The interplay of inflammation, exosomes and Ca2+ dynamics in diabetic cardiomyopathy.

Diabetes mellitus is one of the prime risk factors for cardiovascular complications and is linked with high morbidity and mortality. Diabetic cardiomyopathy (DCM) often manifests as reduced cardiac contractility, myocardial fibrosis, diastolic dysfunction, and chronic heart failure. Inflammation, changes in calcium (Ca2+) handling and cardiomyocyte loss are often implicated in the development and progression of DCM. Although the existence of DCM was established nearly four decades ago, the exact mechanisms underlying this disease pathophysiology is constantly evolving. Furthermore, the complex pathophysiology of DCM is linked with exosomes, which has recently shown to facilitate intercellular (cell-to-cell) communication through biomolecules such as micro RNA (miRNA), proteins, enzymes, cell surface receptors, growth factors, cytokines, and lipids. Inflammatory response and Ca2+ signaling are interrelated and DCM  has been known to adversely affect many of these signaling molecules either qualitatively and/or quantitatively. In this literature review, we have demonstrated that Ca2+ regulators are tightly controlled at different molecular and cellular levels during various biological processes in the heart. Inflammatory mediators, miRNA and exosomes are shown to interact with these regulators, however how these mediators are linked to Ca2+ handling during DCM pathogenesis remains elusive. Thus, further investigations are needed to understand the mechanisms to restore cardiac Ca2+ homeostasis and function, and to serve as potential therapeutic targets in the treatment of DCM.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

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Inhibition of RUNX1 blocks the differentiation of lung fibroblasts to myofibroblasts.

Pathological fibrosis contributes to progression of various diseases, for which the therapeutic options are limited. Idiopathic pulmonary fibrosis (IPF) is one such progressive and fatal interstitial fibrotic disease that is often characterized by excessive accumulation of extracellular matrix (ECM) proteins leading to stiff lung tissue and impaired gas exchange. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined the role of Runt-related transcription factor 1 (RUNX1), an evolutionarily conserved transcription factor, in the differentiation of human lung fibroblasts (HLFs) in vitro and in an animal model of bleomycin (BLM)-induced lung fibrosis. We observed that the expression of RUNX1 was significantly increased in the lungs of BLM-injected mice as compared to saline-treated mice. Furthermore, HLFs stimulated with transforming growth factor ß (TGF-ß) showed significantly higher RUNX1 expression at both mRNA and protein levels, and compartmentalization in the nucleus. Inhibition of RUNX1 in HLFs (using siRNA) showed a significant reduction in the differentiation of fibroblasts into myofibroblasts as evidenced by reduced expression of alpha-smooth muscle actin (α-SMA), TGF-ß and ECM proteins such as fibronectin 1 (FN1), and collagen 1A1 (COL1A1). Mechanistic studies revealed that the increased expression of RUNX1 in TGF-ß-stimulated lung fibroblasts is due to enhanced mRNA stability of RUNX1 through selective interaction with the RNA-binding profibrotic protein, human antigen R (HuR). Collectively, our data demonstrate that increased expression of RUNX1 augments processes involved in lung fibrosis including the differentiation of fibroblasts into collagen-synthesizing myofibroblasts. Our study suggests that targeting RUNX1 could limit the progression of organ fibrosis in diseases characterized by abnormal collagen deposition.

Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice.

Endotoxemia triggers life-threatening immune and cardiovascular response that leads to tissue damage, multi-organ failure, and death. The understanding of underlying molecular mechanisms is still evolving. N6-methyladenosine (m6A)-RNA modification plays key regulatory role in numerous biological processes. However, it remains unclear whether endotoxemia alters RNA methylation in the myocardium. In the current study, we investigated the effect of lipopolysaccharide (LPS)-induced endotoxemia on m6A-RNA methylation and its implications on myocardial inflammation and left ventricular (LV) function. Following LPS administration, mice showed increases in m6A-RNA methylation in the myocardium with a corresponding decrease in the expression of fat mass and obesity-associated protein (FTO, an m6A eraser/demethylase). The changes were associated with a significant increase in expression of myocardial inflammatory cytokine genes, such as IL-6, TNF-α, IL-1ß, and reduced LV function. Moreover, rat cardiomyoblasts (H9c2) exposed to LPS showed similar changes (with increase in m6A-RNA methylation and inflammatory cytokine genes, whereas downregulation of FTO). Furthermore, methylated RNA immunoprecipitation assay showed hypermethylation and increase in the expression of IL-6 and TNF-α genes in LPS-treated H9c2 cells as compared to untreated cells. Interestingly, FTO knockdown in cardiomyocytes mimicked the above effects. Taken together, these data suggest that endotoxemia-induced m6A methylation might play a critical role in expression of cardiac proinflammatory cytokines, and modulation of m6A methylation might limit myocardial inflammation and dysfunction during endotoxemia.

MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages.

OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

Mechanisms of COVID-19-induced cardiovascular disease: Is sepsis or exosome the missing link?

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached a pandemic level, spreading across the globe by affecting over 33 million people and causing over 1,009,270 deaths. SARS-CoV-2 is highly infectious with a high basic reproduction number (R0 ) of 2.2-5.7 that has led to its exponential spread. Besides, very little is known about it in terms of immunogenicity and its molecular targets. SARS-CoV-2 causes acute respiratory distress syndrome, followed by multiple organ failure and death in a small percentage of individuals. Cardiac injury has emerged as another dreaded outcome of COVID-19 complications. However, a thorough understanding of the pathogenesis of SARS-CoV-2 is lacking. In this review, we discuss the virus, possible mechanisms of COVID-19-induced cardiac injury, and potential therapeutic strategies, and we explore if exosomes could be targeted to treat symptoms of COVID-19. Furthermore, we discussed the virus-induced sepsis, which may be the cause of multiple organ failure, including myocardial injury.

Cardiac glycosaminoglycans and structural alterations during chronic stress-induced depression-like behavior in mice.

Major depressive disorder (MDD) is an independent risk factor for cardiovascular disease (CVD) and its complications; however, causal mechanisms remain unclear. In the present study, we investigate cardiac structural and functional alterations and associated changes in myocardial glycosaminoglycans (GAGs) disaccharide profile in mice that exhibit depression-like behavior. Mice were assigned to the chronic mild stress (CMS) group and nonstress control group (CT). The CMS group was exposed to a series of mild, unpredictable stressors for 7 wk. Mice in the CMS group show a significant decrease in protein expression of hippocampal brain-derived neurotrophic factor (BDNF) and exhibit depression-like behavioral changes, such as learned helplessness and decreased exploration behavior, as compared with the control group. Although cardiac function remained unchanged between the groups, echocardiography analysis showed slightly increased left ventricular wall thickness in the CMS group. Furthermore, the CMS group shows an increase in cardiomyocyte cross-sectional area and an associated decrease in BDNF protein expression and increase in IL-6 mRNA expression, when compared with control mice. GAG disaccharide analysis of the left ventricles of the CMS and CT mice revealed an elevation in heparan (HS) and chondroitin sulfate (CS) content in the CMS hearts (35.3% and 17.9%, respectively, vs. control group). Furthermore, we also observed that unsulfated or monosulfated disaccharides were the most abundant units; however, we did not find any significant difference in mole percent or sulfation pattern of HS/CS disaccharides between the groups. The current investigation highlights a need for further research to explore the relationship between cardiac GAGs biology and myocardial remodeling as a causal mechanism that underlie cardiovascular complications in patients with MDD.NEW & NOTEWORTHY Comorbidity between depression and CVD is well established, whereas its etiology, especially the role of nonfibrous components (proteoglycans/GAGs) of the extracellular matrix, is unexplored. To the best of our knowledge, this is the first study to characterize cardiac proteoglycan/glycosaminoglycan profile in response to depression-like behavioral changes in mice. We observed that chronic mild stress (CMS)-induced depression-like behavior and alterations in glycosaminoglycan profile were associated with structural changes in the heart.

Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart.

RNA-binding proteins like human antigen R (HuR) are key regulators in post-transcriptional control of gene expression in several pathophysiological conditions. Diabetes adversely affects monocyte/macrophage biology and function. It is not known whether diabetic milieu affects cellular/exosome-HuR and its implications on cardiac inflammation and fibrosis. Here, we evaluate in vitro and in vivo effects of diabetic milieu on macrophage cellular/exosome-HuR, alterations in intercellular cross talk with fibroblasts, and its impact on cardiac remodeling. Human failing hearts show higher HuR levels. Diabetic milieu activates HuR expression in cardiac- and cultured bone marrow-derived macrophages (BMMØ) and stimulates HuR nuclear-to-cytoplasmic translocation and exosome transfer. Exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice. Intriguingly, Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with BMMØ-derived HuR-deficient exosomes inhibits angiotensin II-induced cardiac fibrosis response and preserves left ventricle function as compared to control-exosome administration. To the best of our knowledge, this is the first study to demonstrate that diabetes activates BMMØ HuR expression and its transfer into exosome. The data suggest that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes.

Convergences of Life Sciences and Engineering in Understanding and Treating Heart Failure.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium, Cardiovascular Bioengineering Symposium, was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present, and >40 scientists presented their latest work on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers, and physicians in the field of cardiovascular sciences met and discussed the most recent advances in their work and proposed future strategies for overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical, and mechanical cues to cells to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here, we present some important perspectives that emerged from this meeting.

DNA damage-free iPS cells exhibit potential to yield competent cardiomyocytes.

DNA damage accrued in induced pluripotent stem cell (iPSC)-derived cardiomyocytes during in vitro culture practices lessens their therapeutic potential. We determined whether DNA-damage-free iPSCs (DdF-iPSCs) can be selected using stabilization of p53, a transcription factor that promotes apoptosis in DNA-damaged cells, and differentiated them into functionally competent DdF cardiomyocytes (DdF-CMs). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA-damaged cells, and the stable DdF cells were cultured further and differentiated into CMs. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species and DNA damage in DdF-iPSCs compared with control (Ctrl) iPSCs. Next-generation RNA sequencing and Ingenuity Pathway Analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 and 60 beats/min accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and, interestingly, cardiac development network was prominent compared with Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs are of ventricular CMs and mature compared with their counterparts. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using a small-molecule inhibitor and differentiated into ventricular DdF-CMs with fine-tuned molecular signatures. These iPSC-derived DdF-CMs show immense clinical potential in repairing injured myocardium.NEW & NOTEWORTHY Culture-stress-induced DNA damage in stem cells lessens their performance. A robust small-molecule-based approach, by stabilizing/activating p53, to select functionally competent DNA-damage-free cells from a heterogeneous population of cells is demonstrated. This protocol can be adopted by clinics to select DNA-damage-free cells before transplanting them to the host myocardium. The intact DNA-damage-free cells exhibited with fine-tuned molecular signatures and improved cellular functions. DNA-damage-free cardiomyocytes compared with control expressed superior cardiomyocyte functional properties, including, but not limited to, enhanced ion channel signatures. These DNA-intact cells would better engraft, survive, and, importantly, improve the cardiac function of the injured myocardium.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

BACKGROUND: Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. METHODS: NOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. RESULTS: Six months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. CONCLUSIONS: Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Sam68 impedes the recovery of arterial injury by augmenting inflammatory response.

OBJECTIVE: The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α-induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury. APPROACH AND RESULTS: The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68-/-) and WT mice. Sam68-/- mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68-/- mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1ß, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68-/- vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction. CONCLUSIONS: Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

OBG-like ATPase 1 inhibition attenuates angiotensin II-induced hypertrophic response in human ventricular myocytes via GSK-3beta/beta-catenin signalling.

Obg-like ATPase 1 (OLA1) that possesses both GTP and ATP hydrolyzing activities has been shown to be involved in translational regulation of cancer cell growth and survival. Also, GSK3ß signalling has been implicated in cardiac development and disease. However, the role of OLA1 in pathological cardiac hypertrophy is unknown. We sought to understand the mechanism by which OLA1 regulates GSK3ß-ß-Catenin signalling and its functional significance in angiotensin-II (ANG II)-induced cardiac hypertrophic response. OLA1 function and its endogenous interaction with GSK3ß/ß-catenin signalling in cultured human ventricular cardiomyocytes (AC16 cells) and mouse hearts (in vivo) was evaluated with/without ANG II-stimulated hypertrophic response. ANG II administration in mice increases myocardial OLA1 protein expression with a corresponding increase in GSK3ß phosphorylation and decrease in ß-Catenin phosphorylation. Cultured cardiomyocytes treated with ANG II show endogenous interaction between OLA1 and GSK3ß, nuclear accumulation of ß-Catenin and significant increase in cell size and expression of hypertrophic marker genes such as atrial natriuretic factor (ANF; NPPA) and ß-myosin heavy chain (MYH7). Intriguingly, OLA1 inhibition attenuates the above hypertrophic response in cardiomyocytes. Taken together, our data suggest that OLA1 plays a detrimental role in hypertrophic response via GSK3ß/ß-catenin signalling. Translation strategies to target OLA1 might potentially limit the underlying molecular derangements leading to left ventricular dysfunction in patients with maladaptive cardiac hypertrophy.

Interleukin-10 Inhibits Bone Marrow Fibroblast Progenitor Cell-Mediated Cardiac Fibrosis in Pressure-Overloaded Myocardium.

BACKGROUND: Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload-induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload-induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC-mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload-induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis. METHODS: Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow. RESULTS: Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow-depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction-induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with α-smooth muscle actin or collagen 1α in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction-induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-ß-induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction-based selective miRNA analysis revealed that transforming growth factor-ß-induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-ß-induced fibrotic signaling in BM-FPCs. CONCLUSIONS: Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad-miRNA-21-mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction.

RATIONALE: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes, however, their effect in the context of the heart is unknown. OBJECTIVE: Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit(+) cardiac progenitor cells (CPCs) function can be enhanced with ESC exosomes. METHODS AND RESULTS: This study demonstrates that mouse ESC-derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival, and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented CPC survival, proliferation, and cardiac commitment concurrent with increased c-kit(+) CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290-295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression, and proliferation. CONCLUSIONS: mES Ex provide a novel cell-free system that uses the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES-derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC-based repair programs in the heart.

MicroRNA-9 inhibits hyperglycemia-induced pyroptosis in human ventricular cardiomyocytes by targeting ELAVL1.

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to heart failure (HF). ELAV-like protein 1 (ELAVL1) plays a critical role in the progression of inflammation and HF. However the role of ELAVL-1 in inflammation induced cardiac cell death (pyroptosis) under hyperglycemic condition remains elusive. Our data demonstrates that ELAVL1 expression augmented with a concomitant increase in caspase-1 and IL-1 beta expression in human hearts and human ventricular cardiomyocytes under hyperglycemic condition. Furthermore, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Interestingly, miRNA-9 expression significantly reduced in high glucose treated cardiomyocytes and in human diabetic hearts. Inhibition of miR-9 upregulates ELAVL1 expression and activates caspase-1. Alternatively, treatment with miR-9 mimics attenuates hyperglycemia-induced ELAVL1 and inhibits cardiomyocyte pyroptosis. Taken together our study highlights the potential therapeutic implications of targeting miR-9/ELAVL1 in preventing cardiomyocyte cell loss during HF in diabetics.

Negative Regulation of miR-375 by Interleukin-10 Enhances Bone Marrow-Derived Progenitor Cell-Mediated Myocardial Repair and Function After Myocardial Infarction.

Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell-based therapies. We have earlier reported that co-administration of interleukin (IL)-10 and BMPAC enhances cell survival and improves left ventricular (LV) functions after acute myocardial infarction (MI) in mice. We hypothesized that IL-10 regulates microRNA-375 (miR-375) signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. miR-375 expression is significantly upregulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 knockout mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knockdown in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization, and LV functions. Our in vitro studies revealed that knockdown of miR-375-enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase-1 (PDK-1) expression and PDK-1-mediated activation of PI3kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.

Alcohol Toxicity in Diabetes and Its Complications: A Double Trouble?

BACKGROUND: Eight percent of the U.S. population has been diagnosed with diabetes mellitus (DM), while another large percentage has gone undiagnosed. As the epidemiology of this disease constitutes a larger percentage of the American population, another factor presents a dangerous dilemma that can exacerbate the hazardous effects imposed by DM. Excessive alcohol consumption concerns the health of more than 50% of all adults. When this heavy-alcohol-drinking population overlaps with DM and its complications, the effects can be dangerous. In this review, we term it as "double trouble." METHODS: We provide evidence of alcohol-induced exacerbation of organ damage in diabetic conditions. In certain cases, we have explained how diabetes and alcohol induce similar pathological effects. RESULTS: Known exacerbated complications include those related to heart diseases, liver damage, kidney dysfunction, as well as retinal and neurological impairment. Often, pathophysiological damage concludes with end-stage disorders and even mortality. The metabolic, cell signaling, and pathophysiological changes associated with "double trouble" would lead to the identification of novel therapeutic targets. CONCLUSIONS: This review summarizes the epidemiology, diagnosis, pathophysiology, metabolic, and cell signaling alterations and finally brushes upon issues and strategies to manage the "double trouble."

Interleukin-10 inhibits chronic angiotensin II-induced pathological autophagy.

BACKGROUND: Although autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is unknown. Here we tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, leads to improve cardiac function. METHODS AND RESULTS: Chronic Ang II strongly induced mortality, cardiac dysfunction in IL-10 Knockout mice. IL-10 deletion exaggerated pathological autophagy in response to Ang II treatment. In isolated cardiac myocytes, IL-10 attenuated Ang II-induced pathological autophagy and activated Akt/mTORC1 signaling. Pharmacological or molecular inhibition of Akt and mTORC1 signaling attenuated IL-10 effects on Ang II-induced pathological autophagy. Furthermore, lysosomal inhibition in autophagic flux experiments further confirmed that IL-10 inhibits pathological autophagy via mTORC1 signaling. CONCLUSION: Our data demonstrate a novel role of IL-10 in regulation of pathological autophagy; thus can act as a potential therapeutic molecule for treatment of chronic heart disease.

Sirtuin-6 deficiency exacerbates diabetes-induced impairment of wound healing.

Delayed wound healing is one of the major complications in diabetes and is characterized by chronic proinflammatory response, and abnormalities in angiogenesis and collagen deposition. Sirtuin family proteins regulate numerous pathophysiological processes, including those involved in promotion of longevity, DNA repair, glycolysis and inflammation. However, the role of sirtuin 6 (SIRT6), a NAD+-dependent nuclear deacetylase, in wound healing specifically under diabetic condition remains unclear. To analyse the role of SIRT6 in cutaneous wound healing, paired 6-mm stented wound was created in diabetic db/db mice and injected siRNA against SIRT6 in the wound margins (transfection agent alone and nonsense siRNA served as controls). Wound time to closure was assessed by digital planimetry, and wounds were harvested for histology, immunohistochemistry and Western blotting. SIRT6-siRNA-treated diabetic wound showed impaired healing, which was associated with reduced capillary density (CD31-staining vessels) when compared to control treatment. Interestingly, SIRT6 deficiency decreased vascular endothelial growth factor expression and proliferation markers in the wounds. Furthermore, SIRT6 ablation in diabetic wound promotes nuclear factor-κB (NF-κB) activation resulting in increased expression of proinflammatory markers (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-α and interleukin-1ß) and increased oxidative stress. Collectively, our findings demonstrate that loss of SIRT6 in cutaneous wound aggravates proinflammatory response by increasing NF-κB activation, oxidative stress and decrease in angiogenesis in the diabetic mice. Based on these findings, we speculate that the activation of SIRT6 signalling might be a potential therapeutic approach for promoting wound healing in diabetics.