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Increasing evidence suggests that maternal cholesterol represents an important risk factor for atherosclerotic disease in offspring already during pregnancy, although the underlying mechanisms have not yet been elucidated. Eighteen human fetal aorta samples were collected from the spontaneously aborted fetuses of normal cholesterolemic and hypercholesterolemic mothers. Maternal total cholesterol levels were assessed during hospitalization. DNA methylation profiling of the whole SREBF2 gene CpG island was performed (p value SREBF2 gene shows 4 significant differentially hypermethylated sites in the 5′UTR-CpG island. This finding indicates that more effective long-term primary cardiovascular prevention programs need to be designed for the offspring of mothers with hypercholesterolemia. Further studies should be conducted to clarify the epigenetic mechanisms underlying the association between early atherogenesis and maternal hypercholesterolemia during pregnancy.

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.

Background The pathogenesis of experimental diabetic cardiomyopathy may involve the activator protein 1 (AP-1) member, JunD. Using non-diabetic heart transplant (HTX) in recipients with diabetes, we examined the effects of the diabetic milieu (hyperglycemia and insulin resistance) on cardiac JunD expression over 12 months. Because sodium/glucose cotransporter-2 inhibitors (SGLT2i) significantly reverse high glucose-induced AP-1 binding in the proximal tubular cell, we investigated JunD expression in a subgroup of type 2 diabetic recipients receiving SGLT2i treatment. Methods We evaluated 77 first HTX recipients (40 and 37 patients with and without diabetes, respectively). Among the recipients with diabetes, 17 (45.9%) were receiving SGLT2i treatment. HTX recipients underwent standard clinical evaluation (metabolic status, echocardiography, coronary computed tomography angiography, and endomyocardial biopsy). In the biopsy samples, we evaluated JunD, insulin receptor substrates 1 and 2 (IRS1 and IRS2), peroxisome proliferator-activated receptor-γ (PPAR-γ), and ceramide levels using real-time polymerase chain reaction and immunofluorescence. The biopsy evaluations in this study were performed at 1–4 weeks (basal), 5–12 weeks (intermediate), and up to 48 weeks (final, end of 12-month follow-up) after HTX. Results There was a significant early and progressive increase in the cardiac expression of JunD/PPAR-γ and ceramide levels, along with a significant decrease in IRS1 and IRS2 in recipients with diabetes but not in those without diabetes. These molecular changes were blunted in patients with diabetes receiving SGLT2i treatment. Conclusion Early pathogenesis in human diabetic cardiomyopathy is associated with JunD/PPAR-γ overexpression and lipid accumulation following HTX in recipients with diabetes. Remarkably, this phenomenon was reduced by concomitant therapy with SGLT2i, which acted directly on diabetic hearts.

The risk of coronary heart disease (CHD) clinical manifestations and patient management is estimated according to risk scores accounting multifactorial risk factors, thus failing to cover the individual cardiovascular risk. Technological improvements in the field of medical imaging, in particular, in cardiac computed tomography angiography and cardiac magnetic resonance protocols, laid the development of radiogenomics. Radiogenomics aims to integrate a huge number of imaging features and molecular profiles to identify optimal radiomic/biomarker signatures. In addition, supervised and unsupervised artificial intelligence algorithms have the potential to combine different layers of data (imaging parameters and features, clinical variables and biomarkers) and elaborate complex and specific CHD risk models allowing more accurate diagnosis and reliable prognosis prediction. Literature from the past 5 years was systematically collected from PubMed and Scopus databases, and 60 studies were selected. We speculated the applicability of radiogenomics and artificial intelligence through the application of machine learning algorithms to identify CHD and characterize atherosclerotic lesions and myocardial abnormalities. Radiomic features extracted by cardiac computed tomography angiography and cardiac magnetic resonance showed good diagnostic accuracy for the identification of coronary plaques and myocardium structure; on the other hand, few studies exploited radiogenomics integration, thus suggesting further research efforts in this field. Cardiac computed tomography angiography resulted the most used noninvasive imaging modality for artificial intelligence applications. Several studies provided high performance for CHD diagnosis, classification, and prognostic assessment even though several efforts are still needed to validate and standardize algorithms for CHD patient routine according to good medical practice.

Objectives: Dilated cardiomyopathy (DCM) is characterized by a specific transcriptome. Since the DCM molecular network is largely unknown, the aim was to identify specific disease-related molecular targets combining an original machine learning (ML) approach with protein-protein interaction network. Methods: The transcriptomic profiles of human myocardial tissues were investigated integrating an original computational approach, based on the Custom Decision Tree algorithm, in a differential expression bioinformatic framework. Validation was performed by quantitative real-time PCR. Results: Our preliminary study, using samples from transplanted tissues, allowed the discovery of specific DCM-related genes, including MYH6, NPPA, MT-RNR1 and NEAT1, already known to be involved in cardiomyopathies Interestingly, a combination of these expression profiles with clinical characteristics showed a significant association between NEAT1 and left ventricular end-diastolic diameter (LVEDD) (Rho = 0.73, p = 0.05), according to severity classification (NYHA-class III). Conclusions: The use of the ML approach was useful to discover preliminary specific genes that could lead to a rapid selection of molecular targets correlated with DCM clinical parameters. For the first time, NEAT1 under-expression was significantly associated with LVEDD in the human heart.

Rationale About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. Objective To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. Methods and results We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. Conclusions The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

Background Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease and the leading cause of death worldwide. Purpose To perform an epigenome-wide analysis in circulating CD4+ and CD8+ T cells of ACS patients and healthy subjects (HS) enrolled in the DIANA clinical trial (NCT04371809) in order to identify differentially methylated genes (DMGs). Methods Genomic DNA was extracted from CD4+ and CD8+ T cells of all subjects and sequenced by the reduced representation bisulfite sequencing (RRBS) platform. Functional pathway analysis was performed and significant DMGs were selected for gene expression validation by qRT-PCR in ACS patients and HS. GeneMANIA was used to built a prediction gene network. Correlation analyses between molecular data and clinical variables were performed. Results In CD4+ T cells we identified 61 differentially methylated regions (DMRs) associated to 57 annotated genes of which 53% (n=32) were hyper- and 47% (n=29) were hypo-methylated in ACS patients vs HS. In CD8+ T cells we identified 613 DMRs associated to 569 annotated genes of which 28% (n=173) were hyper- and 72% (n=440) were hypo-methylated between two groups. In both cell type of ACS patients, 175 DMRs were associated to 157 annotated genes of which 41% (n=72) were hyper- and 59% (n=103) were hypo-methylated. From functional analysis, we selected the top 5 DMGs in the prevalent pathways with the highest differential of methylation values. Specifically, we considered 6 hub genes: NFATC1, TCF7, PDGFA, PRKCB, PRKCZ and ABCA1 and determined their respective expression levels by q-RT-PCR. We found a significant up-regulation of the selected genes in ACS patients vs HS (P Conlusions This study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells, providing specific methylation signatures that could help to clarify the role of aberrant methylation in ACS pathogenesis, and provide the basis for the search of novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health;Italian Ministry of Research and University

Background Glycemic control is a strong predictor of long-term cardiovascular risk in patients with diabetes mellitus, and poor glycemic control influences long-term risk of cardiovascular disease even decades after optimal medical management. This phenomenon, termed glycemic memory, has been proposed to occur due to stable programs of cardiac and endothelial cell gene expression. This transcriptional remodeling has been shown to occur in the vascular endothelium through a yet undefined mechanism of cellular reprogramming. Methods In the current study, we quantified genome-wide DNA methylation of cultured human endothelial aortic cells (HAECs) via reduced-representation bisulfite sequencing (RRBS) following exposure to diabetic (250 mg/dL), pre-diabetic (125 mg/dL), or euglycemic (100 mg/dL) glucose concentrations for 72 h (n = 2). Results We discovered glucose-dependent methylation of genomic regions (DMRs) encompassing 2199 genes, with a disproportionate number found among genes associated with angiogenesis and nitric oxide (NO) signaling-related pathways. Multi-omics analysis revealed differential methylation and gene expression of VEGF (↑5.6% DMR, ↑3.6-fold expression), and NOS3 (↓20.3% DMR, ↓1.6-fold expression), nodal regulators of angiogenesis and NO signaling, respectively. Conclusion In the current exploratory study, we examine glucose-dependent and dose-responsive alterations in endothelial DNA methylation to examine a putative epigenetic mechanism underlying diabetic vasculopathy. Specifically, we uncover the disproportionate glucose-dependent methylation and gene expression of VEGF and NO signaling cascades, a physiologic imbalance known to cause endothelial dysfunction in diabetes. We therefore hypothesize that epigenetic mechanisms encode a glycemic memory within endothelial cells.

Purpose Human leukocyte antigen (HLA)-G is a non-classic major histocompatibility complex HLA class I molecule. HLA-G may have tolerogenic properties which are linked to epigenetic-sensitive pathways. There is a correlation of sHLA-G levels and graft acceptance in transplantation studies. There are previous data on correlation of sHLA-G with graft rejection as well as with viral infections such as hepatitis C virus (HCV) in kidney transplanted patients. Here, we report the sHLA-G expression in patients on the waiting list for kidney transplantation, with and without anti-HCV compared to a control group. Methods Serum of 67 patients on the waiting list for kidney transplantation (n = 43 with anti-HCV and n = 24 without anti-HCV) was analyzed. Among these patients, n = 39 were on the waiting list for the first transplantation, while n = 28 were patients who returned in the list. The control group included n = 23 blood donors with anti-HCV (n = 13) and without anti-HCV (n = 10). Results The expression of sHLA-G was significantly lower in the control group (39.6 ± 34.1 U/ml) compared to both - patients on the waiting list for the first transplantation (62.5 ± 42.4 U/ml, p=0.031) and patients who returned in the list (76.7 ± 53.9 U/ml, p=0.006). No significant differences were observed in all anti-HCV positive groups. A positive linear correlation between sHLA-G and TNF-α, and patient age was observed. Conclusions Serum sHLA-G values were significantly increased in both - patients on the waiting list for the first transplantation and patients who returned in the list, as compared to control group. Our findings confirm the key tolerogenic role of sHLA-G levels as epigenetic-related marker for measuring the state of kidney allograft acceptance.

Aim Hyperglycemia status induces endothelial dysfunction, although the underlying pathogenic mechanisms are not fully understood. There are several studies connecting sugar/sweetened beverages to the cardiovascular disease. Currently, many sweeteners have been extensively introduced into lifestyle to normalize blood glucose levels without altering the sweet taste. However, there is growing concern for their impact on metabolic health. Methods Human endothelial cells were treated with Glucose, Fructose, Aspartame, Rebaudioside A, Stevioside, or Steviol. Morphological characteristics, in vitro angiogenesis and array gene expression were analyzed. Results High-glucose and fructose concentrations significantly decreased cell features such as angiogenic capability. Interestingly, non-caloric sweeteners did not significantly modified all cell characteristics and they did not compromised cell angiogenic ability. Array gene expression analysis revealed that the chemokine fractalkine (CX3CL1) and the enzyme transferase (HPRT1) were always significantly upregulated and downregulated respectively, after glucose and fructose treatments (P > .05), whereas they were non-differentially expressed with all the other sweeteners. Interestingly, both genes are considered as cardiovascular disease risk biomarkers. Specifically, upregulation of CX3CL1/CX3CR1 occurs in the human placenta and serum levels of the ligand are associated with markers of insulin resistance in GDM. Conclusions Differently from glucose and fructose, steviol glycosides do not damage endothelial cells. Prospective preclinical studies and clinical trials are warranted to confirm the long-term safety of such compounds.

AIMS:Immune endothelial inflammation, underlying coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. METHODS:Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score = 0, uninjured coronaries and with no obstructive CHD (no critical stenosis, NCS) were considered as control subjects (n = 18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. RESULTS:For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p = 0.05) of HLA-G gene in CHD patients compared to control group; 2) the hypomethylation level of one specific fragment of 161bp (+616/+777) positively correlated with coronary Ca score, a relevant parameter of CCTA (p

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p p p p

Genome-wide association studies have revealed several genes predisposing to autoimmunity, however, concordance rates in monozygotic twins are significantly below 50% for several autoimmune diseases. The limited presence of a strong genetic association only in some patients supports that other non-genetic mechanisms are active in these pathologies. Epigenetic modifications such as DNA methylation, histone modification, and microRNA signaling regulate gene expression and are sensitive to external stimuli and they might be as bridging between genetic and environmental factors. Some evidence has highlighted the involvement of epigenetic alterations in the pathogenesis of various autoimmune diseases giving rise to great expectations among clinicians and researchers. The direct role of these alterations in the initiation/progression of autoimmune diseases is still unclear. The knowledge in depth of these pathogenic and epigenetic mechanisms will increase the possibility of the control and/or prevention of autoimmune diseases through the use of drugs that target epigenetic pathways. Moreover, we could use epigenetic-related biomarkers to follow this complicated framework (for example H3K4me3 and miRNA-155 are among those proposed biomarkers). This article reviews current understanding of the epigenetic involvement in the field of autoimmune diseases especially in systemic lupus erythematosus, rheumatoid arthritis, sclerosis multiple and type 1 diabetes.

In cardiac fibrosis, following an injury or a stress, non-functional fibrotic tissue substitutes normal myocardium, thus leading to progressive heart failure. Activated fibroblasts are principal determinants of cardiac fibrosis by producing excessive fibrotic extracellular matrix and causing hypertrophy of cardiomyocytes. Epigenetic changes, such as DNA methylation, histone modifications, and miRNAs have been involved in these mechanisms. Therefore, there is a strong interest in reverting such epigenetic transformations in order to arrest myocardial fibrotic degeneration. Demethylating agents, such as 5-aza-2'-deoxycytidine, 5-azacytidine, some selective histone deacetylase inhibitors, including mocetinostat, trichostatin A, and MPT0E014, have a direct action on important inducers of cardiac fibrosis. Also dietary compounds, such as resveratrol, can suppress the differentiation of fibroblasts to myofibroblasts. Although in vivo and in vitro studies suggest specific epigenetic therapies to treat cardiac fibrosis, the related clinical trials are still lacking. A better understanding of the epigenetic effects of dietary compounds (e.g. curcumin and green tea catechins) on the onset and progression of cardiac fibrosis, will allow the identification of protective dietary patterns and/or the generation of novel potential epidrugs.

The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA antibodies have led to continuously develop novel practical and feasible technologies in the field of organ and tissue transplantation. Technologies to identify HLA molecules have evolved from the serological to the molecular methods and a true innovation in the DNA sequencing has taken place with the development of next generation sequencing. An interesting field to explore is how the information resulting from the HLA-DNA sequencing can be applied in the clinical setting by including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce the number of mismatched antigens. Furthermore, high resolution HLA allele typing could help to understand the epitopes against which antibodies are developed. However, the improvements should not only concern the diagnostic tools in the pre-transplantation phase, but also the monitoring of post transplantation outcome. There is a growing interest in developing new non-invasive biomarkers to monitor the rejection. Currently, increasing evidence has focused on miRNAs, epigenetic markers emerged as regulators of molecular events that are differently expressed in biopsies and blood as well as in urinary samples of transplanted recipients. The implementation of next generation sequencing and genome-wide expression analysis together with functional assays may provide useful tools to evaluate the epigenetic modulation in transplantation biology but many efforts are requested for translating in the clinical arena the results obtained in experimental models.

Background Circulating endothelial progenitors cells (EPCs) play a critical role in neovascularization and endothelial repair. There is a growing evidence that hyperglycemia related to Diabetes Mellitus (DM) decreases EPC number and function so promoting vascular complications. Aim of the Study This study investigated whether an intensive glycemic control regimen in Type 2 DM can increase the number of EPCs and restores their function. Methods Sixty-two patients with Type 2 DM were studied. Patients were tested at baseline and after 3 months of an intensive regimen of glycemic control. The Type 2 DM group was compared to control group of subjects without diabetes. Patients with Type 2 DM (mean age 58.2±5.4 years, 25.6% women, disease duration of 15.4±6.3 years) had a baseline HgA1c of 8.7±0.5% and lower EPC levels (CD34+/KDR+) in comparison to healthy controls (p

Epigenetic modifications include DNA methylation, histone modifications, and microRNA. Gene alterations have been found to be associated with cardiovascular diseases, and epigenetic mechanisms are continuously being studied to find new useful strategies for the clinical management of afflicted patients. Numerous cardiovascular disorders are characterized by the abnormal methylation of CpG islands and so specific drugs that could inhibit DNA methyltransferase directly or by reducing its gene expression (e.g., hydralazine and procainamide) are currently under investigation. The anti-proliferative and anti-inflammatory properties of histone deacetylase inhibitors and their cardio-protective effects have been confirmed in preclinical studies. Furthermore, the regulation of the expression of microRNA targets through pharmacological tools is still under development. Indeed, large controlled trials are required to establish whether current possible candidate antisense microRNAs could offer better therapeutic benefits in clinical practice. Here, we updated therapeutic properties, side effects, and feasibility of emerging epigenetic-based strategies in cardiovascular diseases by highlighting specific problematic issues that still affect the development of large scale novel therapeutic protocols.

Inflammation and immune response play a pivotal role in the pathophysiology of ischemic stroke giving their contribution to tissue damage and repair. Emerging evidence supports the involvement of epigenetic mechanisms such as methylation, histone modification and miRNAs in the pathogenesis of stroke. Interestingly, epigenetics can influence the molecular events involved in ischemic injury by controlling the switch from pro- to anti-inflammatory response, however, this is still a field to be fully explored. The knowledge of epigenetic processes could to allow for the discovery of more sensitive and specific biomarkers for risk, onset, and progression of disease as well as further novel tools to be used in both primary prevention and therapy of stroke. Indeed, studies performed in vitro and in small animal models seem to suggest a neuroprotective role of HDAC inhibitors (e.g. valproic acid) and antagomir (e.g. anti-miR-181a) in ischemic condition by modulation of both immune and inflammatory pathways. Thus, the clinical implications of altered epigenetic mechanisms for the prevention of stroke are very promising but clinical prospective studies and translational approaches are still warranted.

Progress in human genetic and genomic research has led to the identification of genetic variants associated with specific cardiovascular diseases (CVDs), but the pathogenic mechanisms remain unclear. Recent studies have analyzed the involvement of epigenetic mechanisms such as DNA methylation and histone modifications in the development and progression of CVD. Preliminary work has investigated the correlations between DNA methylation, histone modifications, and RNA-based mechanisms with CVDs including atherosclerosis, heart failure (HF), myocardial infarction (MI), and cardiac hypertrophy. Remarkably, both in utero programming and postnatal hypercholesterolemia may affect the epigenetic signature in the human cardiovascular system, thereby providing novel early epigenetic-related pharmacological insights. Interestingly, some dietary compounds, including polyphenols, cocoa, and folic acid, can modulate DNA methylation status, whereas statins may promote epigenetic-based control in CVD prevention through histone modifications. We review recent findings on the epigenetic control of cardiovascular system and new challenges for therapeutic strategies in CVDs.