Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

Targeting Adiponectin Receptor 1 Phosphorylation Against Ischemic Heart Failure.

BACKGROUND: Despite significantly reduced acute myocardial infarction (MI) mortality in recent years, ischemic heart failure continues to escalate. Therapeutic interventions effectively reversing pathological remodeling are an urgent unmet medical need. We recently demonstrated that AdipoR1 (APN [adiponectin] receptor 1) phosphorylation by GRK2 (G-protein-coupled receptor kinase 2) contributes to maladaptive remodeling in the ischemic heart. The current study clarified the underlying mechanisms leading to AdipoR1 phosphorylative desensitization and investigated whether blocking AdipoR1 phosphorylation may restore its protective signaling, reversing post-MI remodeling. METHODS: Specific sites and underlying molecular mechanisms responsible for AdipoR1 phosphorylative desensitization were investigated in vitro (neonatal and adult cardiomyocytes). The effects of AdipoR1 phosphorylation inhibition upon APN post-MI remodeling and heart failure progression were investigated in vivo. RESULTS: Among 4 previously identified sites sensitive to GRK2 phosphorylation, alanine substitution of Ser205 (AdipoR1S205A), but not other 3 sites, rescued GRK2-suppressed AdipoR1 functions, restoring APN-induced cell salvage kinase activation and reducing oxidative cell death. The molecular investigation followed by functional determination demonstrated that AdipoR1 phosphorylation promoted clathrin-dependent (not caveolae) endocytosis and lysosomal-mediated (not proteasome) degradation, reducing AdipoR1 protein level and suppressing AdipoR1-mediated cytoprotective action. GRK2-induced AdipoR1 endocytosis and degradation were blocked by AdipoR1S205A overexpression. Moreover, AdipoR1S205E (pseudophosphorylation) phenocopied GRK2 effects, promoted AdipoR1 endocytosis and degradation, and inhibited AdipoR1 biological function. Most importantly, AdipoR1 function was preserved during heart failure development in AdipoR1-KO (AdipoR1 knockout) mice reexpressing hAdipoR1S205A. APN administration in the failing heart reversed post-MI remodeling and improved cardiac function. However, reexpressing hAdipoR1WT in AdipoR1-KO mice failed to restore APN cardioprotection. CONCLUSIONS: Ser205 is responsible for AdipoR1 phosphorylative desensitization in the failing heart. Blockade of AdipoR1 phosphorylation followed by pharmacological APN administration is a novel therapy effective in reversing post-MI remodeling and mitigating heart failure progression.

Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function.

BACKGROUND: Patients with acute myocardial infarction suffer systemic metabolic dysfunction via incompletely understood mechanisms. Adipocytes play critical role in metabolic homeostasis. The impact of acute myocardial infarction upon adipocyte function is unclear. Small extracellular vesicles (sEVs) critically contribute to organ-organ communication. Whether and how small extracellular vesicle mediate post-MI cardiomyocyte/adipocyte communication remain unknown. METHODS: Plasma sEVs were isolated from sham control (Pla-sEVSham) or 3 hours after myocardial ischemia/reperfusion (Pla-sEVMI/R) and incubated with adipocytes for 24 hours. Compared with Pla-sEVSham, Pla-sEVMI/R significantly altered expression of genes known to be important in adipocyte function, including a well-known metabolic regulatory/cardioprotective adipokine, APN (adiponectin). Pla-sEVMI/R activated 2 (PERK-CHOP and ATF6 [transcription factor 6]-EDEM [ER degradation enhancing alpha-mannosidase like protein 1] pathways) of the 3 endoplasmic reticulum (ER) stress pathways in adipocytes. These pathological alterations were also observed in adipocytes treated with sEVs isolated from adult cardiomyocytes subjected to in vivo myocardial ischemia/reperfusion (MI/R) (Myo-sEVMI/R). Bioinformatic/RT-qPCR analysis demonstrates that the members of miR-23-27-24 cluster are significantly increased in Pla-sEVMI/R, Myo-sEVMI/R, and adipose tissue of MI/R animals. Administration of cardiomyocyte-specific miR-23-27-24 sponges abolished adipocyte miR-23-27-24 elevation in MI/R animals, supporting the cardiomyocyte origin of adipocyte miR-23-27-24 cluster. In similar fashion to Myo-sEVMI/R, a miR-27a mimic activated PERK-CHOP and ATF6-EDEM-mediated ER stress. Conversely, a miR-27a inhibitor significantly attenuated Myo-sEVMI/R-induced ER stress and restored APN production. RESULTS: An unbiased approach identified EDEM3 (ER degradation enhancing alpha-mannosidase like protein 3) as a novel downstream target of miR-27a. Adipocyte EDEM3 deficiency phenocopied multiple pathological alterations caused by Myo-sEVMI/R, whereas EDEM3 overexpression attenuated Myo-sEVMI/R-resulted ER stress. Finally, administration of GW4869 or cardiomyocyte-specific miR-23-27-24 cluster sponges attenuated adipocyte ER stress, improved adipocyte endocrine function, and restored plasma APN levels in MI/R animals. CONCLUSIONS: We demonstrate for the first time that MI/R causes significant adipocyte ER stress and endocrine dysfunction by releasing miR-23-27-24 cluster-enriched small extracellular vesicle. Targeting small extracellular vesicle-mediated cardiomyocyte-adipocyte pathological communication may be of therapeutic potential to prevent metabolic dysfunction after MI/R.

Dysfunctional APPL1-Mediated Epigenetic Regulation in Diabetic Vascular Injury.

BACKGROUND: APN (adiponectin) and APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) are potent vasculoprotective molecules, and their deficiency (eg, hypoadiponectinemia) contributes to diabetic vascular complications. However, the molecular mechanisms that govern their vasculoprotective genes as well as their alteration by diabetes remain unknown. METHODS: Diabetic medium-cultured rat aortic endothelial cells, mouse aortic endothelial cells from high-fat-diet animals, and diabetic human aortic endothelial cells were used for molecular/cellular investigations. The in vivo concept-prove demonstration was conducted using diabetic vascular injury and diabetic hindlimb ischemia models. RESULTS: In vivo animal experiments showed that APN replenishment caused APPL1 nuclear translocation, resulting in an interaction with HDAC (histone deacetylase) 2, which inhibited HDAC2 activity and increased H3Kac27 levels. Based on transcriptionome pathway-specific real-time polymerase chain reaction profiling and bioinformatics analysis, Angpt1 (angiopoietin 1), Ocln (occludin), and Cav1 (caveolin 1) were found to be the top 3 vasculoprotective genes suppressed by diabetes and rescued by APN in an APPL1-dependent manner. APN reverses diabetes-induced inhibition of Cav1 interaction with APPL1. APN-induced Cav1 expression was not affected by Angpt1 or Ocln deficiency, whereas APN-induced APPL1 nuclear translocation or upregulation of Angpt1/Ocln expression was abolished in the absence of Cav1 both in vivo and in vitro, suggesting Cav1 is upstream molecule of Angpt1/Ocln in response to APN administration. Chromatin immunoprecipitation-qPCR (quantitative polymerase chain reaction) demonstrated that APN caused significant enrichment of H3K27ac in Angpt1 and Ocln promoter region, an effect blocked by APPL1/Cav1 knockdown or HDAC2 overexpression. The protective effects of APN on the vascular system were attenuated by overexpression of HDAC2 and abolished by knocking out APPL1 or Cav1. The double knockdown of ANGPT1/OCLN blunted APN vascular protection both in vitro and in vivo. Furthermore, in diabetic human endothelial cells, HDAC2 activity is increased, H3 acetylation is decreased, and ANGPT1/OCLN expression is reduced, suggesting that the findings have important translational implications. CONCLUSIONS: Hypoadiponectinemia and dysregulation of APPL1-mediated epigenetic regulation are novel mechanisms leading to diabetes-induced suppression of vasculoprotective gene expression. Diabetes-induced pathological vascular remodeling may be prevented by interventions promoting APPL1 nuclear translocation and inhibiting HDAC2.

Hypoadiponectinemia-induced upregulation of microRNA449b downregulating Nrf-1 aggravates cardiac ischemia-reperfusion injury in diabetic mice.

Diabetes enhances myocardial ischemic/reperfusion (MI/R) injury via an incompletely understood mechanism. Adiponectin (APN) is a cardioprotective adipokine suppressed by diabetes. However, how hypoadiponectinemia exacerbates cardiac injury remains incompletely understood. Dysregulation of miRNAs plays a significant role in disease development. However, whether hypoadiponectinemia alters cardiac miRNA profile, contributing to diabetic heart injury, remains unclear. Methods and Results: Wild-type (WT) and APN knockout (APN-KO) mice were subjected to MI/R. A cardiac microRNA profile was determined. Among 23 miRNAs increased in APN-KO mice following MI/R, miR-449b was most significantly upregulated (3.98-fold over WT mice). Administrating miR-449b mimic increased apoptosis, enlarged infarct size, and impaired cardiac function in WT mice. In contrast, anti-miR-449b decreased apoptosis, reduced infarct size, and improved cardiac function in APN-KO mice. Bioinformatic analysis predicted 73 miR-449b targeting genes, and GO analysis revealed oxidative stress as the top pathway regulated by these genes. Venn analysis followed by luciferase assay identified Nrf-1 and Ucp3 as the two most important miR-449b targets. In vivo administration of anti-miR-449b in APN-KO mice attenuated MI/R-stimulated superoxide overproduction. In vitro experiments demonstrated that high glucose/high lipid and simulated ischemia/reperfusion upregulated miR-449b and inhibited Nrf-1 and Ucp3 expression. These pathological effects were attenuated by anti-miR-449b or Nrf-1 overexpression. In a final attempt to validate our finding in a clinically relevant model, high-fat diet (HFD)-induced diabetic mice were subjected to MI/R and treated with anti-miR-449b or APN. Diabetes significantly increased miR-449b expression and downregulated Nrf-1 and Ucp3 expression. Administration of anti-miR-449b or APN preserved cardiac Nrf-1 expression, reduced cardiac oxidative stress, decreased apoptosis and infarct size, and improved cardiac function. Conclusion: We demonstrated for the first time that hypoadiponectinemia upregulates miR-449b and suppresses Nrf-1/Ucp3 expression, promoting oxidative stress and exacerbating MI/R injury in this population. Dysregulated APN/miR-449b/oxidative stress pathway is a potential therapeutic target against diabetic MI/R injury.

miRNA-Mediated Suppression of a Cardioprotective Cardiokine as a Novel Mechanism Exacerbating Post-MI Remodeling by Sleep Breathing Disorders.

RATIONALE: Obstructive sleep apnea-hypopnea syndrome, a sleep breathing disorder in which chronic intermittent hypoxia (CIH) is the primary pathology, is associated with multiple cardiovascular diseases. However, whether and how CIH may affect cardiac remodeling following myocardial infarction (MI) remains unknown. OBJECTIVE: To determine whether CIH exposure at different periods of MI may exacerbate post-MI heart failure and to identify the mechanisms underlying CIH-exacerbated post-MI remodeling. METHODS AND RESULTS: Adult male mice were subjected to MI (4 weeks) with and without CIH (4 or 8 weeks). CIH before MI (CIH+MI) had no significant effect on post-MI remodeling. However, double CIH exposure (CIH+MI+CIH) or CIH only during the MI period (MI+CIH) significantly exacerbated pathological remodeling and reduced survival rate. Mechanistically, CIH activated TGF-ß (tumor growth factor-ß)/Smad (homologs of both the Drosophila protein MAD and the C. elegans protein SMA) signaling and enhanced cardiac epithelial to mesenchymal transition, markedly increasing post-MI cardiac fibrosis. Transcriptome analysis revealed that, among 15 genes significantly downregulated (MI+CIH versus MI), Ctrp9 (a novel cardioprotective cardiokine) was one of the most significantly inhibited genes. Real-time polymerase chain reaction/Western analysis confirmed that cardiomyocyte CTRP9 expression was significantly reduced in MI+CIH mice. RNA-sequencing, real-time polymerase chain reaction, and dual-luciferase reporter assays identified that microRNA-214-3p is a novel Ctrp9 targeting miRNA. Its upregulation is responsible for Ctrp9 gene suppression in MI+CIH. Finally, AAV9 (adeno-associated virus 9)-mediated cardiac-specific CTRP9 overexpression or rCTRP9 (recombinated CTRP9) administration inhibited TGF-ß/Smad and Wnt/ß-catenin pathways, attenuated interstitial fibrosis, improved cardiac function, and enhanced survival rate in MI+CIH animals. CONCLUSIONS: This study provides the first evidence that MI+CIH upregulates miR-214-3p, suppresses cardiac CTRP9 (C1q tumor necrosis factor-related protein-9) expression, and exacerbates cardiac remodeling, suggesting that CTRP9 may be a novel therapeutic target against pathological remodeling in MI patients with obstructive sleep apnea-hypopnea syndrome.

Small Extracellular Microvesicles Mediated Pathological Communications Between Dysfunctional Adipocytes and Cardiomyocytes as a Novel Mechanism Exacerbating Ischemia/Reperfusion Injury in Diabetic Mice.

BACKGROUND: Diabetes mellitus exacerbates myocardial ischemia/reperfusion (MI/R) injury by incompletely understood mechanisms. Adipocyte dysfunction contributes to remote organ injury. However, the molecular mechanisms linking dysfunctional adipocytes to increased MI/R injury remain unidentified. The current study attempted to clarify whether and how small extracellular vesicles (sEV) may mediate pathological communication between diabetic adipocytes and cardiomyocytes, exacerbating MI/R injury. METHODS: Adult male mice were fed a normal or a high-fat diet for 12 weeks. sEV (from diabetic serum, diabetic adipocytes, or high glucose/high lipid-challenged nondiabetic adipocytes) were injected intramyocardially distal of coronary ligation. Animals were subjected to MI/R 48 hours after injection. RESULTS: Intramyocardial injection of diabetic serum sEV in the nondiabetic heart significantly exacerbated MI/R injury, as evidenced by poorer cardiac function recovery, larger infarct size, and greater cardiomyocyte apoptosis. Similarly, intramyocardial or systemic administration of diabetic adipocyte sEV or high glucose/high lipid-challenged nondiabetic adipocyte sEV significantly exacerbated MI/R injury. Diabetic epididymal fat transplantation significantly increased MI/R injury in nondiabetic mice, whereas administration of a sEV biogenesis inhibitor significantly mitigated MI/R injury in diabetic mice. A mechanistic investigation identified that miR-130b-3p is a common molecule significantly increased in diabetic serum sEV, diabetic adipocyte sEV, and high glucose/high lipid-challenged nondiabetic adipocyte sEV. Mature (but not primary) miR-130b-3p was significantly increased in the diabetic and nondiabetic heart subjected to diabetic sEV injection. Whereas intramyocardial injection of a miR-130b-3p mimic significantly exacerbated MI/R injury in nondiabetic mice, miR-130b-3p inhibitors significantly attenuated MI/R injury in diabetic mice. Molecular studies identified AMPKα1/α2, Birc6, and Ucp3 as direct downstream targets of miR-130b-3p. Overexpression of these molecules (particularly AMPKα2) reversed miR-130b-3p induced proapoptotic/cardiac harmful effect. Finally, miR-130b-3p levels were significantly increased in plasma sEV from patients with type 2 diabetes mellitus. Incubation of cardiomyocytes with diabetic patient sEV significantly exacerbated ischemic injury, an effect blocked by miR-130b-3p inhibitor. CONCLUSIONS: We demonstrate for the first time that miR-130b-3p enrichment in dysfunctional adipocyte-derived sEV, and its suppression of multiple antiapoptotic/cardioprotective molecules in cardiomyocytes, is a novel mechanism exacerbating MI/R injury in the diabetic heart. Targeting miR-130b-3p mediated pathological communication between dysfunctional adipocytes and cardiomyocytes may be a novel strategy attenuating diabetic exacerbation of MI/R injury.

Withaferin A Prevents Myocardial Ischemia/Reperfusion Injury by Upregulating AMP-Activated Protein Kinase-Dependent B-Cell Lymphoma2 Signaling.

BACKGROUND: Withaferin A (WFA), an anticancer constituent of the plant Withania somnifera, inhibits tumor growth in association with apoptosis induction. However, the potential role of WFA in the cardiovascular system is little-studied and controversial.Methods and Results:Two different doses of WFA were tested to determine their cardioprotective effects in myocardial ischemia/reperfusion (MI/R) injury through evaluation of cardiofunction in wild-type and AMP-activated protein kinase domain negative (AMPK-DN) gentransgenic mice. Surprisingly, cardioprotective effects (improved cardiac function and reduced infarct size) were observed with low-dose WFA (1 mg/kg) delivery but not high-dose (5 mg/kg). Mechanistically, low-dose WFA attenuated myocardial apoptosis. It decreased MI/R-induced activation of caspase 9, the indicator of the intrinsic mitochondrial pathway, but not caspase 8. It also upregulated the level of AMP-activated protein kinase (AMPK) phosphorylation and increased the MI/R inhibited ratio of Bcl2/Bax. In AMPK-deficient mice, WFA did not ameliorate MI/R-induced cardiac dysfunction, attenuate infarct size, or restore the Bcl2/Bax (B-cell lymphoma2/Mcl-2-like protein 4) ratio. CONCLUSIONS: These results demonstrated for the first time that low-dose WFA is cardioprotective via upregulation of the anti-apoptotic mitochondrial pathway in an AMPK-dependent manner.

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.

COVID-19 and cardiovascular complications: updates of emergency medicine.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV-2 variants, has become a global pandemic resulting in significant morbidity and mortality. Severe cases of COVID-19 are characterized by hypoxemia, hyper-inflammation, cytokine storm in lung. Clinical studies have reported an association between COVID-19 and cardiovascular disease (CVD). Patients with CVD tend to develop severe symptoms and mortality if contracted COVID-19 with further elevations of cardiac injury biomarkers. Furthermore, COVID-19 itself can induce and promoted CVD development, including myocarditis, arrhythmia, acute coronary syndrome, cardiogenic shock, and venous thromboembolism. Although the direct etiology of SARS-CoV-2 induced cardiac injury remains unknown and under-investigated, it is suspected that it is related to myocarditis, cytokine-mediated injury, microvascular injury, and stress-related cardiomyopathy. Despite vaccinations having provided the most effective approach to reducing mortality overall, an adapted treatment paradigm and regular monitoring of cardiac injury biomarkers is critical for improving outcomes in vulnerable populations at risk for severe COVID-19. In this review, we focus on the latest progress in clinic and research on the cardiovascular complications of COVID-19 and provide a perspective of treating cardiac complications deriving from COVID-19 in Emergency Medicine.

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.

C1q/TNF-Related Protein 3 Prevents Diabetic Retinopathy via AMPK-Dependent Stabilization of Blood-Retinal Barrier Tight Junctions.

Background The impairment of the inner blood-retinal barrier (iBRB) increases the pathological development of diabetic retinopathy (DR), a severe complication in diabetic patients. Identifying approaches to preserving iBRB integrity and function is a significant challenge in DR. C1q/tumor necrosis factor-related protein-3 (CTRP3) is a newly discovered adipokine and a vital biomarker, predicting DR severity. We sought to determine whether and how CTRP3 affects the pathological development of non-proliferative diabetic retinopathy (NPDR). Methods To clarify the pathophysiologic progress of the blood-retinal barrier in NPDR and explore its potential mechanism, a mouse Type 2 diabetic model of diabetic retinopathy was used. The capillary leakage was assessed by confocal microscope with fluorescent-labeled protein in vivo. Furthermore, the effect of CTRP3 on the inner blood-retinal barrier (iBRB) and its molecular mechanism was clarified. Results The results demonstrated that CTRP3 protects iBRB integrity and resists the vascular permeability induced by DR. Mechanistically, the administration of CTRP3 activates the AMPK signaling pathway and enhances the expression of Occludin and Claudin-5 (tight junction protein) in vivo and in vitro. Meanwhile, CTRP3 improves the injury of human retinal endothelial cells (HRMECs) induced by high glucose/high lipids (HG/HL), and its protective effects are AMPK-dependent. Conclusions In summary, we report, for the first time, that CTRP3 prevents diabetes-induced retinal vascular permeability via stabilizing the tight junctions of the iBRB and through the AMPK-dependent Occludin/Claudin-5 signaling pathway, thus critically affecting the development of NPDR.

Reduced vascular responsiveness to adiponectin in hyperlipidemic rats--mechanisms and significance.

Deficiency of adiponectin (APN), an adipocyte-derived vascular protective molecule, contributes to diabetic vascular injury. The current study determined whether obesity/hyperlipidemia may alter the vascular response to APN, and investigated the involved mechanisms and pathologic significance. Adult male Sprague-Dawley rats were fed a regular or high-fat diet (HF) for 4-16 weeks. Circulating APN levels, aortic pAMPK/AMPK, peNOS/eNOS, and APN receptor expression levels were determined. Compared to time-matched animals fed control diet, plasma APN levels in HF-diet animals were significantly increased at 8 weeks, and rapidly declined thereafter. Despite unchanged or elevated circulating APN levels, phosphorylated AMPK and eNOS in vascular tissue were significantly reduced at all observed time points. Recombinant full-length APN (rAPN)-induced AMPK/eNOS phosphorylation and vasodilatation were significantly reduced in 16-week obese/hyperlipidemic aortic segments. Vascular APN receptor 1 (AdipoR1) and receptor 2 (AdipoR2) expression were significantly reduced 16 weeks after HF-diet. Pre-incubation of rAPN with obese/hyperlipidemic plasma, but not with normal plasma, significantly reduced its AMPK and eNOS activation effect, and blunted its protective effect against TNFalpha-induced HUVEC apoptosis. This study demonstrated for the first time that obesity/hyperlipidemia reduces vascular responsiveness to APN. Modification/inactivation of APN by unidentified factors present in obese/hyperlipidemic plasma, decreased vascular AdipoR1/R2 expression, and reduced circulating APN levels contribute to reduced vascular responsiveness to APN at different stages of the obese condition. Reduced APN bioactivity allows unmitigated TNFalpha pro-apoptotic and pro-inflammatory actions, contributing to vascular injury in obesity/hyperlipidemia.

AMP-activated protein kinase deficiency enhances myocardial ischemia/reperfusion injury but has minimal effect on the antioxidant/antinitrative protection of adiponectin.

BACKGROUND: Diabetes increases the morbidity/mortality of ischemic heart disease, but the underlying mechanisms are incompletely understood. Deficiency of both AMP-activated protein kinase (AMPK) and adiponectin occurs in diabetes, but whether AMPK is cardioprotective or a central mediator of adiponectin cardioprotection in vivo remains unknown. METHODS AND RESULTS: Male adult mice with cardiomyocyte-specific overexpression of a mutant AMPKalpha2 subunit (AMPK-DN) or wild-type (WT) littermates were subjected to in vivo myocardial ischemia/reperfusion (MI/R) and treated with vehicle or adiponectin. In comparison to WT, AMPK-DN mice subjected to MI/R endured greater cardiac injury (larger infarct size, more apoptosis, and poorer cardiac function) likely as a result of increased oxidative stress in these animals. Treatment of AMPK-DN mice with adiponectin failed to phosphorylate cardiac acetyl-CoA carboxylase as it did in WT mouse heart. However, a significant portion of the cardioprotection of adiponectin against MI/R injury was retained in AMPK-DN mice. Furthermore, treatment of AMPK-DN mice with adiponectin reduced MI/R-induced cardiac oxidative and nitrative stress to the same degree as that seen in WT mice. Finally, treating AMPK-DN cardiomyocytes with adiponectin reduced simulated MI/R-induced oxidative/nitrative stress and decreased cell death (P<0.01). CONCLUSIONS: Collectively, our results demonstrated that AMPK deficiency significantly increases MI/R injury in vivo but has minimal effect on the antioxidative/antinitrative protection of adiponectin.

Methylglyoxal increases cardiomyocyte ischemia-reperfusion injury via glycative inhibition of thioredoxin activity.

Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality, but the specific molecular basis linking DM with increased vulnerability to cardiovascular injury remains incompletely understood. Methylglyoxal (MG), a precursor to advanced glycation end products (AGEs), is increased in diabetic patient plasma, but its role in diabetic cardiovascular complications is unclear. Thioredoxin (Trx), a cytoprotective molecule with antiapoptotic function, has been demonstrated to be vulnerable to glycative inhibition, but whether Trx is glycatively inhibited by MG, thus contributing to increased cardiac injury, has never been investigated. Cultured H9c2 cardiomyocytes were treated with MG (200 muM) for 6 days. The following were determined pre- and post-simulated ischemia-reperfusion (SI-R; 8 h of hypoxia followed by 3 h of reoxygenation): cardiomyocyte death/apoptosis, Trx expression and activity, AGE formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity. Compared with vehicle, MG significantly increased SI-R-induced cardiomyocyte LDH release and apoptosis (P < 0.01). Prior to SI-R, Trx activity was reduced in MG-treated cells, but Trx expression was increased moderately. Moreover, Trx-ASK1 complex formation was reduced, and both p38 MAPK activity and phosphorylation were increased. To investigate the effects of MG on Trx directly, recombinant human Trx (hTrx) was incubated with MG in vitro. Compared with vehicle, MG incubation markedly increased CML formation (a glycation footprint) and inhibited Trx activity. Finally, glycation inhibitor aminoguanidine administration during MG treatment of cultured cells reduced AGE formation, increased Trx activity, restored Trx-ASK1 interaction, and reduced p38 MAPK phosphorylation and activity, caspase-3 activation, and LDH release (P < 0.01). We demonstrated for the first time that methylglyoxal sensitized cultured cardiomyocytes to SI-R injury by posttranslational modification of Trx via glycation. Therapeutic interventions scavenging AGE precursors may attenuate ischemic-reperfusion injury in hyperglycemic state diseases such as diabetes.

The future of emergency medicine.

BACKGROUND: The specialty of emergency medicine (EM) continues to experience a significant workforce shortage in the face of increasing demand for emergency care. SUMMARY: In July 2009, representatives of the leading EM organizations met in Dallas for the Future of Emergency Medicine Summit. Attendees at the Future of Emergency Medicine Summit agreed on the following: 1) Emergency medical care is an essential community service that should be available to all; 2) An insufficient emergency physician workforce also represents a potential threat to patient safety; 3) Accreditation Council for Graduate Medical Education/American Osteopathic Association (AOA)-accredited EM residency training and American Board of Medical Specialties/AOA EM board certification is the recognized standard for physician providers currently entering a career in emergency care; 4) Physician supply shortages in all fields contribute to-and will continue to contribute to-a situation in which providers with other levels of training may be a necessary part of the workforce for the foreseeable future; 5) A maldistribution of EM residency-trained physicians persists, with few pursuing practice in small hospital or rural settings; 6) Assuring that the public receives high quality emergency care while continuing to produce highly skilled EM specialists through EM training programs is the challenge for EM's future; 7) It is important that all providers of emergency care receive continuing postgraduate education.

The future of emergency medicine.

Physician shortages are being projected for most medical specialties. The specialty of emergency medicine continues to experience a significant workforce shortage in the face of increasing demand for emergency care. The limited supply of emergency physicians, emergency nurses, and other resources is creating an urgent, untenable patient care problem. In July 2009, representatives of the leading emergency medicine organizations met in Dallas, TX, for the Future of Emergency Medicine Summit. This consensus document, agreed to and cowritten by all participating organizations, describes the substantive issues discussed and provides a foundation for the future of the specialty.

Adiponectin inhibits cardiac arrest/cardiopulmonary resuscitation­induced apoptosis in brain by increasing autophagy involved in AdipoR1­AMPK signaling.

Emerging evidence suggests that both apoptosis and autophagy contribute to global cerebral ischemia­reperfusion (GCIR)­induced neuronal death, which results from cardiac arrest (CA). However, the mechanism of how GCIR may affect the balance between apoptosis and autophagy resulting from CA remains to be elucidated. Additionally, the role of adiponectin (APN) in reversing the apoptosis and autophagy induced by GCIR following cardiac arrest­cardiopulmonary resuscitation (CA­CPR) is unclear. Thus, the aim of the present study was to investigate how GCIR affect the apoptosis and autophagy in response to CA and to clarify whether APN may alter the apoptosis and autophagy of neuronal death in GCIR­injured brain post­CA­CPR. Using normal controls (Sham group) and two experimental groups [CA­CPR­induced GCIR injury (PCAS) group and exogenous treatment with adiponectin post­CA­CPR (APN group)], it was demonstrated that both apoptosis and autophagy were observed simultaneously in the brain subjected to GCIR, but apoptosis appeared to be more apparent. Exogenous administration of APN significantly reduced the formation of malondialdehyde, a marker of oxidative stress and increased the expression of superoxide dismutase, an anti­oxidative enzyme, resulting in the stimulation of autophagy, inhibition of apoptosis and reduced brain tissue injury (P<0.05 vs. PCAS). APN treatment increased the expression of APN receptor 1 (AdipR1) and the phosphorylation of AMP­activated protein kinase (AMPK; Ser182) in brain tissues. In conclusion, GCIR induced apoptosis and inhibited autophagy, contributing to brain injury in CA­CPR. By contrast, APN reduced the brain injury by reversing the changes of neuronal autophagy and apoptosis induced by GCIR. The possible mechanism might owe to its effects on the activation of AMPK after combining with AdipR1 on neurons, which suggests a novel intervention against GCIR injury in CA­CPR conditions.

C1q/TNF-related protein 5 contributes to diabetic vascular endothelium dysfunction through promoting Nox-1 signaling.

OBJECTIVE: Dysregulated adipokine profiles contribute to the pathogenesis of diabetic cardiovascular complications. Endothelial cell (EC) dysfunction, a common pathological alteration in cardiovascular disorders, is exaggerated in diabetes. However, it is unclear whether and how dysregulated adipokines may contribute to diabetic EC dysfunction. METHODS AND RESULTS: Serum C1q/TNF-Related Protein 5 (CTRP5) were determined in control/diabetes patients, and control/diabetic mice (high-fat diet, HFD). We observed for the first time that serum total CTRP5 was increased, high molecular weight (HMW) form was decreased, but the globular form (gCTRP5) was significantly increased in diabetic patients. These pathological alterations were reproduced in diabetic mice. To determine the pathological significance of increased gCTRP5 in diabetes, in vivo, ex vivo and in vitro experiments were performed. Diabetic atherosclerosis and EC dysfunction were significantly attenuated by the in vivo administration of CTRP5 neutralization antibody (CTRP5Ab). EC apoptosis was significantly increased in diabetic EC (isolated from HFD animal aorta) or high glucose high lipid (HGHL) cultured HUVECs. These pathological alterations were further potentiated by gCTRP5 and attenuated by CTRP5Ab. Pathway specific discovery-driven approach revealed that Nox1 expression was one of the signaling molecules commonly activated by HFD, HGHL, and gCTRP5. Treatment with CTRP5Ab reversed HFD-induced Nox1 upregulation. Finally, Nox1siRNA was used to determine the causative role of Nox1 in gCTRP5 induced EC apoptosis in diabetes. Results showed that gCTRP5 activated the mitochondrial apoptotic signal of EC in diabetes, which was blocked by the silencing Nox1 gene. CONCLUSION: We demonstrated for the first time that gCTRP5 is a novel molecule contributing to diabetic vascular EC dysfunction through Nox1-mediated mitochondrial apoptosis, suggesting that interventions blocking gCTRP5 may protect diabetic EC function, ultimately attenuate diabetic cardiovascular complications.

Cardioprotective effect of adiponectin is partially mediated by its AMPK-independent antinitrative action.

Adiponectin (APN) exerts its metabolic regulation largely through AMP-dependent protein kinase (AMPK). However, the role of AMPK in APN's antiapoptotic effect in ischemic-reperfused (I/R) adult cardiomyocytes remains incompletely understood. The present study was designed to determine the involvement of AMPK in the antiapoptotic signaling of APN. Cardiomyocytes from adult male mice overexpressing a dominant-negative alpha(2)-subunit of AMPK (AMPK-DN) or wild-type (WT) littermates were subjected to simulated I/R (SI/R) and pretreated with 2 microg/ml globular domain of APN (gAPN) or vehicle. SI/R-induced cardiomyocyte apoptosis was modestly increased in AMPK-DN cardiomyocytes (P < 0.05). Treatment with gAPN significantly reduced SI/R-induced apoptosis in WT cardiomyocytes as well as in AMPK-DN cardiomyocytes, indicating that the antiapoptotic effect of gAPN is partially AMPK independent. Furthermore, gAPN-induced endothelial nitric oxide synthase (eNOS) phosphorylation was significantly reduced in AMPK-DN cardiomyocytes, suggesting that the APN-eNOS signaling axis is impaired in AMPK-DN cardiomyocytes. Additional experiments demonstrated that treatment of AMPK-DN cardiomyocytes with gAPN reduced SI/R-induced NADPH oxidase overexpression, decreased superoxide generation, and blocked peroxynitrite formation to the same extent as that observed in WT cardiomyocytes. Collectively, our present study demonstrated that although the metabolic and eNOS activation effect of APN is largely mediated by AMPK, the superoxide-suppressing effect of APN is not mediated by AMPK, and this AMPK-independent antioxidant property of APN increased nitric oxide bioavailability and exerted significant antiapoptotic effect.