Cardiovascular Adiponectin Resistance: The Critical Role of Adiponectin Receptor Modification.

For the past two decades, a great deal of research has been published concerning adiponectin (APN), an abundant protein responsible for regulating numerous biologic functions including antioxidative, antinitrative, anti-inflammatory, and cardioprotective effects. A review of APN and its two major receptors is timely because of new findings concerning the mechanisms by which APN signaling may be altered in pathologic processes such as diabetes and heart failure. In this review we elaborate on currently known information regarding the physiologic role of APN and the known mechanisms underlying pathologic APN resistance - namely, APN receptor downregulation and phosphorylation - and provide insight regarding the future directions of APN research including an assessment of the clinical applicability of preventing pathologic post-translational modification of the APN receptor.

Effective glycaemic control critically determines insulin cardioprotection against ischaemia/reperfusion injury in anaesthetized dogs.

AIMS: Experimental evidence has shown significant cardioprotective effects of insulin, whereas clinical trials produced mixed results without valid explanations. This study was designed to examine the effect of hyperglycaemia on insulin cardioprotective action in a preclinical large animal model of myocardial ischaemia/reperfusion (MI/R). METHODS AND RESULTS: Anaesthetized dogs were subjected to MI/R (30 min/4 h) and randomized to normal plasma insulin/euglycaemia (NI/NG), normal-insulin/hyperglycaemia (NI/HG), high-insulin/euglycaemia (HI/NG), and high-insulin/hyperglycaemia (HI/HG) achieved by controlled glucose/insulin infusion. Endogenous insulin production was abolished by peripancreatic vessel ligation. Compared with the control animals (NI/NG), hyperglycaemia (NI/HG) significantly aggravated MI/R injury. Insulin elevation at clamped euglycaemia (HI/NG) protected against MI/R injury as evidenced by reduced infarct size, decreased necrosis and apoptosis, and alleviated inflammatory and oxidative stress (leucocyte infiltration, myeloperoxidase, and malondialdehyde levels). However, these cardioprotective effects of insulin were markedly blunted in hyperglycaemic animals (HI/HG). In vitro mechanistic study in neonatal rat cardiomyocytes revealed that insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt was significantly attenuated by high glucose, accompanied by markedly increased IRS-1 O-GlcNAc glycosylation following hypoxia/reoxygenation. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine abrogated high glucose-induced O-GlcNAc modification and inactivation of IRS-1/Akt as well as cell injury. CONCLUSIONS: Our results, derived from a canine model of MI/R, demonstrate that hyperglycaemia blunts insulin protection against MI/R injury via hyperglycaemia-induced glycosylation and subsequent inactivation of insulin-signalling proteins. Our findings suggest that prevention of hyperglycaemia is critical for achieving maximal insulin cardioprotection for the ischaemic/reperfused hearts.

Lymphotoxin-α is a novel adiponectin expression suppressor following myocardial ischemia/reperfusion.

Recent clinical observations demonstrate adiponectin (APN), an adipocytokine with potent cardioprotective actions, is significantly reduced following myocardial ischemia/reperfusion (MI/R). However, mechanisms responsible for MI/R-induced hypoadiponectinemia remain incompletely understood. Adult male mice were subjected to 30-min MI followed by varying reperfusion periods. Adipocyte APN mRNA and protein expression and plasma APN and TNFα concentrations were determined. APN expression/production began to decline 3 h after reperfusion (reaching nadir 12 h after reperfusion), returning to control levels 7 days after reperfusion. Plasma TNFα levels began to increase 1 h after reperfusion, peaking at 3 h and returning to control levels 24 h after reperfusion. TNFα knockout significantly increased plasma APN levels 12 h after reperfusion but failed to improve APN expression/production 72 h after reperfusion. In contrast, TNF receptor-1 (TNFR1) knockout significantly restored APN expression 12 and 72 h after reperfusion, suggesting that other TNFR1 binding cytokines contribute to MI/R-induced APN suppression. Among many cytokines increased after MI/R, lymphotoxin-α (LTα) was the only cytokine remaining elevated 24-72 h after reperfusion. LTα knockout did not augment APN levels 12 h post-reperfusion, but did so by 72 h. Finally, in vitro treatment of adipocytes with TNFα and LTα at concentrations seen in MI/R plasma additively inhibited APN expression/production in TNFR1-dependent fashion. Our study demonstrates for the first time that LTα is a novel suppressor of APN expression and contributes to the sustained hypoadiponectinemia following MI/R. Combining anti-TNFα with anti-LTα strategies may achieve the best effects restoring APN in MI/R patients.

Regulation of Abro1/KIAA0157 during myocardial infarction and cell death reveals a novel cardioprotective mechanism for Lys63-specific deubiquitination.

Abro1 (also known as KIAA0157) is a scaffold protein that recruits polypeptides to assemble the BRISC (BRCC36-containing isopeptidase complex) deubiquitinating (DUB) enzyme. The four subunits of BRISC enzyme include Abro1, NBA1, BRE, and BRCC36 proteins. The DUB activity of the BRISC enzyme is exclusively directed against Lys63-linked polyubiquitin that does not have a proteolytic role but regulates protein function. In this report, we identified Abro1 as a specific interactor of THAP5, a zinc finger transcription factor that is involved in G2/M control and apoptosis. Abro1 was predominantly expressed in the heart and its protein level was regulated following experimentally induced myocardial ischemia/reperfusion (MI/R) injury. Furthermore, in patients with coronary artery disease (CAD), there was a dramatic increase in Abro1 protein level in the myocardial infarction (MI) area. Increase in Abro1 leads to a significant reduction in Lys63-linked ubiquitination of specific protein targets. Reducing the Abro1 protein level exacerbated cellular damage and cell death of cardiomyocytes due to MI/R injury. Additionally, overexpression of Abro1 in a heterologous system provided significant protection against oxidative stress-induced apoptosis. In conclusion, our results demonstrate that Abro1 protein level substantially increases in myocardial injury and coronary artery disease and this up-regulation is part of a novel cardioprotective mechanism. In addition, our data suggest a potential new link between Lys63-specific ubiquitination, its modulation by the BRISC DUB enzyme, and the development and progression of heart disease.

Systemic adiponectin malfunction as a risk factor for cardiovascular disease.

Adiponectin (Ad) is an abundant protein hormone regulatory of numerous metabolic processes. The 30 kDa protein originates from adipose tissue, with full-length and globular domain circulatory forms. A collagenous domain within Ad leads to spontaneous self-assemblage into various oligomeric isoforms, including trimers, hexamers, and high-molecular-weight multimers. Two membrane-spanning receptors for Ad have been identified, with differing concentration distribution in various body tissues. The major intracellular pathway activated by Ad includes phosphorylation of AMP-activated protein kinase, which is responsible for many of Ad's metabolic regulatory, anti-inflammatory, vascular protective, and anti-ischemic properties. Additionally, several AMP-activated protein kinase-independent mechanisms responsible for Ad's anti-inflammatory and anti-ischemic (resulting in cardioprotective) effects have also been discovered. Since its 1995 discovery, Ad has garnered considerable attention for its role in diabetic and cardiovascular pathology. Clinical observations have demonstrated the association of hypoadiponectinemia in patients with obesity, cardiovascular disease, and insulin resistance. In this review, we elaborate currently known information about Ad malfunction and deficiency pertaining to cardiovascular disease risk (including atherosclerosis, endothelial dysfunction, and cardiac injury), as well as review evidence supporting Ad resistance as a novel risk factor for cardiovascular injury, providing insight about the future of Ad research and the protein's potential therapeutic benefits.

Long-term active immunization with a synthetic peptide corresponding to the second extracellular loop of ß1-adrenoceptor induces both morphological and functional cardiomyopathic changes in rats.

BACKGROUND: ß1-adrenoceptors (ß1-ARs) are the predominant receptors in regulating heart functions. ß1-ARs contain 7-transmembrane domains (7TM), 3 extracellular loops and 3 intracellular loops. Among these loops, the second extracellular loop of ß1-AR (ß1-AR-ECII) plays an important role in the pathogenesis of heart failure. METHODS: (1) Select the sera-negative rats for antibodies against ß1-AR-ECII. (2) Detect the level of antibodies against ß1-AR-ECII in the process of active immunization with artificial synthetic peptides according to the sequence of human ß1-AR-ECII. (3) Observe its long-term role on cardiac structure and function in vivo by immunizing rats. (4) Detect the changes of T lymphocytes subsets in the process of active immunization. RESULTS: The peptides induced the production of specific autoantibody against ß1-AR-ECII. Furthermore, immunization with ß1-AR-ECII peptide induced both morphological and functional alterations in the hearts of rats, which potentially results in heart failure. In addition, induction of the autoantibodies against ß1-AR-ECII increased the CD4+/CD8+ ratio in the peripheral blood of rats. DISCUSSION: In this study, we determined the effect of anti-ß1-AR-ECII autoantibody on morphological and functional cardiomyopathic alterations by immunization of rats with a synthetic peptide corresponding to the ß1-AR-ECII for 18 months. These results provide further evidence that autoantibody against ß1-AR-ECII is involved in the pathogenesis of heart failure. But the underlying mechanisms need further study.

Triage during the week of the Sichuan earthquake: a review of utilized patient triage, care, and disposition procedures.

OBJECTIVES: There exists no standard hospital emergency department (ED) triage procedure model for earthquake victims. This study provides an overview of the hospital triage procedure used for patients evaluated and treated at the West China Hospital of Sichuan University, Chengdu in the Sichuan province of China, following the May 12, 2008 Wenchuan earthquake. METHODS: Emergency triage and treatment teams were comprised of senior emergency medicine (EM) attending physician, junior EM attending physician, EM residents, and specialty surgeons. Retrospective analysis of the hospital medical records of 2283 earthquake victims was performed. Victims' demographic data, triage process and group assignments, diagnoses and dispositions were reviewed. RESULTS: In the 2 weeks following the Wenchuan earthquake, 2283 total patients with earthquake-related injuries were admitted to our hospital. 54 victims (2.4%) were lost to follow up. Patients were triaged into four main groups: resuscitation (n=6), urgent treatment (n=369), delayed treatment (n=1502), and minor injuries (n=406). 68.9% (1572/2283) of the patients were admitted to the hospital during the 15 days after the earthquake. The overall hospital mortality rate was 1.0% (15/1572). 1304 victims were transferred to nearby hospitals after initial treatment, stabilization, or surgery. CONCLUSIONS: Proper triage strategy should be established prior to the onset of a mass casualty event and should be appropriate to both the severity of the disaster and the accepting facility resource availability. Triage methods utilizing multi-specialty treatment teams and dynamic hospital-wide coordination are critical for efficient, efficacious patient management. Hopefully, sharing with the emergency medicine community the arduous challenges we faced in the wake of the Wenchuan earthquake will be useful for planning the response to future disasters.

Insulin says NO to cardiovascular disease.

It is well recognized that insulin resistance found in patients with type 2 diabetes and obesity is a major risk factor for cardiovascular disease. Since its discovery in the 1920s, insulin has been used as an essential therapeutic agent in diabetes for blood glucose management. Recent studies demonstrate that insulin signalling is essential for normal cardiovascular function, and lack of it (i.e. insulin resistance) will result in cardiovascular dysfunction and disease. Moreover, insulin is the key component of glucose-insulin-potassium cocktail and exerts significant cardiovascular protective effect via a phosphatidylinositol 3'-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-Akt-eNOS)-dependent signalling mechanism in addition to its metabolic modulation, which renders it a potent organ protector in multiple clinical applications. This review focuses on insulin-initiated PI3K-Akt-eNOS survival signalling, with nitric oxide as an 'end effector' delivering cardioprotection in health and disease (especially in ischaemic heart disease), and highlights the impairment of this survival signalling as a key link between insulin resistance and cardiovascular disease.

A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse.

RATIONALE: coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. OBJECTIVE: the purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. METHODS AND RESULTS: male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22 ± 0.05 minutes, whereas the classic method requires 23.2 ± 0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. CONCLUSIONS: this new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.

Adiponectin: an indispensable molecule in rosiglitazone cardioprotection following myocardial infarction.

RATIONALE: Patients treated with peroxisome proliferator-activated receptor (PPAR)-gamma agonist manifest favorable metabolic profiles associated with increased plasma adiponectin (APN). However, whether increased APN production as a result of PPAR-gamma agonist treatment is an epiphenomenon or is causatively related to the cardioprotective actions of PPAR-gamma 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(+ or -) and APN(- or -)) 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 + or - 0.02% versus 0.47 + or - 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(+ or -), and completely lost in APN(- or -) (P>0.05 versus vehicle-treated APN(- or -) 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-gamma 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-gamma agonists may outweigh its cardioprotective benefits.

The effects of autoantibodies against the second extracellular loop of alpha(1)-adrenoceptor on vasoconstriction.

Recent studies have demonstrated the presence of autoantibodies against alpha(1)-adrenoceptor (alpha(1)-AAB) in the serum of patients with primary hypertension, and that these autoantibodies exert adrenergic-agonist-like effects. However, their role in the development of hypertension remains unclear. The current study determined whether alpha(1)-AAB can cause vascular contraction, and further investigated the cellular receptors that mediate their vasoactivity. Enzyme-linked immunosorbent assay (ELISA) was used to detect alpha(1)-AAB in blood samples collected from 73 patients with primary hypertension and 86 normotensive patients. IgGs were purified from mixed sera from 25 alpha(1)-AAB-positive hypertensive patients and 20 alpha(1)-AAB-negative normotensives, respectively. The vasoconstrictive effect of purified IgG from the sera of either hypertensive or normotensive patients was observed in isolated rat thoracic aorta, coronary artery, renal artery, middle cerebral artery, and mesenteric artery. The effects of alpha(1)-AAB administration on mean arterial blood pressure in vivo were also assessed. The frequency of alpha(1)-AAB presence was considerably higher in patients with primary hypertension than normotensive subjects (34.2 vs. 10.5%, P < 0.01). In isolated thoracic aortic rings, renal artery, middle cerebral artery, and coronary artery segments, alpha(1)-AAB induced vasoconstriction in a concentration-dependent fashion, which can be completely blocked by prazosin, a selective alpha(1)-adrenoceptor antagonist. The mean arterial pressure significantly increased after the administration of alpha(1)-AAB in vivo. Our results demonstrated that the alpha(1)-AAB (which exhibited remarkable effects of vascular contraction in vitro, elevated blood pressure in vivo, and showed no desensitization phenomena) may play a role in both elevating vascular resistance and the development and maintenance of hypertension.

Protective vascular and myocardial effects of adiponectin.

Adiponectin is an abundant plasma protein secreted from adipocytes that elicits protective effects in the vasculature and myocardium. In obesity and insulin-resistant states, adiponectin levels are reduced and loss of its protective effects might contribute to the excess cardiovascular risk observed in these conditions. Adiponectin ameliorates the progression of macrovascular disease in rodent models, consistent with its correlation with improved vascular outcomes in epidemiological studies. The mechanisms of adiponectin signaling are multiple and vary among its cellular sites of action. In endothelial cells, adiponectin enhances production of nitric oxide, suppresses production of reactive oxygen species, and protects cells from inflammation that results from exposure to high glucose levels or tumor necrosis factor, through activation of AMP-activated protein kinase and cyclic AMP-dependent protein kinase (also known as protein kinase A) signaling cascades. In the myocardium, adiponectin-mediated protection from ischemia-reperfusion injury is linked to cyclo-oxygenase-2-mediated suppression of tumor necrosis factor signaling, inhibition of apoptosis by AMP-activated protein kinase, and inhibition of excess peroxynitrite-induced oxidative and nitrative stress. In this Review, we provide an update of studies of the signaling effects of adiponectin in endothelial cells and cardiomyocytes.

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.

Autoantibody against AT1 receptor from preeclamptic patients induces vasoconstriction through angiotensin receptor activation.

Preeclampsia is a serious pathologic complication during pregnancy, and its pathogenesis remains poorly understood. Recent studies have demonstrated that autoantibodies against angiotensin II type 1A receptor (AT1-AA) are present in women with preeclampsia. However, their role in the development of hypertension in preeclamptic patients has never been previously investigated. The present study was designed to determine whether AT1-AA isolated from the sera of preeclamptic patients causes vascular constriction and, if so, to further investigate the cellular receptors that mediate their vasoactivity. Blood samples were collected from 49 pregnant women (preeclampsia = 31, control = 18) and AT1-AA was detected using enzyme-linked immunosorbent assay. Vasoconstrictive effect of purified IgG from the sera of either preeclamptic patients or normal pregnant women was determined in isolated rat thoracic aorta, arteriae cerebri media and coronary artery. Compared with normal pregnant women, frequency of AT1-AA positive samples was markedly increased in preeclamptic patients (80.7 vs. 5.6%, P < 0.01). In isolated thoracic aortic rings, middle cerebral artery and coronary artery segments, AT1-AA induced vasoconstriction in a concentration-dependent fashion (P < 0.01). The vasoconstrictive effect of AT1-AA was completely blocked by losartan, an AT1-receptor antagonist. These data demonstrate that the AT1-AA causes significant vascular constriction in large conduit vessel as well as small resistant vessels though activation of the AT1 receptor. These results suggest that overproduction of AT1-AA is a novel risk factor in pregnant women and may play a causative role in the development of hypertension and vascular injury in preeclamptic patients.

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.

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity.

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.

TNFalpha-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals.

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFalpha. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFalpha plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFalpha overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFalpha with etanercept or TNFalpha gene deletion reduced post-trauma myocyte apoptosis (P<0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and O2-, and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFalpha-/- mice, suggesting that oxidative/nitrative stress may contribute to TNFalpha-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P<0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFalpha production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.

Tumor necrosis factor-alpha in mechanic trauma plasma mediates cardiomyocyte apoptosis.

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation (P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) or TNF-alpha alone, but not with IL-1beta or IFN-gamma alone, caused significant caspase-3 activation (P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-alpha antibody, and TP isolated from TNF-alpha(-/-) mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-alpha (anti-TNF-alpha antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) (P < 0.01). Taken together, our study demonstrated that there exists a TNF-alpha-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.