Cardioprotective Cytokine Research Articles

Primitive macrophages enable long-term vascularization of human heart-on-a-chip platforms

The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. Invivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.

The role of charme in the phenotype and function of resident cardiac mesenchymal stromal cells

Abstract Background Charme is a murine long non-coding RNA with specific expression in striated muscles, presenting a human orthologue with 45% identity. Its depletion leads to incomplete muscle differentiation. Charme-/- mice display an altered cardiac phenotype with increased wall thickness and reduced chamber dimensions due to myocyte hyperplasia. Left ventricular dilatation and reduced fractional shortening are detected in aged Charme-/- mice. Embryonic myocardial architecture reveals aberrant compaction and ventricular hypotrabeculation, resembling some congenital cardiomyopathies. Cardiac mesenchymal stromal cells (CMSCs) play key roles in tissue homeostasis and pathophysiology, by maintaining the extracellular matrix and by paracrine action on all myocardial cells. Purpose To elucidate the phenotype, paracrine function, and myofibroblast differentiation capacity of resident CMSCs in the Charme-/- mouse model. Methods CMSCs were isolated from 5-week-old wild-type (WT) and Charme-/- mice by explant culture, pooling at least 4 different hearts per culture. Results Charme-/- CMSCs showed increased features of primitive phenotypes, such as enhanced spontaneous spheroid growth (123,6±11,9 vs 51,0±6,4 spheroids/well; N=8, p<0,0001) and clonogenesis efficiency (2,7±0,5-fold; N=7, p<0,05) compared to WT cells. Charme-/- CMSCs also showed increased migration ability (Charme-/-: 68,5±2,7% vs WT: 80,2±0,7% wound area; N=2). Flow cytometry identified a reduced proportion of lin-/Sca1+/CD90+ primitive mesenchymal cells in Charme-/- versus WT CMSCs (7,4±1,6% vs 56,9±6,5%; N=6, p<0,05). Western blot analysis on Charme-/- whole cardiac tissue showed reduced collagen I and collagen I/III protein ratio compared to WT hearts (0,17±0,03 vs 0,85±0,10 normalized OD; N=4, p<0,005). Stimulation with TGFb-1 resulted in lower expression of fibroblast activation markers compared to WT CMSCs, as assessed by immunofluorescence staining for aSMA (0,49±0,05 vs 0,76±0,10 mean fluorescence intensity; N=6-12, p<0,05) and collagen I/III mRNA expression ratio (1,3±0,1 vs 2,2±0,4 2^-DCt; N=3, p<0,01). The secretome of Charme-/- CMSCs revealed a general depletion in many cardioprotective cytokines, included in the KEGG term "PI3K-Akt signalling pathway" (FDR<0,0005) and classified in Gene Ontology categories of myoblast differentiation and fusion (GO:1901739, GO:1901741, GO:0045663, GO:0045661; FDR<0,05). In line with this, the secretome of TGFb1-stimulated Charme-/- CMSCs was less effective in sustaining cardiomyocyte survival versus the WT CMSC secretome (1,70±0,03 vs 1,90±0,04 normalized OD at 48h; N=5, p<0,005). Conclusions Charme-/- CMSCs show a less mature phenotype associated to reduced collagen I presence in situ, reduced activation and differentiation upon stimulation, and impaired cardioprotective paracrine functions, suggesting a key role of Charme in the physiology of the cardiac stroma, possibly mediating in part the altered cardiac phenotype of Charme-/- mice.

AAV9-mediated functional screening for cardioprotective cytokines in Coxsackievirus-B3-induced myocarditis

Viral myocarditis (VM) is an important cause of heart failure (HF) in children and adults. However, the molecular determinants involved in cardiac inflammation and cardiomyocyte necrosis remain poorly characterized, and cardioprotective molecules are currently missing. Here, we applied an in vivo method based on the functional selection (FunSel) of cardioprotective factors using AAV vectors for the unbiased identification of novel immunomodulatory molecules in a Coxsackievirus B3 (CVB3)-induced myocarditis mouse model. Two consecutive rounds of in vivo FunSel using an expression library of 60 cytokines were sufficient to identify five cardioprotective factors (IL9, IL3, IL4, IL13, IL15). The screening also revealed three cytokines (IL18, IL17b, and CCL11) that were counter-selected and likely to exert a detrimental effect. The pooled overexpression of the five most enriched cytokines using AAV9 vectors decreased inflammation and reduced cardiac dilatation, persisting at 1 month after treatment. Individual overexpression of IL9, the top ranking in our functional selection, markedly reduced cardiac inflammation and injury, concomitant with an increase of anti-inflammatory Th2-cells and a reduction of pro-inflammatory Th17- and Th22-cells at 14 days post-infection. AAV9-mediated FunSel cardiac screening identified IL9 and other four cytokines (IL3, IL4, IL13, and IL15) as cardioprotective factors in CVB3-induced VM in mice.

The Microenvironment of Decellularized Extracellular Matrix from Heart Failure Myocardium Alters the Balance between Angiogenic and Fibrotic Signals from Stromal Primitive Cells.

Cardiac adverse remodeling is characterized by biological changes that affect the composition and architecture of the extracellular matrix (ECM). The consequently disrupted signaling can interfere with the balance between cardiogenic and pro-fibrotic phenotype of resident cardiac stromal primitive cells (CPCs). The latter are important players in cardiac homeostasis and can be exploited as therapeutic cells in regenerative medicine. Our aim was to compare the effects of human decellularized native ECM from normal (dECM-NH) or failing hearts (dECM-PH) on human CPCs. CPCs were cultured on dECM sections and characterized for gene expression, immunofluorescence, and paracrine profiles. When cultured on dECM-NH, CPCs significantly upregulated cardiac commitment markers (CX43, NKX2.5), cardioprotective cytokines (bFGF, HGF), and the angiogenesis mediator, NO. When seeded on dECM-PH, instead, CPCs upregulated pro-remodeling cytokines (IGF-2, PDGF-AA, TGF-β) and the oxidative stress molecule H2O2. Interestingly, culture on dECM-PH was associated with impaired paracrine support to angiogenesis, and increased expression of the vascular endothelial growth factor (VEGF)-sequestering decoy isoform of the KDR/VEGFR2 receptor. Our results suggest that resident CPCs exposed to the pathological microenvironment of remodeling ECM partially lose their paracrine angiogenic properties and release more pro-fibrotic cytokines. These observations shed novel insights on the crosstalk between ECM and stromal CPCs, suggesting also a cautious use of non-healthy decellularized myocardium for cardiac tissue engineering approaches.

Synthetic Prostacyclin Agonist Attenuates Pressure-Overloaded Cardiac Fibrosis by Inhibiting FMT

Fibroblast-to-myofibroblast transition (FMT) is the primary inducer of cardiac fibrosis. ONO-1301, a synthetic prostacyclin agonist, reportedly promotes tissue fibrosis repair by enhancing anti-fibrotic cytokine production. We hypothesized that ONO-1301 attenuates pressure-overloaded cardiac fibrosis by modulating FMT and generated a pressure-overloaded murine model via transverse aortic constriction (TAC) to evaluate the in vivo effects of ONO-1301. Cardiac fibrosis, left ventricular dilatation, and systolic dysfunction were established 4 weeks after TAC; however, ONO-1301 treatment initiated 2 weeks after TAC significantly attenuated those effects. Furthermore, ONO-1301 treatment significantly upregulated expression levels of cardioprotective cytokines such as vascular endothelial growth factor and hepatocyte growth factor in TAC hearts, whereas FMT-related factors, including transforming growth factor (TGF)-β1 and connective tissue growth factor, were significantly downregulated. The number of α-smooth muscle actin (α-SMA)- and vimentin-positive cells, representing fibroblast-originated cells transitioned into myofibroblasts, was significantly reduced in ONO-1301-treated TAC hearts. We isolated cardiac fibroblasts (CFs) from the left ventricles of adult male mice and assessed the effects of ONO-1301 on CFs stimulated by TGF-β. Results showed that ONO-1301 co-incubation significantly suppressed TGF-β-induced α-SMA expression and collagen synthesis, and significantly inhibited TGF-β-induced CF proliferation and migration. Our findings suggest that ONO-1301 ameliorates pressure overloaded cardiac fibrosis by inhibiting TGF-β-induced FMT.

Interleukin‐22 Directly Activates Myocardial STAT3 (Signal Transducer and Activator of Transcription‐3) Signaling Pathway and Prevents Myocardial Ischemia Reperfusion Injury

BACKGROUNDInterleukin (IL)‐22, a member of the IL‐10 cytokine family, is the only known cytokine that is secreted by immune cells but does not target immune cells; it mainly targets epithelial cells. In this study, we aimed to determine whether IL‐22 administration could activate the myocardial STAT3 (signal transducer and activator of transcription‐3) signaling pathway, and thus prevent myocardial injury, in a mouse model of ischemia reperfusion injury.METHODS AND RESULTSWe evaluated the STAT3 activation after IL‐22 injection by Western blot analysis and immunostaining for phosphorylated STAT3 in the heart and found that STAT3 activation in heart tissue rapidly peaked after IL‐22 injection. Coimmunostaining of phosphorylated STAT3 and α‐actinin revealed that STAT3 activation occurred in cardiomyocytes after IL‐22 administration. In heart tissue from intact mice, real‐time PCR demonstrated significant expression of IL‐22 receptor subunit 1, and coimmunostaining of IL‐22 receptor subunit 1 and α‐actinin showed IL‐22 receptor subunit 1 expression in cardiomyocytes. In cultured cardiomyocytes, IL‐22 activated STAT3, and we detected IL‐22 receptor subunit 1 expression. Overall, these results indicated that IL‐22 directly activated the myocardial IL‐22‐receptor subunit 1–STAT3 signaling pathway. Following ischemia reperfusion, compared with PBS‐treated mice, IL‐22‐treated mice exhibited a significantly reduced infarct size, significantly reduced myocardial apoptosis, and significantly enhanced phosphorylated STAT3 expression. Moreover, heart tissue from IL‐22‐treated mice exhibited a significantly reduced expression ratio of phosphorylated p53 to p53.CONCLUSIONSOur present findings suggest that IL‐22 directly activated the myocardial STAT3 signaling pathway and acted as a cardioprotective cytokine to ameliorate acute myocardial infarction after ischemia reperfusion.

Growth differentiating factor-15 and its association with traditional cardiovascular risk factors: The African-PREDICT study

Background and aimsGrowth differentiating factor-15 (GDF-15) is a stress-induced and cardio-protective cytokine, reported to be influenced by a number of cardiovascular risk factors. In older adults, GDF-15 associated with age, black ethnicity and smoking. It is important to determine if GDF-15 could potentially be used as an early marker of cardiovascular disease, especially in young populations. We investigated whether GDF-15 associated with traditional cardiovascular risk factors (age, sex, ethnicity, blood pressure (BP), socio-economic status, waist-to-hip ratio, cholesterol, physical inactivity, smoking and alcohol use) in young apparently healthy adults. Methods and resultsWe included 1189 black and white participants (aged between 20 and 30 years). Questionnaires were used to collect demographic and physical activity data. We measured serum GDF-15, and performed 24-h ambulatory BP and pulse wave analysis. The following risk factors increased with increasing GDF-15 quartiles: age, black ethnicity, central systolic BP, 24-h diastolic BP, tumour necrosis factor-alpha, lipids, cotinine, smoking and alcohol use (all p trend ≤ 0.013). Socio-economic status and physical activity (p trend ≤ 0.014) were the lowest in the highest quartile. In multi-variable adjusted regression analyses GDF-15 associated with central systolic BP (β = 0.076; p = 0.027), age (β = 0.096; p = 0.006), low socio-economic status (β = −0.12; p = 0.003), physical inactivity (β = −0.18; p < 0.0001), tumour necrosis factor-alpha (β = 0.28; p < 0.0001) and cotinine (β = 0.12; p < 0.0001). ConclusionIn young adults, GDF-15 associated independently with multiple traditional cardiovascular risk factors including higher central systolic blood pressure, older age, lower socio-economic status, physical inactivity, inflammation and smoking. These results suggest that GDF-15 is a promising biomarker for early identification of cardiovascular risk.

Cardioprotective cytokine interleukin-33 is up-regulated by statins in human cardiac tissue.

Interleukin (IL)‐33 is a member of the IL‐1 family and is able to act cardioprotective. The aim of this study was to investigate the regulation of IL‐33 by 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A (HMG‐CoA) reductase inhibitors (statins) and bisphosphonates (BPs) in human cardiac tissue. The lipophilic fluvastatin, simvastatin, atorvastatin, and lovastatin as well as the nitrogenous BPs alendronate and ibandronate, but not hydrophilic pravastatin increased IL‐33 mRNA and intracellular IL‐33 protein levels in both human adult cardiac myocytes (HACM) and fibroblasts (HACF). Additionally, fluvastatin reduced soluble ST2 secretion from HACM. IL‐33 was also up‐regulated by the general inhibitor of prenylation perillic acid, a RhoA kinase inhibitor Y‐27632, and by latrunculin B, but statin‐induced IL‐33 expression was inhibited by mevalonate, geranylgeranyl pyrophosphate (GGPP) and RhoA activator U‐46619. The IL‐33 promoter was 2.3‐fold more accessible in statin‐treated HACM compared to untreated cells (P = 0.037). In explanted hearts of statin‐treated patients IL‐33 protein was up‐regulated as compared with the hearts of non‐statin‐treated patients (P = 0.048). As IL‐33 was previously shown to exert cardioprotective effects, one could speculate that such up‐regulation of IL‐33 expression in human cardiac cells, which might happen mainly through protein geranylgeranylation, could be a novel mechanism contributing to known cardioprotective effects of statins and BPs.

Macrophage migration inhibitory factor serves a pivotal role inthe regulation of radiation-induced cardiac senescencethrough rebalancing the microRNA-34a/sirtuin 1 signaling pathway.

Radiotherapy significantly increases survival innumerous cancer patients, although it may have delayed adverse effects, including significant short‑ and long‑term effects on cardiovascular function, leading to significant morbidity and mortality. However, the mechanisms underlying these effects remain unclear. Cardiomyocyte senescence contributes to cardiovascular disease via impaired cardiac function. MicroRNA‑34a (miR‑34a) is a senescence‑associated miR involved in the pathology of cardiovascular diseases, while macrophage migration inhibitory factor (MIF) is a cardioprotective cytokine with an important role in cardiovascular diseases. The present study aimed to determine whether MIF has a cytoprotective effect in cardiomyocytes exposed to radiation through modulating miR‑34a. Human cardiomyocytes (HCMs) were incubated with MIF and then exposed to radiation. Cellular proliferation was measured using a Cell Counting Kit‑8, while cellular senescence was evaluated based on the senescence‑associated β‑galactosidase activity and the gene expression levels of cyclin‑dependent kinase inhibitor 1a (Cdkn1a) and Cdkn2c. Oxidative stress was evaluated by measuring the generation of reactive oxygen species and malondialdehyde, as well as the expression of antioxidant genes. In addition, HCMs were treated with small interfering RNA against sirtuin1 (SIRT1) to examine the role of this gene in MIF‑associated rejuvenation following radiation‑associated senescence. miR‑34a was significantly increased in HCMs exposed to radiation, while MIF inhibited senescence by suppressing miR‑34a. SIRT1 was identified as a target gene of miR‑34a, mediating the anti‑senescence effect induced by MIF. Furthermore, MIF rejuvenation involved rebalancing the oxidation process disturbed by radiation. These results provided direct evidence that inhibition of miR‑34a by MIF protected against radiation‑induced cardiomyocyte senescence via targeting SIRT1. Inhibition of miR‑34a by MIF may thus be a novel strategy for combating cardiac radiation‑associated damage.

Left Ventricular Unloading Before Reperfusion Promotes Functional Recovery After Acute Myocardial Infarction

BackgroundHeart failure after an acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. We recently reported that activation of a transvalvular axial-flow pump in the left ventricle and delaying myocardial reperfusion, known as primary unloading, limits infarct size after AMI. The mechanisms underlying the cardioprotective benefit of primary unloading and whether the acute decrease in infarct size results in a durable reduction in LV scar and improves cardiac function remain unknown. ObjectivesThis study tested the importance of LV unloading before reperfusion, explored cardioprotective mechanisms, and determined the late-term impact of primary unloading on myocardial function. MethodsAdult male swine were subjected to primary reperfusion or primary unloading after 90 min of percutaneous left anterior descending artery occlusion. ResultsCompared with primary reperfusion, 30 min of LV unloading was necessary and sufficient before reperfusion to limit infarct size 28 days after AMI. Compared with primary reperfusion, primary unloading increased expression of genes associated with cellular respiration and mitochondrial integrity within the infarct zone. Primary unloading for 30 min further reduced activity levels of proteases known to degrade the cardioprotective cytokine, stromal-derived factor (SDF)-1α, thereby increasing SDF-1α signaling via reperfusion injury salvage kinases, which limits apoptosis within the infarct zone. Inhibiting SDF-1α activity attenuated the cardioprotective effect of primary unloading. Twenty-eight days after AMI, primary unloading reduced LV scar size, improved cardiac function, and limited expression of biomarkers associated with heart failure and maladaptive remodeling. ConclusionsThe authors report for the first time that first mechanically reducing LV work before coronary reperfusion with a transvalvular pump is necessary and sufficient to reduce infarct size and to activate a cardioprotective program that includes enhanced SDF-1α activity. Primary unloading further improved LV scar size and cardiac function 28 days after AMI.

Epicardial Fat: Pathophysiology and Clinical Significance.

Over the last decade and a half there has been much interest in understanding the role of epicardial adipose tissue (EAT) in cardiac pathology. EAT is a visceral adipose deposit with putative paracrine function. In the nondiseased state, EAT releases cardioprotective cytokines and chemokines to the coronary vasculature. In pathological states, EAT releases an inflammatory cytokine profile that is believed to contribute to the development and progression of coronary artery disease (CAD). EAT imaging with echocardiography, computed tomography, and magnetic resonance imaging has demonstrated a correlation between EAT size and CAD. Small interventional studies have found evidence that the pathological state of EAT is at least somewhat reversible. The relationship between EAT size and the development and/or progression of CAD may present future clinicians with a new tool for risk assessment and intervention response monitoring. In this article we review current basic science and clinical research, comment on the role of EAT imaging in the management of patients at risk for CAD, and suggest areas for future investigation.

Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

Four cases of investigational therapy with interleukin-11 against acute myocardial infarction

We describe four cases of the patients with ST-elevation myocardial infarction (STEMI) that were treated with interleukin-11 (IL-11), a cardioprotective cytokine. Recombinant human IL-11 (rhIL-11), was intravenously administered to two cases at low dose (6µg/kg) and to two at high dose (25µg/kg). The cytokine administration started just after the coronary occlusion was confirmed by coronary angiography (CAG), taking 3h. Following CAG, percutaneous coronary intervention (PCI) was performed as a standard therapy. No serious adverse drug reactions were observed. All the cases left the hospital without the symptom of heart failure. We discuss the possibility of the clinical use of rhIL-11 as an adjunct therapy to PCI for the STEMI patients.

Abstract 151: Cell-based Therapies for Heart Failure: Changes in Cytokine Expression From Mesenchymal Stem Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

Mesenchymal stem cells (MSCs) use paracrine signaling to modulate the cellular microenvironment via expression of cytokines, chemokines, and adhesion molecules to aid and promote endogenous repair. Induced pluripotent stem cell derived cardiomyocyte (iPSC-CMs) and mesenchymal stem cells (MSCs) together may play a synergistic role in changing the microenvironment milieu to allow for endogenous cellular repair through paracrine signaling. Cytokine expression is involved in the progression of heart failure (HF). Using a rat model of HF, cell based therapies with a fibroblast embedded patch only, iPSC-CM patch, and MSCs via tail vein (IV) or intracardiac injections (IC) to the left ventricle (LV) were administered, and RNA was subsequently isolated, and real-time polymerase chain reaction (PCR) was performed for analysis of gene expression. Evaluation of gene expression revealed significant increases in the expression of connexin 43 with iPSC-CMs (p&lt;0.05). Expression of MMP9 was decreased with MSCs alone (p&lt;0.05) but with the use of the patch with both cell types, its levels were significantly increased (p&lt;0.05). Myosin heavy chain was seen to increase significantly with increasing cell numbers in iPSC-CM therapy (p&lt;0.05). Markers of angiogenesis including vascular endothelial growth factor, angiopoietin, and insulin like growth factor were significantly increased with iPSC-CM patch therapy (p&lt;0.05). Up-regulation of angiogenic cytokines and cardio-protective cytokines may help in slowing progression of HF. Interestingly, we also observed an increase in some markers, which were associated with HF. In conclusion, both iPSC-CM patch and MSCs altered signaling in the setting of HF, perhaps leading to improvement of at the cellular level. An iPSC-CM-based patch and MSCs as an adjuvant therapy may be able to play a role in the setting of HF as cellular therapeutic approach.

Akt protects the heart against ischaemia-reperfusion injury by modulating mitochondrial morphology.

The mechanism through which the protein kinase Akt (also called PKB), protects the heart against acute ischaemia-reperfusion injury (IRI) is not clear. Here, we investigate whether Akt mediates its cardioprotective effect by modulating mitochondrial morphology. Transfection of HL-1 cardiac cells with constitutively active Akt (caAkt) changed mitochondrial morphology as evidenced by an increase in the proportion of cells displaying predominantly elongated mitochondria (73 ± 5.0 % caAkt vs 49 ± 5.8 % control: N=80 cells/group; p< 0.05). This effect was associated with delayed time taken to induce mitochondrial permeability transition pore (MPTP) opening (by 2.4 ± 0.5 fold; N=80 cells/group: p< 0.05); and reduced cell death following simulated IRI (32.8 ± 1.2 % caAkt vs 63.8 ± 5.6 % control: N=320 cells/group: p< 0.05). Similar effects on mitochondrial morphology, MPTP opening, and cell survival post-IRI, were demonstrated with pharmacological activation of Akt using the known cardioprotective cytokine, erythropoietin (EPO). The effect of Akt on inducing mitochondrial elongation was found to be dependent on the mitochondrial fusion protein, Mitofusin-1 (Mfn1), as ablation of Mfn1 in mouse embryonic fibroblasts (MEFs) abrogated Akt-mediated mitochondrial elongation. Finally, in vivo pre-treatment with EPO reduced myocardial infarct size (as a % of the area at risk) in adult mice subjected to IRI (26.2 ± 2.6 % with EPO vs 46.1 ± 6.5 % in control; N=7/group: p< 0.05), and reduced the proportion of cells displaying myofibrillar disarray and mitochondrial fragmentation observed by electron microscopy in adult murine hearts subjected to ischaemia from 5.8 ± 1.0 % to 2.2 ± 1.0 % (N=5 hearts/group; p< 0.05). In conclusion, we found that either genetic or pharmacological activation of Akt protected the heart against acute ischaemia-reperfusion injury by modulating mitochondrial morphology.

Association between plasma adiponectin levels and decline in forced expiratory volume in 1 s in a general Japanese population: the Takahata study.

Background:Adiponectin is an anti-inflammatory and cardio-protective cytokine. However, several studies have demonstrated that plasma adiponectin levels were inversely associated with pulmonary function in patients with chronic obstructive pulmonary disease, suggesting a proinflammatory or pulmonary-destructive role. It is still unclear whether adiponectin is a potent biomarker predicting declines in pulmonary function. The aim of this study was to investigate the association between adiponectin and pulmonary function among Japanese individuals who participated in an annual health check-up.Methods:Spirometry and blood sampling, including measurements of plasma adiponectin, were performed for 3,253 subjects aged 40 years or older who participated in a community-based annual health check-up in Takahata, Japan from 2004 to 2006. In 2011, spirometry was re-performed, and the data from 872 subjects (405 men and 467 women) were available for a longitudinal analysis.Results:Plasma adiponectin levels were found to be significantly associated with age, body mass index (BMI), and alanine aminotransferase (ALT), triglycerides (TG), and high-density lipoprotein-cholesterol (HDL-c) levels among both men and women in the study population. Plasma adiponectin levels were found to be associated with lifetime cigarette consumption (Brinkman index, BI) in men only. Plasma adiponectin levels were inversely correlated with forced expiratory volume in 1 s (FEV1) per forced vital capacity in both men and women. In addition, the annual change in FEV1 was inversely associated with plasma adiponectin levels in both genders. A multiple linear regression analysis revealed that this association was independent of other confounding factors such as age, BMI, BI, ALT, TG, and HDL-c.Conclusions:The results of the present study suggest that adiponectin levels are predictive of declines in FEV1 in the general population.

Pleiotropic effect of factor Xa inhibitor: fondaparinux inhibites ROS-induced cell proliferation and augments cardioprotective cytokine production in mouse cardiac-derived fibroblast

Purpose: Factor Xa inhibitors are anticoagulants to reduce in stroke and other thromboembolic events. Fondaparinux (FDP) is an approved indirect factor Xa inhibitor to prevent venous thromboembolism. It is known that the proliferation of cardiac fibroblasts leads the fibrosis of coronary vessels, which are deeply associated with the progression of coronary atherosclerosis, and cardiac remodeling to cause heart failure in clinical settings. However, the effect of FDP in cardiac fibroblasts for the development of atherosclerosis and cardiac remodeling is still remaining unclear. Therefore, we aim to evaluate the functional role of FDP in mouse cardiac fibroblasts stimulated by hydrogen peroxide (H2O2). Materials and methods: Confluent fibroblasts were pretreated with or without FDP (0.34 μg/ml) for 6 hours, and then stimulated by H2O2 in various times. Reactive oxygen species (ROS) were measured by DCFDA method. Cell proliferation was measured by MTT assay after 12 and 24 hours of H2O2 stimulation (25 μM). Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and tissue growth factor (TGF)-β productions were evaluated by ELISA after 12, 24 and 48 hours of H2O2 stimulation (25 μM), respectively. Results: H2O2 stimulation by itself induced excessive ROS production and cell proliferation in mouse cardiac fibroblast. H2O2 (25 mM) induced cell proliferation was significantly decreased in FDP-pretreated cardiac fibroblasts compared to cells without FDP (86% decrease in 24 hours). Although H2O2 had no effect in TNF-α and TGF-β production, IL-6 production was significantly enhanced by FDP pretreatment (84% increase in 48 hours). Conclusion: These data suggest that the pleiotropic effects of factor Xa inhibitor may provide favorable effects on cardiovascular disease prevention, at least part of in vitro model.

Differential regulation of TNF receptor 1 and receptor 2 in adiponectin expression following myocardial ischemia

In vitro experiments demonstrate that adiponectin, a cardioprotective cytokine, is inhibited by tumor necrosis factor-alpha (TNFα). However, the role of TNFα in post-myocardial infarction (post-MI) adiponectin reduction remains unclear. More importantly, the TNF receptor type (TNFR1 or TNFR2) responsible for TNFα-mediated suppression of adiponectin production is unknown. The current study determined the role of TNFα in post-myocardial infarction (post-MI) adiponectin reduction, and identified the receptor type responsible for TNFα-mediated suppression of adiponectin production. Adult male wild type (WT) and three knockout variety (TNFα(-/-), TNFR1(-/-), and TNFR2(-/-)) mice were subjected to MI via coronary artery occlusion. Histological and biochemical analyses were performed 3 and 7days post-MI. In WT mice, MI significantly increased plasma TNFα, reduced adipocyte adiponectin mRNA, and decreased plasma adiponectin levels. TNFα deletion had no significant effect upon basal adiponectin level, and partially restored adiponectin expression/production post-MI (P<0.01 vs. WT). Basal adiponectin levels were significantly increased in TNFR1(-/-) (P<0.05 vs. WT), and unchanged in TNFR2(-/-) mice. Importantly, suppressed adiponectin expression/production by MI or TNFα administration was markedly decreased by TNFR1 deletion (P<0.01 vs. WT), but exacerbated by TNFR2 deletion (P<0.05 vs. WT). Mechanistically, TNFR1 knockout significantly inhibited, whereas TNFR2 knockout further enhanced TNFα-induced mRNA and protein expression of ATF3, a transcriptional factor known to significantly inhibit adiponectin expression. Our study demonstrates that TNFα overproduction is responsible for reduced adiponectin expression/production following MI. Furthermore, we show that TNFR1/TNFR2 exerts opposite effects upon adiponectin expression/production via differential regulation of ATF3.

Activation of Natural Killer T Cells Ameliorates Postinfarct Cardiac Remodeling and Failure in Mice

Chronic inflammation in the myocardium is involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Invariant natural killer T (iNKT) cells have been shown to produce inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have determined the pathophysiological role of iNKT cells in post-MI LV remodeling. The purpose of this study was to examine whether the activation of iNKT cells might affect the development of LV remodeling and failure. After creation of MI, mice received the injection of either α-galactosylceramide (αGC; n=27), the activator of iNKT cells, or phosphate-buffered saline (n=31) 1 and 4 days after surgery, and were followed during 28 days. Survival rate was significantly higher in MI+αGC than MI+PBS (59% versus 32%, P<0.05). LV cavity dilatation and dysfunction were significantly attenuated in MI+αGC, despite comparable infarct size, accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. The infiltration of iNKT cells were increased during early phase in noninfarcted LV from MI and αGC further enhanced them. It also enhanced LV interleukin (IL)-10 gene expression at 7 days, which persisted until 28 days. AntienIL-10 receptor antibody abrogated these protective effects of αGC on MI remodeling. The administration of αGC into iNKT cell-deficient Jα18(-/-) mice had no such effects, suggesting that αGC was a specific activator of iNKT cells. iNKT cells play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as IL-10.

Abstract 8829: The Disruption of Natural Killer T Cell Exacerbates Cardiac Hypertrophy and Heart Failure Due to Pressure Overload in Mice

Background Chronic inflammation has been reported to be involved in the development of cardiac remodeling and failure. Natural killer T (NKT) cells, a subset of T lymphocytes, have capacity to produce various inflammatory cytokines and orchestrate tissue inflammation. Our previous study demonstrated that the pharmacological activation of NKT cells attenuated the development of heart failure (HF), which was mediated by the enhanced expression of interleukin-10. However, no previous studies have examined the pathophysiological role of NKT cells in pressure overload-induced HF. We thus hypothesized that the disruption of NKT cell could exacerbate cardiac hypertrophy and HF after pressure overload in mice. Methods Transverse aortic constriction (TAC) operation was performed in male C57BL/6J wild-type(WT) and NKT cell knockout (KO) mice, and sham operation was also performed; WT+Sham (n=5), KO+sham (n=5), WT+TAC (n=29), and KO+TAC (n=34). After 14 days, echocardiography was performed, and gene expressions in left ventricle (LV) tissues were quantified by RT-PCR. Results Survival rate during 28 days was significantly lower in KO+TAC than WT+TAC (16% vs. 61%, P&lt;0.05), and no mice died in sham groups. NKT cell receptor was increased by 60% in LV from TAC compared to Sham at 14days. LV end-diastolic dimension (4.60±0.14vs3.57±0.08 mm, p&lt;0.05) were greater, and LV fractional shortening (17.1±0.8 vs 30.4±4.3 %, p&lt;0.05) was lower in KO+TAC than in WT+TAC. LV weight and lung weight/body weight were significantly lower in KO+TAC than in WT+TAC. No such changes were observed in either WT+Sham or KO+Sham. In parallel, myocyte cross-sectional area and collagen volume fraction were greater in KO+TAC than in WT+TAC. Interleukin-10 (IL-10) mRNA expression was completely abolished in KO+TAC compared to TAC. Conclusions The disruption of NKT cell exacerbated the development of HF after TAC through the diminished expression of cardioprotective cytokine, IL-10. NKT cells play a critical role in the development of pressure-overload hypertrophy and failure.