Inhibition of de novo ceramide synthesis by Sirtuin-1 improves beta-cell function and glucose metabolism in type 2 diabetes.

BACKGROUND: Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular diseases (CVD). Dysregulated pro-apoptotic ceramide synthesis reduces ß-cell insulin secretion, thereby promoting hyperglycemic states which may manifest as T2D. Pro-apoptotic ceramides modulate insulin sensitivity and glucose tolerance while being linked to poor cardiovascular outcomes. Sirtuin-1 (SIRT1) is a NAD + - dependent deacetylase that protects against pancreatic ß-cell dysfunction; however, systemic levels are decreased in obese T2D mice and may promote pro-apoptotic ceramide synthesis and hyperglycemia. Herein, we aimed to assess the effects of restoring circulating SIRT1 levels to prevent metabolic imbalance in obese and diabetic mice. METHODS AND RESULTS: Circulating SIRT1 levels were reduced in obese diabetic mice (db/db) as compared to age-matched non-diabetic db/+ controls. Restoration of SIRT1 plasma levels with recombinant murine SIRT1 for 4-weeks prevented body weight gain, improved glucose tolerance, insulin sensitivity and vascular function in mice models of obesity and T2D. Untargeted lipidomics revealed that SIRT1 restored insulin-secretory function of ß-cells by reducing synthesis and accumulation of pro-apoptotic ceramides. Molecular mechanisms involved direct binding to and deacetylation of Toll-like receptor 4 (TLR4) by SIRT1 in ß-cells thereby decreasing the rate limiting enzymes of sphingolipid synthesis SPTLC1/2 via AKT/NF-κB. Among T2D patients, those with high baseline plasma levels of SIRT1 prior to metabolic surgery displayed restored ß-cell function (HOMA2- ß) and were more likely to have T2D remission during follow-up. CONCLUSION: Acetylation of TLR4 promotes ß-cell dysfunction via ceramide synthesis in T2D, which is blunted by systemic SIRT1 replenishment. Hence, restoration of systemic SIRT1 may provide a novel therapeutic strategy to counteract toxic ceramide synthesis and mitigate cardiovascular complications of T2D.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

Endothelial SIRT6 deficiency promotes arterial thrombosis in mice.

OBJECTIVE: Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD+-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. APPROACH AND RESULTS: Using a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6-/- mice as compared to Sirt6fl/fl controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. CONCLUSIONS: Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.

Among biomarkers of neutrophil activity, matrix metalloproteinases 8 independently predicts remission of metabolic syndrome.

BACKGROUND AND AIMS: Inflammation due to the excess of nutrient intake plays an important role in the pathophysiology of metabolic syndrome (MetS). Here, the potential influence of neutrophils and their degranulation markers on MetS improvement upon dietary and behavioral counselling, has been investigated. Specifically, we aimed at investigating their role as potential predictors of metabolic syndrome improvements. METHODS AND RESULTS: patients with MetS (n = 127) received behavioral and dietary recommendations before follow-up at 6 months. Serum levels of matrix metalloproteinases (MMP)8, MMP9, myeloperoxidase (MPO), tissue inhibitor of MMP (TIMP)-1, TIMP-2, TIMP-3 and resistin were tested at baseline. In the whole cohort, baseline levels of proinflammatory MMP8, MMP9 and MPO increased together with the number of MetS criteria. Seventy-three (57%) patients experienced a reduction in MetS-defining criteria at follow-up. With respect to those with no improvement, such individuals showed lower weight and waist circumference at enrolment, less frequent smoking habits, higher levels of triglycerides and lower circulating MMP8. At logistic regression analysis, baseline MMP8 showed negative predictive ability (odds ratio (OR) 0.979 [0.961-0.997]; p = 0.025) against MetS improvement. Such findings hold true even when included in the backward stepwise logistic regression model confirming MMP8 as an independent predictor (OR 0.970 [0.949-0.993]; p = 0.009). Receiver operating characteristic (ROC) curve confirmed the predictive ability of MMP8 combined in a model including baseline MetS criteria and waist circumference. Bootstrap resampling analysis internally validated our findings. CONCLUSION: Improvement of MetS is independently associated with baseline low MMP-8 levels, suggesting a pivotal role for inflammation in metabolic alteration.

Effects of acute administration of trimethylamine N-oxide on endothelial function: a translational study.

Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery (Roux-en-Y gastric bypass, RYGB), stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg-1) 1.5-h before sacrifice and ex-vivo 30-min incubation of thoracic aortas with 10-6 M TMAO did not impair vasodilation in response to acetylcholine (Ach), glucagon-like peptide 1, or insulin. Similarly, in lean WT mice (n = 5-6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10-6 M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO.

Supervised and unsupervised learning to define the cardiovascular risk of patients according to an extracellular vesicle molecular signature.

Cardiovascular (CV) disease represents the most common cause of death in developed countries. Risk assessment is highly relevant to intervene at individual level and implement prevention strategies. Circulating extracellular vesicles (EVs) are involved in the development and progression of CV diseases and are considered promising biomarkers. We aimed at identifying an EV signature to improve the stratification of patients according to CV risk and likelihood to develop fatal CV events. EVs were characterized by nanoparticle tracking analysis and flow cytometry for a standardized panel of 37 surface antigens in a cross-sectional multicenter cohort (n = 486). CV profile was defined by presence of different indicators (age, sex, body mass index, hypertension, hyperlipidemia, diabetes, coronary artery disease, cardiac heart failure, chronic kidney disease, smoking habit, organ damage) and according to the 10-year risk of fatal CV events estimated using SCORE charts of European Society of Cardiology. By combining expression levels of EV antigens using unsupervised learning, patients were classified into 3 clusters: Cluster-I (n = 288), Cluster-II (n = 83), Cluster-III (n = 30). A separate analysis was conducted on patients displaying acute CV events (n = 82). Prevalence of hypertension, diabetes, chronic heart failure, and organ damage (defined as left ventricular hypertrophy and/or microalbuminuria) increased progressively from Cluster-I to Cluster-III. Several EV antigens, including markers for platelets (CD41b-CD42a-CD62P), leukocytes (CD1c-CD2-CD3-CD4-CD8-CD14-CD19-CD20-CD25-CD40-CD45-CD69-CD86), and endothelium (CD31-CD105) were independently associated with CV risk indicators and correlated to age, blood pressure, glucometabolic profile, renal function, and SCORE risk. EV profiling, obtained from minimally invasive blood sampling, allows accurate patient stratification according to CV risk profile.

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies.

AIMS: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. METHODS AND RESULTS: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. CONCLUSIONS: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.

The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

Calcific aortic valve disease: from molecular and cellular mechanisms to medical therapy.

Calcific aortic valve disease (CAVD) is a highly prevalent condition that comprises a disease continuum, ranging from microscopic changes to profound fibro-calcific leaflet remodelling, culminating in aortic stenosis, heart failure, and ultimately premature death. Traditional risk factors, such as hypercholesterolaemia and (systolic) hypertension, are shared among atherosclerotic cardiovascular disease and CAVD, yet the molecular and cellular mechanisms differ markedly. Statin-induced low-density lipoprotein cholesterol lowering, a remedy highly effective for secondary prevention of atherosclerotic cardiovascular disease, consistently failed to impact CAVD progression or to improve patient outcomes. However, recently completed phase II trials provide hope that pharmaceutical tactics directed at other targets implicated in CAVD pathogenesis offer an avenue to alter the course of the disease non-invasively. Herein, we delineate key players of CAVD pathobiology, outline mechanisms that entail compromised endothelial barrier function, and promote lipid homing, immune-cell infiltration, and deranged phospho-calcium metabolism that collectively perpetuate a pro-inflammatory/pro-osteogenic milieu in which valvular interstitial cells increasingly adopt myofibro-/osteoblast-like properties, thereby fostering fibro-calcific leaflet remodelling and eventually resulting in left ventricular outflow obstruction. We provide a glimpse into the most promising targets on the horizon, including lipoprotein(a), mineral-binding matrix Gla protein, soluble guanylate cyclase, dipeptidyl peptidase-4 as well as candidates involved in regulating phospho-calcium metabolism and valvular angiotensin II synthesis and ultimately discuss their potential for a future therapy of this insidious disease.

Adeno-Associated Virus-Mediated Gain-of-Function mPCSK9 Expression in the Mouse Induces Hypercholesterolemia, Monocytosis, Neutrophilia, and a Hypercoagulative State.

Hypercholesterolemia has previously been induced in the mouse by a single intravenous injection of adeno-associated virus (AAV)-based vector harboring gain-of-function pro-protein convertase subtilisin/kexin type 9. Despite the recent emergence of the PCSK9-AAV model, the profile of hematological and coagulation parameters associated with it has yet to be characterized. We injected 1.0 × 1011 viral particles of mPCSK9-AAV or control AAV into juvenile male C57BL/6N mice and fed them with either a Western-type high-fat diet (HFD) or standard diet over the course of 3 weeks. mPCSK9-AAV mice on HFD exhibited greater plasma PCSK9 concentration and lower low-density lipoprotein levels, concomitant with increased total cholesterol and non-high-density lipoprotein (non-HDL)-cholesterol concentrations, and lower HDL-cholesterol concentrations than control mice. Furthermore, mPCSK9-AAV-injected mice on HFD exhibited no signs of atherosclerosis at 3 weeks after the AAV injection. Hypercholesterolemia was associated with a thromboinflammatory phenotype, as neutrophil levels, monocyte levels, and neutrophil-to-lymphocyte ratios were higher and activated partial thromboplastin times (aPTTs) was lower in HFD-fed mPCSK9-AAV mice. Therefore, the mPCSK9-AAV is a suitable model of hypercholesterolemia to examine the role of thromboinflammatory processes in the pathogenesis of cardiovascular and cerebrovascular diseases.

Resilience of the Internal Mammary Artery to Atherogenesis: Shifting From Risk to Resistance to Address Unmet Needs.

Fueled by the global surge in aging, atherosclerotic cardiovascular disease reached pandemic dimensions putting affected individuals at enhanced risk of myocardial infarction, stroke, and premature death. Atherosclerosis is a systemic disease driven by a wide spectrum of factors, including cholesterol, pressure, and disturbed flow. Although all arterial beds encounter a similar atherogenic milieu, the development of atheromatous lesions occurs discontinuously across the vascular system. Indeed, the internal mammary artery possesses unique biological properties that confer protection to intimal growth and atherosclerotic plaque formation, thus making it a conduit of choice for coronary artery bypass grafting. Its endothelium abundantly expresses nitric oxide synthase and shows accentuated nitric oxide release, while its vascular smooth muscle cells exhibit reduced tissue factor expression, high tPA (tissue-type plasminogen activator) production and blunted migration and proliferation, which may collectively mitigate intimal thickening and ultimately the evolution of atheromatous plaques. We aim here to provide insights into the anatomy, physiology, cellular, and molecular aspects of the internal mammary artery thereby elucidating its remarkable resistance to atherogenesis. We propose a change in perspective from risk to resilience to decipher mechanisms of atheroresistance and eventually identification of novel therapeutic targets presently not addressed by currently available remedies.

TNF-α antagonism rescues the effect of ageing on stroke: Perspectives for targeting inflamm-ageing.

AIMS: Epidemiologic evidence links ischemic stroke to age, yet the mechanisms that underlie the specific and independent effects of age on stroke remain elusive, impeding the development of targeted treatments. This study tested the hypothesis that age directly aggravates stroke outcomes and proposes inflamm-aging as a mediator and potential therapeutic target. METHODS: 3 months- (young) and 18-20 months-old (old) mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 minutes followed by 48 hours of reperfusion. Old animals received weekly treatment with the TNF-α neutralizing antibody adalimumab over 4 weeks before tMCAO in a separate set of experiments. Plasma levels of TNF- α were assessed in patients with ischemic stroke and correlated with age and outcome. RESULTS: Old mice displayed larger stroke size than young ones with increased neuromotor deficit. Immunohistochemical analysis revealed impairment of the blood-brain barrier in old mice, i.e. increased post-stroke degradation of endothelial tight junctions and expression of tight junctions-digesting and neurotoxic matrix metalloproteinases. At baseline, old animals showed a broad modulation of several circulating inflammatory mediators. TNF-α displayed the highest increase in old animals and its inhibition restored the volume of stroke, neuromotor performance, and survival rates of old mice to the levels observed in young ones. Patients with ischemic stroke showed increased TNF-α plasma levels which correlated with worsened short-term neurological outcome as well as with age. CONCLUSIONS: This study identifies TNF-α as a causative contributor to the deleterious effect of aging on stroke and points to inflamm-aging as a mechanism of age-related worsening of stroke outcomes and potential therapeutic target in this context. Thus, this work provides a basis for tailoring novel stroke therapies for the particularly vulnerable elderly population.

Corrigendum: Adeno-Associated Virus-Mediated Gain-of-Function mPCSK9 Expression in the Mouse Induces Hypercholesterolemia, Monocytosis, Neutrophilia, and a Hypercoagulative State.

[This corrects the article DOI: 10.3389/fcvm.2021.718741.].

The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.

Role of somatic cell sources in the maturation degree of human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are a unique source of human cardiomyocytes for cardiac disease modeling. Incomplete functional maturation remains a major limitation, however. One of the determinants of iPSC-CM maturation is somatic cell origin. We therefore compared iPSC-CMs derived from different somatic cell sources. METHODS: Cardiac-derived mesenchymal progenitor cells (CPCs), bone marrow-derived mesenchymal stem cells (BMCs), and human dermal fibroblasts (HDFs) from same patients were reprogrammed into iPSCs and differentiated into iPSC-CMs. Expression of cardiac-specific genes, caffeine-responsive cells, and electrophysiological properties of differentiated cells were analyzed. To assess the contribution of epigenetic memory toward differences in gene expression observed during cardiac differentiation, DNA methylation patterns were determined in the early mesodermal cardiac promoter NKX2-5 and KCNQ1, which encodes for the pore-forming α-subunit of the slow component of delayed-rectifier potassium current (IKs). RESULTS: Cardiac genes (MYH6, TNNI3, KCNQ1, KCNE1) were upregulated in CPC-vs. BMC- and HDF-iPSC-CMs. At early differentiation stages, CPC-iPSC-CMs displayed higher numbers of caffeine-responsive cells than BMC- and HDF-iPSC-CMs. The hERG1 (KV11.1) blocker, E4031, followed by the IKs blocker, JNJ303, increased extracellular field potential duration in CPC-iPSC-CMs to a greater extent than in BMC- and HDF-iPSC-CMs. The promoter region of NKX2-5 was more highly methylated in BMCs and HDFs compared to CPCs, and to a lesser extent in BMC-iPSCs compared to CPC-iPSCs. CONCLUSIONS: These results suggest that human iPSCs from cardiac somatic cell sources may display enhanced capacity toward cardiac re-differentiation compared to non-cardiac cell sources, and that epigenetic mechanisms may play a role in this regard.

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

Apold1 deficiency associates with increased arterial thrombosis in vivo.

BACKGROUND: Endothelial cells regulate the formation of blood clots; thus, genes selectively expressed in these cells could primarily determine thrombus formation. Apold1 (apolipoprotein L domain containing 1) is a gene expressed by endothelial cells; whether Apold1 directly contributes to arterial thrombosis has not yet been investigated. Here, we assessed the effect of Apold1 deletion on arterial thrombus formation using an in vivo model of carotid thrombosis induced by photochemical injury. MATERIAL AND METHODS: Apold1 knockout (Apold1-/- ) mice and wild-type (WT) littermates underwent carotid thrombosis induced by photochemical injury, and time to occlusion was recorded. Tissue factor (TF) activity and activation of mitogen-activated protein kinases (MAPKs) and phosphatidyl-inositol-3 kinase (PI3K)/Akt pathways were analysed by colorimetric assay and Western blotting in both Apold1-/- and WT mice. Finally, platelet reactivity was assessed using light transmission aggregometry. RESULTS: After photochemical injury, Apold1-/- mice exhibited shorter time to occlusion as compared to WT mice. Moreover, TF activity was increased in carotid arteries of Apold1-/- when compared to WT mice. Underlying mechanistic markers such as TF mRNA and MAPKs activation were unaffected in Apold1-/- mice. In contrast, phosphorylation of Akt was reduced in Apold1-/- as compared to WT mice. Additionally, Apold1-/- mice displayed increased platelet reactivity to stimulation with collagen compared with WT animals. CONCLUSIONS: Deficiency of Apold1 results in a prothrombotic phenotype, accompanied by increased vascular TF activity, decreased PI3K/Akt activation and increased platelet reactivity. These findings suggest Apold1 as an interesting new therapeutic target in the context of arterial thrombosis.

fMRI Reveals Mitigation of Cerebrovascular Dysfunction by Bradykinin Receptors 1 and 2 Inhibitor Noscapine in a Mouse Model of Cerebral Amyloidosis.

Functional magnetic resonance imaging (fMRI) techniques can be used to assess cerebrovascular dysfunction in Alzheimer's disease, an important and early contributor to pathology. We hypothesized that bradykinin receptor inhibition alleviates the vascular dysfunction in a transgenic arcAß mouse model of cerebral amyloidosis and that fMRI techniques can be used to monitor the treatment response. Transgenic arcAß mice, and non-transgenic littermates of 14 months-of-age were either treated with the bradykinin receptors 1 and 2 blocker noscapine or received normal drinking water as control over 3 months (n = 8-11/group) and all mice were assessed using fMRI at the end of the treatment period. Perfusion MRI using an arterial spin labeling technique showed regional hypoperfusion in arcAß compared to non-transgenic controls, which was alleviated by noscapine treatment. Similarly, measuring cerebral blood volume changes upon pharmacological stimulation using vessel dilator acetazolamide revealed recovery of regional impairment of cerebral vascular reactivity in arcAß mice upon noscapine treatment. In addition, we assessed with immunohistochemistry beta-amyloid (Aß) and inflammation levels in brain sections. Immunohistological stainings for Aß deposition (6E10) and related microgliosis (Iba1) in the cortex and hippocampus were found comparable between noscapine-treated and untreated arcAß mice. In addition, levels of soluble and insoluble Aß38, Aß40, Aß42 were found to be similar in brain tissue homogenates of noscapine-treated and untreated arcAß mice using electro-chemiluminescent based immunoassay. In summary, bradykinin receptors blockade recovered cerebral vascular dysfunction in a mouse model of cerebral amyloidosis. fMRI methods revealed the functional deficit in disease condition and were useful tools to monitor the treatment response.

AP-1 (Activated Protein-1) Transcription Factor JunD Regulates Ischemia/Reperfusion Brain Damage via IL-1ß (Interleukin-1ß).

Background and Purpose- Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD-a member of the AP-1 (activated protein-1) family of transcription factors-was recently shown to regulate inflammation by targeting IL (interleukin)-1ß synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods- WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1ß was investigated by treating JunD siRNA mice with an anti-IL-1ß monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results- In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1ß levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti-IL-1ß antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions- JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1ß.

Obesity-induced activation of JunD promotes myocardial lipid accumulation and metabolic cardiomyopathy.

AIMS: Metabolic cardiomyopathy (MC)-characterized by intra-myocardial triglyceride (TG) accumulation and lipotoxic damage-is an emerging cause of heart failure in obese patients. Yet, its mechanisms remain poorly understood. The Activator Protein 1 (AP-1) member JunD was recently identified as a key modulator of hepatic lipid metabolism in obese mice. The present study investigates the role of JunD in obesity-induced MC. METHODS AND RESULTS: JunD transcriptional activity was increased in hearts from diet-induced obese (DIO) mice and was associated with myocardial TG accumulation and left ventricular (LV) dysfunction. Obese mice lacking JunD were protected against MC. In DIO hearts, JunD directly binds PPARγ promoter thus enabling transcription of genes involved in TG synthesis, uptake, hydrolysis, and storage (i.e. Fas, Cd36, Lpl, Plin5). Cardiac-specific overexpression of JunD in lean mice led to PPARγ activation, cardiac steatosis, and dysfunction, thereby mimicking the MC phenotype. In DIO hearts as well as in neonatal rat ventricular myocytes exposed to palmitic acid, Ago2 immunoprecipitation, and luciferase assays revealed JunD as a direct target of miR-494-3p. Indeed, miR-494-3p was down-regulated in hearts from obese mice, while its overexpression prevented lipotoxic damage by suppressing JunD/PPARγ signalling. JunD and miR-494-3p were also dysregulated in myocardial specimens from obese patients as compared with non-obese controls, and correlated with myocardial TG content, expression of PPARγ-dependent genes, and echocardiographic indices of LV dysfunction. CONCLUSION: miR-494-3p/JunD is a novel molecular axis involved in obesity-related MC. These results pave the way for approaches to prevent or treat LV dysfunction in obese patients.