Circulating Long Noncoding RNA Signatures Associate With Incident Diabetes in Older Adults: A Prospective Analysis From the VITA Cohort Study.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are involved in diabetogenesis in experimental models, yet their role in humans is unclear. We investigated whether circulating lncRNAs associate with incident type 2 diabetes in older adults. RESEARCH DESIGN AND METHODS: A preselected panel of lncRNAs was measured in serum of individuals without diabetes (n = 296) from the Vienna Transdanube Aging study, a prospective community-based cohort study. Participants were followed up over 7.5 years. A second cohort of individuals with and without type 2 diabetes (n = 90) was used to validate our findings. RESULTS: Four lncRNAs (ANRIL, MIAT, RNCR3, and PLUTO) were associated with incident type 2 diabetes and linked to hemoglobin A1c trajectories throughout the 7.5-year follow-up. Similar results (for MIAT and PLUTO also in combined analysis) were obtained in the validation cohort. CONCLUSIONS: We found a set of circulating lncRNAs that independently portends incident type 2 diabetes in older adults years before disease onset.

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.

Methylation of the Hippo effector YAP by the methyltransferase SETD7 drives myocardial ischaemic injury: a translational study.

AIMS: Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has shown to methylate and alter the function of a variety of proteins in vitro; however, its function in the heart is poorly understood. The present study investigates the role of SETD7 in myocardial ischaemic injury. METHODS AND RESULTS: Experiments were performed in neonatal rat ventricular myocytes (NRVMs), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischaemia/reperfusion (I/R) injury, left ventricular (LV) myocardial samples from patients with ischaemic cardiomyopathy (ICM), and peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes manganese superoxide dismutase (MnSOD) and catalase (CAT). Such impairment of antioxidant defence was associated with mitochondrial reactive oxygen species (mtROS), organelle swelling, and apoptosis. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization, thus preventing mtROS, mitochondrial damage, and apoptosis in NRVMs. In mice, genetic deletion of SETD7 attenuated myocardial I/R injury, mtROS, and LV dysfunction by restoring YAP-dependent transcription of MnSOD and CAT. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 prevented mtROS accumulation, while improving Ca2+-activated tension. Finally, SETD7 was up-regulated in PBMCs from STEMI patients and negatively correlated with MnSOD and CAT. CONCLUSION: We show a methylation-dependent checkpoint regulating oxidative stress during myocardial ischaemia. SETD7 inhibition may represent a valid therapeutic strategy in this setting.

Epi-Drugs in Heart Failure.

Unveiling the secrets of genome's flexibility does not only foster new research in the field, but also gives rise to the exploration and development of novel epigenetic-based therapies as an approach to alleviate disease phenotypes. A better understanding of chromatin biology (DNA/histone complexes) and non-coding RNAs (ncRNAs) has enabled the development of epigenetic drugs able to modulate transcriptional programs implicated in cardiovascular diseases. This particularly applies to heart failure, where epigenetic networks have shown to underpin several pathological features, such as left ventricular hypertrophy, fibrosis, cardiomyocyte apoptosis and microvascular dysfunction. Targeting epigenetic signals might represent a promising approach, especially in patients with heart failure with preserved ejection fraction (HFpEF), where prognosis remains poor and breakthrough therapies have yet to be approved. In this setting, epigenetics can be employed for the development of customized therapeutic approaches thus paving the way for personalized medicine. Even though the beneficial effects of epi-drugs are gaining attention, the number of epigenetic compounds used in the clinical practice remains low suggesting that more selective epi-drugs are needed. From DNA-methylation changes to non-coding RNAs, we can establish brand-new regulations for drug targets with the aim of restoring healthy epigenomes and transcriptional programs in the failing heart. In the present review, we bring the timeline of epi-drug discovery and development, thus highlighting the emerging role of epigenetic therapies in heart failure.

Epigenetic remodeling in heart failure with preserved ejection fraction.

PURPOSE OF REVIEW: In this review, we critically address the role of epigenetic processing and its therapeutic modulation in heart failure with preserved ejection fraction (HFpEF). RECENT FINDINGS: HFpEF associates with a poor prognosis and the identification of novel molecular targets and therapeutic approaches are in high demand. Emerging evidence indicates a key involvement of epigenetic signals in the regulation of transcriptional programs underpinning features of HFpEF. The growing understanding of chromatin dynamics has led to the development of selective epigenetic drugs able to reset transcriptional changes thus delaying or preventing the progression toward HFpEF. Epigenetic information in the setting of HFpEF can be employed to: (i) dissect novel epigenetic networks and chromatin marks contributing to HFpEF; (ii) unveil circulating and cell-specific epigenetic biomarkers; (iii) build predictive models by using computational epigenetics and deep machine learning; (iv) develop new chromatin modifying drugs for personalized management of HFpEF. SUMMARY: Acquired epigenetic signatures during the lifetime can contribute to derail molecular pathways involved in HFpEF. A scrutiny investigation of the individual epigenetic landscape will offer opportunities to develop personalized epigenetic biomarkers and therapies to fight HFpEF in the decades to come.

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.

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.

Inflammation in Metabolic Cardiomyopathy.

Overlapping pandemics of lifestyle-related diseases pose a substantial threat to cardiovascular health. Apart from coronary artery disease, metabolic disturbances linked to obesity, insulin resistance and diabetes directly compromise myocardial structure and function through independent and shared mechanisms heavily involving inflammatory signals. Accumulating evidence indicates that metabolic dysregulation causes systemic inflammation, which in turn aggravates cardiovascular disease. Indeed, elevated systemic levels of pro-inflammatory cytokines and metabolic substrates induce an inflammatory state in different cardiac cells and lead to subcellular alterations thereby promoting maladaptive myocardial remodeling. At the cellular level, inflammation-induced oxidative stress, mitochondrial dysfunction, impaired calcium handling, and lipotoxicity contribute to cardiomyocyte hypertrophy and dysfunction, extracellular matrix accumulation and microvascular disease. In cardiometabolic patients, myocardial inflammation is maintained by innate immune cell activation mediated by pattern recognition receptors such as Toll-like receptor 4 (TLR4) and downstream activation of the NLRP3 inflammasome and NF-κB-dependent pathways. Chronic low-grade inflammation progressively alters metabolic processes in the heart, leading to a metabolic cardiomyopathy (MC) phenotype and eventually to heart failure with preserved ejection fraction (HFpEF). In accordance with preclinical data, observational studies consistently showed increased inflammatory markers and cardiometabolic features in patients with HFpEF. Future treatment approaches of MC may target inflammatory mediators as they are closely intertwined with cardiac nutrient metabolism. Here, we review current evidence on inflammatory processes involved in the development of MC and provide an overview of nutrient and cytokine-driven pro-inflammatory effects stratified by cell type.

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.].

Disentangling the epigenetic landscape in cardiovascular patients: a path toward personalized medicine.

Despite significant advances in our understanding of cardiovascular disease (CVD) we are still far from having developed breakthrough strategies to combat coronary atherosclerosis and heart failure, which account for most of CV deaths worldwide. Available cardiovascular therapies have failed to show to be equally effective in all patients, suggesting that inter-individual diversity is an important factor when it comes to conceive and deliver effective personalized treatments. Genome mapping has proved useful in identifying patients who could benefit more from specific drugs depending on genetic variances; however, our genetic make-up determines only a limited part of an individual's risk profile. Recent studies have demonstrated that epigenetic changes - defined as dynamic changes of DNA and histones which do not affect DNA sequence - are key players in the pathophysiology of cardiovascular disease and may participate to delineate cardiovascular risk trajectories over the lifetime. Epigenetic modifications include changes in DNA methylation, histone modifications and non-coding RNAs and these epigenetic signals have shown to cooperate in modulating chromatin accessibility to transcription factors and gene expression. Environmental factors such as air pollution, smoking, psychosocial context, and unhealthy diet regimens have shown to significantly modify the epigenome thus leading to altered transcriptional programs and CVD phenotypes. Therefore, the integration of genetic and epigenetic information might be invaluable to build individual maps of cardiovascular risk and hence, could be employed for the design of customized diagnostic and therapeutic strategies. In the present review, we discuss the growing importance of epigenetic information and its putative implications in cardiovascular precision medicine.

Epigenetic Remodeling in Obesity-Related Vascular Disease.

Significance: The prevalence of obesity and cardiometabolic phenotypes is alarmingly increasing across the globe and is associated with atherosclerotic vascular complications and high mortality. In spite of multifactorial interventions, vascular residual risk remains high in this patient population, suggesting the need for breakthrough therapies. The mechanisms underpinning obesity-related vascular disease remain elusive and represent an intense area of investigation. Recent Advances: Epigenetic modifications-defined as environmentally induced chemical changes of DNA and histones that do not affect DNA sequence-are emerging as a potent modulator of gene transcription in the vasculature and might significantly contribute to the development of obesity-induced endothelial dysfunction. DNA methylation and histone post-translational modifications cooperate to build complex epigenetic signals, altering transcriptional networks that are implicated in redox homeostasis, mitochondrial function, vascular inflammation, and perivascular fat homeostasis in patients with cardiometabolic disturbances. Critical Issues: Deciphering the epigenetic landscape in the vasculature is extremely challenging due to the complexity of epigenetic signals and their function in regulating transcription. An overview of the most important epigenetic pathways is required to identify potential molecular targets to treat or prevent obesity-related endothelial dysfunction and atherosclerotic disease. This would enable the employment of precision medicine approaches in this setting. Future Directions: Current and future research efforts in this field entail a better definition of the vascular epigenome in obese patients as well as the unveiling of novel, cell-specific chromatin-modifying drugs that are able to erase specific epigenetic signals that are responsible for maladaptive transcriptional alterations and vascular dysfunction in obese patients. Antioxid. Redox Signal. 34, 1165-1199.

The vascular epigenome in patients with obesity and type 2 diabetes: opportunities for personalized therapies.

Our genetic background provides limited information on individual risk of developing vascular complications overtime. New biological layers, namely epigenetic modifications, are now emerging as potent regulators of gene expression thus leading to altered transcriptional programs and vascular disease phenotypes. Such epigenetic modifications, defined as changes to the genome that do not involve changes in DNA sequence, are generally induced by environmental factors and poor lifestyle habits. Of note, adverse epigenetic signals acquired during life can be transmitted to the offspring thus leading to premature alterations of the epigenetic and transcriptional landscape eventually leading to early endothelial dysfunction and vascular senescence. Modifications of the epigenome play a pivotal role in the pathophysiology of cardiometabolic disturbances such as obesity and type 2 diabetes. In these patients, changes of DNA methylation and chromatin structure contribute to alter pathways regulating insulin sensitivity, glucose homeostasis, adipogenesis and vascular function. In this perspective, unveiling the 'epigenetic landscape' in cardiometabolic patients may help to identify new players implicated in obesity and diabetes-related vascular dysfunction and may pave the way for personalized therapies in this setting. In the present review, we discuss current knowledge of the epigenetic routes implicated in vascular damage and cardiovascular disease in patients with metabolic alterations.

New Mechanisms of Vascular Dysfunction in Cardiometabolic Patients: Focus on Epigenetics.

Epigenetic processing takes centre stage in cardiometabolic diseases (obesity, metabolic syndrome, type 2 diabetes, hypertension), where it participates in adiposity, inflammation, endothelial dysfunction, vascular insulin resistance and atherosclerosis. Epigenetic modifications, defined as heritable changes in gene expression that do not entail mutation in the DNA sequence, are mainly induced by environmental stimuli (stress, pollution, cigarette smoking) and are gaining considerable interest due to their causal role in cardiovascular disease, and their amenability to pharmacological intervention. Importantly, epigenetic modifications acquired during life can be transmitted to the offspring and exert their biological effects across multiple generations. Indeed, such transgenerational transmission of epigenetic signals may contribute to anticipating cardiovascular and metabolic disease phenotypes already in children and young adults. A deeper understanding of environmental factors and their effects on the epigenetic machinery and transcriptional programs is warranted to develop effective mechanism-based therapeutic strategies. The clinical application of epigenetic drugs-also known as "epi-drugs"-is currently exploding in the field of cardiovascular disease. The present review describes the main epigenetic networks underlying cardiometabolic alterations and sheds light on specific points of intervention for pharmacological reprogramming in this setting.

Epigenetic Control of Mitochondrial Function in the Vasculature.

The molecular signatures of epigenetic regulation and chromatin architecture are emerging as pivotal regulators of mitochondrial function. Recent studies unveiled a complex intersection among environmental factors, epigenetic signals, and mitochondrial metabolism, ultimately leading to alterations of vascular phenotype and increased cardiovascular risk. Changing environmental conditions over the lifetime induce covalent and post-translational chemical modification of the chromatin template which sensitize the genome to establish new transcriptional programs and, hence, diverse functional states. On the other hand, metabolic alterations occurring in mitochondria affect the availability of substrates for chromatin-modifying enzymes, thus leading to maladaptive epigenetic signatures altering chromatin accessibility and gene transcription. Indeed, several components of the epigenetic machinery require intermediates of cellular metabolism (ATP, AcCoA, NADH, α-ketoglutarate) for enzymatic function. In the present review, we describe the emerging role of epigenetic modifications as fine tuners of gene transcription in mitochondrial dysfunction and vascular disease. Specifically, the following aspects are described in detail: (i) mitochondria and vascular function, (ii) mitochondrial ROS, (iii) epigenetic regulation of mitochondrial function; (iv) the role of mitochondrial metabolites as key effectors for chromatin-modifying enzymes; (v) epigenetic therapies. Understanding epigenetic routes may pave the way for new approaches to develop personalized therapies to prevent mitochondrial insufficiency and its complications.

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.

Epigenetic processing in cardiometabolic disease.

Albeit a consistent body of evidence supports the notion that genes influence cardiometabolic features and outcomes, the "non-genetic regulation" of this process is gaining increasing attention. Plastic chemical changes of DNA/histone complexes - known as epigenetic changes - critically determine gene activity by rapidly modifying chromatin accessibility to transcription factors. In this review, we describe the emerging role of chromatin modifications as fine tuners of gene transcription in adipogenesis, insulin resistance, macrophage polarization, immuno-metabolism, endothelial dysfunction and metabolic cardiomyopathy. Epigenetic processing participates in the dynamic interplay among different organs in the cardiometabolic patient. DNA methylation and post-translational histone modifications in both visceral and subcutaneous adipose tissue enable the transcription of genes implicated in lipo- and adipogenesis, inflammation and insulin resistance. Along the same line, complex networks of chromatin modifying enzymes are responsible for impaired nitric oxide bioavailability and defective insulin signalling in the vasculature, thus leading to reduced capillary recruitment and insulin delivery in the liver, skeletal muscle and adipose tissue. Furthermore, changes in methylation status of IL-4, IFNγ and Forkhead box P3 (Foxp3) gene loci are crucial for the polarization of immune cells, thus leading to adipose tissue inflammation and atherosclerosis. Cell-specific epigenetic information could advance our understanding of cardiometabolic processes, thus leading to individualized risk assessment and personalized therapeutic approaches in patients with cardiometabolic disturbances. The development of new chromatin modifying drugs indicates that targeting epigenetic changes is a promising approach to reduce the burden of cardiovascular disease in this setting.