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.

A Chinese scoring system for predicting successful retrograde collateral traverse in patients with chronic total coronary occlusion.

BACKGROUND: Retrograde approach technique has been challenging in percutaneous coronary interventional treatment of chronic total occlusion (CTO) coronary disease. The present study endeavors to determine a novel Chinese scoring system for predicting successful collateral channels traverse via retrograde approach. METHODS: The demographic characteristics and angiographic characteristics of 309 CTO patient were analyzed by univariable and multivariable analysis for selecting potential predictors. And the nomogram was used to establish the scoring system. Then it was evaluated by the internal and external validation. RESULTS: The predictors of Age, Connections between collateral channels and recipient vessels, and Channel Tortuosity (ACT) were identified with univariable and multivariable analysis and employed to the ACT score system. With acceptable calibrations, the area under curve of the scoring system and the external validation were 0.826 and 0.816 respectively. Based on score, the predictors were divided into three risk categories and it showed a consistent prediction power in the validation cohort. CONCLUSIONS: The novel Chinese ACT score is a reliable tool for predicting successful retrograde collateral traverse.

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.

Serum CTRP9 and high-molecular weight adiponectin are associated with ischemic stroke.

BACKGROUND: C1q/TNF-related protein 9 (CTRP9) and adiponectin (APN) have beneficial metabolic regulatory and vasoprotective effects. This study explored alteration of CTRP9 and APN multimers during onset of ischemic stroke and development, to provide novel clinical and experimental basis for recognition and prevention of ischemic stroke. METHODS: There were 269 patients with ischemic stroke and 182 control subjects included in this study. Serum levels of CTRP9 and APN multimers in different disease stages were measured. RESULTS: Serum CTRP9, total APN (tAPN), and high-molecular weight (HMW) APN decreased gradually in stage I (acute stage, within 72 h of onset) of ischemic stroke and increased during stage III (11th day to one month) and stage IV (1 month after), compared to control. In the non-hyperlipidemia group, serum CTRP9, tAPN, and HMW were decreased in ischemic stroke patients compared to control (P < 0.05). Serum CTRP9 is closely related to serum tAPN and HMW (r = 0.992, 0.991). Serum CTRP9 are protective against ischemic stroke (OR = 0.400, 95% CI 0.197-0.810, P < 0.05). CONCLUSIONS: Lower serum CTRP9, tAPN, LMW, and HMW are significantly associated with increased ischemic stroke risk in non-hyperlipidemia subjects. CTRP9, tAPN, and HMW isoforms may be valuable clinical indicators for patients with ischemic stroke.

Extracellular vesicle-derived miR-144 as a novel mechanism for chronic intermittent hypoxia-induced endothelial dysfunction.

Rationale: Extracellular vesicles (EVs) play a significant role in cell-cell communication. However, whether and how extracellular vesicles are involved in chronic intermittent hypoxia-induced endothelial dysfunction is unknown. Methods: Comparative transcriptomics analysis and miRNA screening were used to identify the possible pathways or target molecules mediating chronic intermittent hypoxia-induced endothelial function. Serum- or erythrocyte-derived EVs were isolated through ultracentrifugation plus filtration. After in vitro or in vivo treatment with EVs, aortic rings were treated with dihydroethidium staining for superoxidative anion measurement or mounted with wire myography to measure isometric forces. Immunoblotting and qPCR were used for evaluating the molecular mechanism mediating EV miR-144-induced endothelial function under intermittent hypoxia. Results: We revealed a previously undefined importance of circulating extracellular vesicles in regulating endothelial function via delivery of miR-144 to endothelial cells, reducing nuclear factor erythroid 2-related factor 2 expression. Additionally, we identified that erythrocytes were the primary cellular source of miR-144-enriched serum-derived extracellular vesicles and that erythrocyte-derived extracellular vesicles were largely responsible for chronic intermittent hypoxia-impaired endothelial function. Furthermore, silencing of miR-144 by anti-miR-144 confirmed its essential role in endothelial dysfunction elicited by erythrocyte-derived extracellular vesicles from chronic intermittent hypoxia-exposed C57BL/6 mice. Conclusion: The results expand the scope of blood-borne substances involved in vascular homeostasis and suggest that anti-miR-144-loaded extracellular vesicles may represent a promising therapeutic approach against obstructive sleep apnea or chronic intermittent hypoxia-associated endothelial dysfunction.

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.

Hyperlipidemic plasma molecules bind and inhibit adiponectin activity.

INTRODUCTION: Adiponectin is a potent vascular protective molecule. Recent findings have suggested adiponectin resistance during early diabetes. However, the molecular mechanisms responsible remain unidentified. Here, we took an unbiased approach to identify whether hyperlipidemic plasma molecules exist that bind and inhibit adiponectin function, contributing to adiponectin resistance and diabetic vascular injury. METHODS: Adult rats were randomly assigned to receive either a normal or a high-fat diet for 8 weeks. Plasma was co-immunoprecipitated with anti-APN antibody and analyzed by mass spectrometry. The APN binding molecules and their effect upon APN biological activity were determined. RESULTS: As expected, the high-fat-diet increased plasma triglyceride, total cholesterol, and low-density lipoprotein. Importantly, the circulating APN level was significantly increased at this time point. Mass spectrometry identified 18 proteins with increased APN binding in hyperlipidemic plasma, among which four proteins critical in lipid metabolism, including apolipoprotein A1 (APOA1), APOA4, APOC1, and paraoxonase 1, were further investigated. Incubating recombinant APN with APOA1 markedly (P < 0.01), and incubating with APOC1 significantly (P < 0.05), inhibited APN activity as evidenced by the reduced AMPK activation in HUVECs. APOA4 and paraoxonase 1 incubation had no effect upon APN activity. Finally, plasma APOA1 was significantly increased (P < 0.05) in hyperlipidemic plasma compared with the control plasma. CONCLUSIONS: It was demonstrated for the first time that increased APOA1 and APOC1 in hyperlipidemic plasma binds and inhibits APN activity. This result not only identifies a novel molecular mechanism responsible for adiponectin resistance during early stage diabetes, but also provides additional new insight into the diverse/controversial (protective and harmful) functions of high-density lipoprotein.

lncRNA NONHSAT069381 and NONHSAT140844 Increase in Aging Human Blood, Regulating Cardiomyocyte Apoptosis.

Aging augments postischemic apoptosis via incomplete mechanisms. Our previous animal study suggests that in addition to proapoptotic effects, lncRNAs also exert antiapoptotic effects in cardiomyocytes. However, whether this unexpected phenomenon exists in humans is unknown. In the present study, we investigated the relationship between aging and apoptosis regulation in human blood samples and confirmed their role by utilizing the cardiomyocyte lines (AC16 cells). Human blood samples were collected from 20 pairs of older adult and young volunteers. Age-different apoptotic regulatory lncRNAs and miRNAs were identified by microarray and bioinformatics analysis. The results indicated that lncRNA (NONHSAT069381 and NONHSAT140844) and miRNA (hsa-miR-124-5p and hsa-miR-6507-5p) were increased in aging human blood, confirmed by both bioinformatics analysis and polymerase chain reaction (PCR). Overexpression of NONHSAT069381 in AC16 cells increased caspase-3 levels and increased cardiomyocyte apoptotic cell death (determined by TUNEL staining and caspase activity assays) after hypoxia/reoxygenation (H/R), while overexpression of NONHSAT140844 increased X-chromosome-linked inhibitor of apoptosis protein (XIAP) content and decreased the myocardial apoptotic cell death. Furthermore, luciferase reporter assay revealed that hsa-miR-124-5p might be a mediator between NONHSAT069381 and mCASP3 and hsa-miR-6507-5p might be a mediator between NONHSAT140844 and mXIAP. Overexpression of hsa-miR-124-5p decreased caspase-3 levels and overexpression of hsa-miR-6507-5p decreased XIAP content in AC16 cells. We have found evidence that lncRNAs are important regulatory molecules in aging-mediated effects upon apoptosis. More interestingly, besides apoptosis-promoting effects, aging also inhibits myocardial apoptosis after H/R. This phenomenon also exists in the human cardiomyocyte line.

Atg16L1 as a Novel Biomarker and Autophagy Gene for Diabetic Retinopathy.

OBJECTIVE: Accumulating evidence suggests the critical role of autophagy in the pathogenesis of diabetic retinopathy (DR). In the current study, we aim to identify autophagy genes involved in DR via microarray analyses. METHODS: Gene microarrays were performed to identify differentially expressed lncRNAs/mRNAs between normal and DR retinas. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of lncRNA-coexpressed mRNAs were used to determine the related pathological pathways and biological modules. Real-time polymerase chain reactions (PCR) were conducted to validate the microarray analyses. RESULTS: A total of 2474 significantly dysregulated lncRNAs and 959 differentially expressed mRNAs were identified in the retina of DR. Based upon Signalnet analysis, Bcl2, Gabarapl2, Atg4c, and Atg16L1 participated the process of cell death in DR. Moreover, real-time PCR revealed significant upregulation of Atg16L1. CONCLUSION: This study indicated the importance and potential role of Atg16L1, one of the autophagy genes, as a biomarker in DR development and progression.

Identification of a CTRP9 C-Terminal polypeptide capable of enhancing bone-derived mesenchymal stem cell cardioprotection through promoting angiogenic exosome production.

BACKGROUND: Mesenchymal stem cell therapy improves ischemic heart failure via incompletely understood mechanisms. C1q-TNFα related protein-9 (CTRP9) is a novel anti-oxidative cardiokine capable of improving the local microenvironment and cell survival by its c-terminal active globular domain (gCTRP9). The current study attempted to: 1) identify active gCTRP9 c-terminal polypeptides with stem cell protective function; 2) determine whether a lead polypeptide may enable/enhance cortical bone-derived mesenchymal stem cell (CBSC) cardioprotection against post-myocardial infarction (post-MI) remodeling; and 3) define the responsible underlying cellular/molecular mechanisms. METHODS AND RESULTS: Utilizing I-TASSER structure prediction and 3-D active site modeling, we cloned and purified 3 gCTRP9 fragments (CTRP9-237, CTRP9-277, and CTRP9-281). Their activation of cell salvage kinase was compared against gCTRP9. Among the three fragments, CTRP9-281 (a 45 residue-containing polypeptide) exerted comparable or greater ERK1/2 activation compared to gCTRP9. Treatment with CTRP9-281 or gCTRP9 significantly increased CBSC proliferation and migration, and attenuated oxidative stress-induced CBSC apoptosis. CTRP9-281 and gCTRP9 comparably upregulated SOD2 and SOD3 expression. However, CTRP9-281, not gCTRP9, upregulated FGF2 and VEGFA expression/secretion in an ERK1/2 dependent manner. Administration of gCTRP9 or CTRP9-281 alone attenuated post-MI cardiac dysfunction and improved CBSC retention in the infarcted heart in similar fashion. However, CTRP9-281 exerted greater synergistic effect with CBSC than gCTRP9 related to pro-angiogenic, anti-fibrotic, and anti-remodeling effects. Mechanistically, CTRP9-281 significantly increased SOD2-rich and VEGFA-rich exosome production by CBSC. Exosomes from CTRP9-281 treated CBSC significantly attenuated oxidative stress-induced cardiomyocyte apoptosis in vitro. An exosome generation inhibitor attenuated CTRP9-281 enhancement of CBSC cardioprotection in vivo. CONCLUSION: We identified a CTRP9 polypeptide that upregulates SOD2/SOD3 expression and improves CBSC survival/retention, similar to gCTRP9. Moreover, CTRP9-281 stimulates VEGFA-rich exosome production by CBSC, exerting superior pro-angiogenic, anti-fibrotic, and cardioprotective actions.

"Know Diabetes by Heart": role of adipocyte-cardiomyocyte communications.

Cardiovascular disease is the leading cause of morbidity in patients with diabetes mellitus. In 2019, the American Heart Association and the American Diabetes Association (along with industry leaders) launched the groundbreaking collaborative initiative "Know Diabetes by Heart™" to reduce cardiovascular deaths in type 2 diabetic patients. The molecular basis linking diabetes with cardiovascular complications has not yet been fully defined. Recent clinical and experimental studies strongly suggest that adipocyte dysfunction and subsequent pathological communications between adipocyte and cardiomyocytes play important roles in diabetic cardiac injury. This perspective article will review recent development concerning adipocyte-cardiomyocyte communications, and identify the most critical questions remain to be answered in this filed.

mir15a/mir16-1 cluster and its novel targeting molecules negatively regulate cardiac hypertrophy.

In response to pathological stimuli, the heart develops ventricular hypertrophy that progressively decompensates and leads to heart failure. miRNAs are increasingly recognized as pathogenic factors, clinically relevant biomarkers, and potential therapeutic targets. We identified that mir15a/mir16-1 cluster was negatively correlated with hypertrophic severity in patients with hypertrophic cardiomyopathy. The mir15a/mir16-1 expression was enriched in cardiomyocytes (CMs), decreased in hypertrophic human hearts, and decreased in mouse hearts after transverse aortic constriction (TAC). CM-specific mir15a/mir16-1 knockout promoted cardiac hypertrophy and dysfunction after TAC. CCAAT/enhancer binding protein (C/EBP)ß was responsible for the downregulation of mir15a/mir16-1 cluster transcription. Mechanistically, mir15a/mir16-1 cluster attenuated the insulin/IGF1 signal transduction cascade by inhibiting multiple targets, including INSR, IGF-1R, AKT3, and serum/glucocorticoid regulated kinase 1 (SGK1). Pro-hypertrophic response induced by mir15a/mir16-1 inhibition was abolished by knockdown of insulin receptor (INSR), insulin like growth factor 1 receptor (IGF1R), AKT3, or SGK1. In vivo systemic delivery of mir15a/mir16-1 by nanoparticles inhibited the hypertrophic phenotype induced by TAC. Importantly, decreased serum mir15a/mir16-1 levels predicted the occurrence of left ventricular hypertrophy in a cohort of patients with hypertension. Therefore, mir15a/mir16-1 cluster is a promising therapeutic target and biomarker for cardiac hypertrophy.

Nicotine induces cardiac toxicity through blocking mitophagic clearance in young adult rat.

Since an outbreak of vaping-related deaths in the US has been reported as a public health crisis, the cardiovascular safety of nicotine nowadays receives increasing attention due to use of tobacco cigarette alternatives, such as electronic cigarettes. However, whether and how nicotine contributes to cardiac detrimental effects are in great controversy, especially less understood in young adult population. We report that chronic nicotine exposure, a major component of Electronic cigarettes, resulted in directly inhibited cardiomyocytes viability, increased cardiac fibrosis, and markedly suppressed cardiac function compared with sham. Gene array combined with bioinformatics analysis identified cardiac apoptosis and mitophagy were the key signals responsible for nicotine induced cardiac detrimental effect. Mechanistically, nicotine exposure markedly increased cleaved Caspase 3 and cleaved Caspase 9 indicating the involvement of intrinsic apoptotic pathway (mitochondrial cell death pathway). Meanwhile, nicotine-induced ROS outbreak promoted lysomal alkalization, furthermore blocked mitophagic degradation, thereby disrupted mitophagic flux promoted mitochondrial cell death cascade. Taken together, these findings indicate that nicotine confers cardiotoxicity via ROS-induced mitophagic flux blockage and provide the first demonstration of a causative link between nicotine and cardiac toxicity in young adult rat which may suggest nicotine induces cardiomyocytes impairment leading to cardiotoxicity in young adult population.

THE PRESENT AND FUTURE OF THE MASS SPECTROMETRY-BASED INVESTIGATION OF THE EXOSOME LANDSCAPE.

Exosomes are critical intercellular messengers released upon the fusion of multivesicular bodies with the cellular plasma membrane that deliver their cargo in the form of extracellular vesicles. Containing numerous nonrandomly packed functional proteins, lipids, and RNAs, exosomes are vital intercellular messengers that contribute to the physiologic processes of the healthy organism. During the post-genome era, exosome-oriented proteomics have garnered great interest. Since its establishment, mass spectrometry (MS) has been indispensable for the field of proteomics research and has advanced rapidly to interrogate biological samples at a higher resolution and sensitivity. Driven by new methodologies and more advanced instrumentation, MS-based approaches have revolutionized our understanding of protein biology. As the access to online proteomics database platforms has blossomed, experimental data processing occurs with more speed and accuracy. Here, we review recent advances in the technological progress of MS-based proteomics and several new detection strategies for MS-based proteomics research. We also summarize the use of integrated online databases for proteomics research in the era of big data. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

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.

N-Cadherin Overexpression Mobilizes the Protective Effects of Mesenchymal Stromal Cells Against Ischemic Heart Injury Through a ß-Catenin-Dependent Manner.

RATIONALE: Mesenchymal stromal cell-based therapy is promising against ischemic heart failure. However, its efficacy is limited due to low cell retention and poor paracrine function. A transmembrane protein capable of enhancing cell-cell adhesion, N-cadherin garnered attention in the field of stem cell biology only recently. OBJECTIVE: The current study investigates whether and how N-cadherin may regulate mesenchymal stromal cells retention and cardioprotective capability against ischemic heart failure. METHODS AND RESULTS: Adult mice-derived adipose tissue-derived mesenchymal stromal cells (ADSC) were transfected with adenovirus harboring N-cadherin, T-cadherin, or control adenovirus. CM-DiI-labeled ADSC were intramyocardially injected into the infarct border zone at 3 sites immediately after myocardial infarction (MI) or myocardial ischemia/reperfusion. ADSC retention/survival, cardiomyocyte apoptosis/proliferation, capillary density, cardiac fibrosis, and cardiac function were determined. Discovery-driven/cause-effect analysis was used to determine the molecular mechanisms. Compared with ADSC transfected with adenovirus-control, N-cadherin overexpression (but not T-cadherin) markedly increased engrafted ADSC survival/retention up to 7 days post-MI. Histological analysis revealed that ADSC transfected with adenovirus-N-cadherin significantly preserved capillary density and increased cardiomyocyte proliferation and moderately reduced cardiomyocyte apoptosis 3 days post-MI. More importantly, ADSC transfected with adenovirus-N-cadherin (but not ADSC transfected with adenovirus-T-cadherin) significantly increased left ventricular ejection fraction and reduced fibrosis in both MI and myocardial ischemia/reperfusion mice. In vitro experiments demonstrated that N-cadherin overexpression promoted ADSC-cardiomyocyte adhesion and ADSC migration, enhancing their capability to increase angiogenesis and cardiomyocyte proliferation. MMP (matrix metallopeptidases)-10/13 and HGF (hepatocyte growth factor) upregulation is responsible for N-cadherin's effect upon ADSC migration and paracrine angiogenesis. N-cadherin overexpression promotes cardiomyocyte proliferation by HGF release. Mechanistically, N-cadherin overexpression significantly increased N-cadherin/ß-catenin complex formation and active ß-catenin levels in the nucleus. ß-catenin knockdown abolished N-cadherin overexpression-induced MMP-10, MMP-13, and HGF expression and blocked the cellular actions and cardioprotective effects of ADSC overexpressing N-cadherin. CONCLUSIONS: We demonstrate for the first time that N-cadherin overexpression enhances mesenchymal stromal cells-protective effects against ischemic heart failure via ß-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention.

Implications of C1q/TNF-related protein superfamily in patients with coronary artery disease.

The C1q complement/TNF-related protein superfamily (CTRPs) displays differential effects on the regulation of metabolic homeostasis, governing cardiovascular function. However, whether and how they may serve as predictor/pro-diagnosis factors for assessing the risks of coronary artery disease (CAD) remains controversial. Therefore, we performed a clinical study to elaborate on the implication of CTRPs (CTRP1, CTRP5, CTRP7, and CTRP15) in CAD. CTRP1 were significantly increased, whereas CTRP7 and CTRP15 levels were decreased in CAD patients compared to the non-CAD group. Significant differences in CTRP1 levels were discovered between the single- and triple-vascular-vessel lesion groups. ROC analysis revealed that CTRP7 and CTRP15 may serve as CAD markers, while CTRP1 may serve as a marker for the single-vessel lesion of CAD. CTRP1 and CTRP5 can serve as markers for the triple-vessel lesion. CTRP1 may serve as an independent risk predictor for triple-vessel lesion, whereas CTRP15 alteration may serve for a single-vessel lesion of CAD. CTRP1 may serve as a novel superior biomarker for diagnosis of severity of vessel-lesion of CAD patients. CTRP7, CTRP15 may serve as more suitable biomarker for the diagnosis of CAD patients, whereas CTRP5 may serve as an independent predictor for CAD. These findings suggest CTRPs may be the superior predictive factors for the vascular lesion of CAD and represent novel therapeutic targets against CAD.

Sevoflurane Pre-conditioning Ameliorates Diabetic Myocardial Ischemia/Reperfusion Injury Via Differential Regulation of p38 and ERK.

Diabetes mellitus (DM) significantly increases myocardial ischemia/reperfusion (MI/R) injury. During DM, cardioprotection induced by conventional pre-conditioning (PreCon) is decreased due to impaired AMP-activated protein kinase (AMPK) signaling. The current study investigated whether PreCon with inhaled anesthetic sevoflurane (SF-PreCon) remains cardioprotective during DM, and identified the involved mechanisms. Normal diet (ND) and high-fat diet (HFD)-induced DM mice were randomized into control and SF-PreCon (3 cycles of 15-minute period exposures to 2% sevoflurane) groups before MI/R. SF-PreCon markedly reduced MI/R injury in DM mice, as evidenced by improved cardiac function (increased LVEF and ±Dp/dt), decreased infarct size, and decreased apoptosis. To determine the relevant role of AMPK, the effect of SF-PreCon was determined in cardiac-specific AMPKα2 dominant negative expressing mice (AMPK-DN). SF-PreCon decreased MI/R injury in AMPK-DN mice. To explore the molecular mechanisms responsible for SF-PreCon mediated cardioprotection in DM mice, cell survival molecules were screened. Interestingly, in ND mice, SF-PreCon significantly reduced MI/R-induced activation of p38, a pro-death MAPK, without altering ERK and JNK. In DM and AMPK-DN mice, the inhibitory effect of SF-PreCon upon p38 activation was significantly blunted. However, SF-PreCon significantly increased phosphorylation of ERK1/2, a pro-survival MAPK in DM and AMPK-DN mice. We demonstrate that SF-PreCon protects the heart via AMPK-dependent inhibition of pro-death MAPK in ND mice. However, SF-PreCon exerts cardioprotective action via AMPK-independent activation of a pro-survival MAPK member in DM mice. SF-PreCon may be beneficial compared to conventional PreCon in diabetes or clinical scenarios in which AMPK signaling is impaired.

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.