Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66^Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66^Shc contributes to obesity-related vascular disease. ROS-driven endothelial dysfunction was observed in visceral fat arteries (VFAs) isolated from obese subjects when compared with normal weight controls. Gene profiling of chromatin-modifying enzymes in VFA revealed a significant dysregulation of methyltransferase SUV39H1 (fold change, −6.9, P < 0.01), demethylase JMJD2C (fold change, 3.2, P < 0.01), and acetyltransferase SRC-1 (fold change, 5.8, P < 0.01) in obese vs. control VFA. These changes were associated with reduced di-(H3K9me2) and trimethylation (H3K9me3) as well as acetylation (H3K9ac) of histone 3 lysine 9 (H3K9) on p66^Shc promoter. Reprogramming SUV39H1, JMJD2C, and SRC-1 in isolated endothelial cells as well as in aortas from obese mice (Lep^Ob/Ob) suppressed p66^Shc-derived ROS, restored nitric oxide levels, and rescued endothelial dysfunction. Consistently, in vivo editing of chromatin remodellers blunted obesity-related vascular p66^Shc expression. We show that SUV39H1 is the upstream effector orchestrating JMJD2C/SRC-1 recruitment to p66^Shc promoter. Indeed, SUV39H1 overexpression in obese mice erased H3K9-related changes on p66^Shc promoter, while SUV39H1 genetic deletion in lean mice recapitulated obesity-induced H3K9 remodelling and p66^Shc transcription. These results uncover a novel epigenetic mechanism underlying endothelial dysfunction in obesity. Targeting SUV39H1 may attenuate oxidative transcriptional programmes and thus prevent vascular disease in obese individuals.

Rationale: Hyperglycemia -induced reactive oxygen species are key mediators of cardiac dysfunction. JunD (Jund proto-oncogene subunit), a member of the AP-1 (activator protein-1) family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to redox state and inflammation in the diabetic heart remains to be elucidated. Objective: The present study investigates the role of JunD in hyperglycemia-induced and reactive oxygen species–driven myocardial dysfunction. Methods and Results: JunD mRNA and protein expression were reduced in the myocardium of mice with streptozotocin-induced diabetes mellitus as compared to controls. JunD downregulation was associated with oxidative stress and left ventricular dysfunction assessed by electron spin resonance spectroscopy as well as conventional and 2-dimensional speckle-tracking echocardiography. Furthermore, myocardial expression of free radical scavenger superoxide dismutase 1 and aldehyde dehydrogenase 2 was reduced, whereas the NOX2 (NADPH [nicotinamide adenine dinucleotide phosphatase] oxidase subunit 2) and NOX4 (NADPH [nicotinamide adenine dinucleotide phosphatase] oxidase subunit 4) were upregulated. The redox changes were associated with increased NF-κB (nuclear factor kappa B) binding activity and expression of inflammatory mediators. Interestingly, mice with cardiac-specific overexpression of JunD via the α MHC (α- myosin heavy chain) promoter (α MHC JunD ^tg ) were protected against hyperglycemia-induced cardiac dysfunction. We also showed that JunD was epigenetically regulated by promoter hypermethylation, post-translational modification of histone marks, and translational repression by miRNA (microRNA)-673/menin. Reduced JunD mRNA and protein expression were confirmed in left ventricular specimens obtained from patients with type 2 diabetes mellitus as compared to nondiabetic subjects. Conclusions: Here, we show that a complex epigenetic machinery involving DNA methylation, histone modifications, and microRNAs mediates hyperglycemia-induced JunD downregulation and myocardial dysfunction in experimental and human diabetes mellitus. Our results pave the way for tissue-specific therapeutic modulation of JunD to prevent diabetic cardiomyopathy.

Background: Histone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium. Methods: We studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O₂⁻) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice. Results: Incubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O₂⁻ generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice. Conclusions: EZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes. Graphical

Impairments of retinal vessel diameter are associated with major adverse cardiovascular (CV) events. Promoter DNA methylation is a repressor of the mitochondrial adaptor p66^Shc gene transcription, a key driver of ageing-induced reactive oxygen species. The study aimed to investigate whether high-intensity interval training (HIIT) affects retinal microvascular phenotype as well as p66^Shc expression and oxidative stress in ageing subjects with increased CV risk from the EXAMIN AGE cohort. Eighty-four sedentary subjects (mean age 59.4 ± 7.0 years) with ≥2 CV risk factors were randomized into either a 12-week HIIT or standard physical activity recommendations. Retinal arteriolar and venular diameters were measured by use of a retinal vessel analyser. As a marker of oxidative stress plasma 3-nitrotyrosine (3-NT) level was determined by ELISA. Gene expression of p66^Shc and DNA methylation were assessed in mononuclear cells by RT-qPCR and methylated-DNA capture (MethylMiner Enrichment Kit) coupled with qPCR, respectively. High-intensity interval training reduced body mass index, fat mass, low-density lipoprotein and increased muscle mass, as well as maximal oxygen uptake (VO₂max). Moreover, HIIT restored microvascular phenotype by inducing retinal arteriolar widening (pre: 175 ± 14 µm vs. post: 181 ± 13 µm, P = 0.001) and venular narrowing (pre: 222 ± 14 µm vs. post: 220 ± 14 µm, P = 0.007). After HIIT, restoration of p66^Shc promoter methylation (P = 0.034) reduced p66^Shc gene expression (P = 0.037) and, in turn, blunted 3-NT plasma levels (P = 0.002). High-intensity interval training rescues microvascular dysfunction in ageing subjects at increased CV risk. Exercise-induced reprogramming of DNA methylation of p66^Shc gene may represent a putative mechanistic link whereby exercise protects against age-related oxidative stress. ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

Type 2 diabetes (T2D) is strongly associated with early cognitive decline and may facilitate the development of neurodegenerative diseases. Despite the overwhelming amount of indirect evidence pointing toward the presence of cerebral neurodegeneratio