subject to AMI by left anterior descending coronary artery ligation or sham surgery and treated with saline (vehicle) or globular CTRP9 via peritoneal implant osmotic pumps for 6 weeks. H9C2 cardiac cell lines were used in vitro for determining underlying mechanisms. Adipocyte CTRP9 expression and plasma CTRP9 levels were both significantly reduced after AMI. Compared with vehicle, CTRP9 treatment improved animal survival rate (P<0.05), restored cardiac function (P<0.05), attenuated adverse remodeling (P<0.01), and ameliorated cardiomyocyte apoptosis and fibrosis after AMI (P<0.01). Among the multiple antiremodeling molecules determined, AMP-activated protein kinase, protein kinase A (PKA), and Akt were significantly activated in CTRP9-treated heart. Surprisingly, CTRP9 remains cardioprotective in mice with cardiomyocyte-specific overexpression of a mutant AMP-activated protein kinase &agr;2 subunit (AMPK-DN). Additional in vitro experiments demonstrated that administration of either PKA inhibitor or PKA-specific small interfering RNA virtually abolished the antiapoptotic effect of CTRP9 (P<0.05), whereas inhibition of Akt is less effective in blocking CTRP9 cardioprotection. Finally, CTRP9 phosphorylates BCL-2-associated agonist of cell death at its multiple antiapoptotic sites, an effect blocked by PKA inhibitor. Conclusions— We demonstrate that adipokine CTRP9 attenuates adverse cardiac remodeling after AMI, largely via a PKA-dependent pathway.

Reduced Cardioprotective Action of Adiponectin in High-Fat Diet–Induced Type II Diabetic Mice and Its Underlying Mechanisms

Diabetes exacerbates ischemic heart disease morbidity and mortality via incompletely understood mechanisms. Although adiponectin (APN) reduces myocardial ischemia/reperfusion (MI/R) injury in nondiabetic animals, whether APNs cardioprotective actions are altered in diabetes, a pathologic condition with endogenously reduced APN, has never been investigated. High-fat diet (HD)-induced diabetic mice and normal diet (ND) controls were subjected to MI via coronary artery ligation, and given vehicle or APN globular domain (gAPN, 2 μg/g) 10 min before reperfusion. Compared to ND mice (where gAPN exerted pronounced cardioprotection), HD mice manifested greater MI/R injury, and a tripled gAPN dose was requisite to achieve cardioprotective extent seen in ND mice (i.e., infarct size, apoptosis, and cardiac function). APN reduces MI/R injury via AMP-activated protein kinase (AMPK)-dependent metabolic regulation and AMPK-independent antioxidative/antinitrative pathways. Compared to ND, HD mice manifested significantly blunted gAPN-induced AMPK activation, basally and after MI/R (p<0.05). Although both low- and high-dose gAPN equally attenuated MI/R-induced oxidative stress (i.e., NADPH oxidase expression and superoxide production) and nitrative stress (i.e., inducible nitric oxide synthase expression, nitric oxide production, and peroxynitrite formation) in ND mice, only high-dose gAPN efficaciously did so in HD mice. We demonstrate for the first time that HD-induced diabetes diminished both AMPK-dependent and AMPK-independent APN cardioprotection, suggesting an unreported diabetic heart APN resistance.

Adiponectin Inhibits Tumor Necrosis Factor-α–Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.