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Pathogenic DNA methylation changes may be involved in pulmonary arterial hypertension (PAH) onset and its progression, but there is no data on potential associations with patient-derived hemodynamic parameters. The reduced representation bisulfite sequencing (RRBS) platform identified N = 631 differentially methylated CpG sites which annotated to N = 408 genes (DMGs) in circulating CD4+ T cells isolated from PAH patients vs. healthy controls (CTRLs). A promoter-restricted network analysis established the PAH subnetwork that included 5 hub DMGs (SOCS3, GNAS, ITGAL, NCOR2, NFIC) and 5 non-hub DMGs (NR4A2, GRM2, PGK1, STMN1, LIMS2). The functional analysis revealed that the SOCS3 gene was the most recurrent among the top ten significant pathways enriching the PAH subnetwork, including the growth hormone receptor and the interleukin-6 signaling. Correlation analysis showed that the promoter methylation levels of each network-oriented DMG were associated individually with hemodynamic parameters. In particular, SOCS3 hypomethylation was negatively associated with right atrial pressure (RAP) and positively associated with cardiac index (CI) (|r|≥ 0.6). A significant upregulation of the SOCS3, ITGAL, NFIC, NCOR2, and PGK1 mRNA levels (qRT-PCR) in peripheral blood mononuclear cells from PAH patients vs. CTRLs was found (P ≤ 0.05). By immunoblotting, a significant upregulation of the SOCS3 protein was confirmed in PAH patients vs. CTRLs (P + T cell DNA methylation signatures, hemodynamic parameters, and validation experiments in PAH patients at first diagnosis or early follow-up. Our data suggests that SOCS3 gene might be involved in PAH pathogenesis and serve as potential prognostic biomarker. Graphical abstract

DNA methylation is associated with gene silencing, but its clinical role in cardiovascular diseases (CVDs) remains to be elucidated. We hypothesized that extracellular vesicles (EVs) may carry epigenetic changes, showing themselves as a potentially valuable non-invasive diagnostic liquid biopsy. We isolated and characterized circulating EVs of acute coronary syndrome (ACS) patients and assessed their role on DNA methylation in epigenetic modifications.EVs were recovered from plasma of 19 ACS patients and 50 healthy subjects (HS). Flow cytometry, qRT-PCR, and Western blot (WB) were performed to evaluate both intra-vesicular and intra-cellular signals. ShinyGO, PANTHER, and STRING tools were used to perform GO and PPI network analyses.ACS-derived EVs showed increased levels of DNA methyltransferases (DNMTs) (p0.001) and Ten-eleven translocation (TET) genes reduction. Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B, were significantly increased in plasma ACS-EVs. DNA methylation analysis on PBMCs from healthy donors treated with HS- and ACS-derived EVs showed an important role of DNMTs carried by EVs. PPI network analysis evidenced that ACS-EVs induced changes in PBMC methylome. In the most enriched subnetwork, the hub gene SRC was connected to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, BAIAP2 genes that were showed to have many molecular effects on various cell types into onset of several CVDs. Modulation in gene expression after ACS-EVs treatment was confirmed for SRC, NOTCH1, FOXO3, RXRA, DGKG and BAIAP2 (p0.05).Our data showed an important role for ACS-derived EVs in gene expression modulation through de novo DNA methylation signals, and modulating signalling pathways in target cells.

Supplementary Figure 1 from Deletion of Yin Yang 1 Protein in Osteosarcoma Cells on Cell Invasion and CXCR4/Angiogenesis and Metastasis

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth. [Cancer Res 2008;68(6):1797–808]

Supplementary Tables 1-2 from Deletion of Yin Yang 1 Protein in Osteosarcoma Cells on Cell Invasion and CXCR4/Angiogenesis and Metastasis

Both progression from the early pathogenic events to clinically manifest cardiovascular diseases (CVD) and cancer impact the integrity of the vascular system. Pathological vascular modifications are affected by interplay between endothelial cells and their microenvironment. Soluble factors, extracellular matrix molecules and extracellular vesicles (EVs) are emerging determinants of this network that trigger specific signals in target cells. EVs have gained attention as package of molecules with epigenetic reversible activity causing functional vascular changes, but their mechanisms are not well understood. Valuable insights have been provided by recent clinical studies, including the investigation of EVs as potential biomarkers of these diseases. In this paper, we review the role and the mechanism of exosomal epigenetic molecules during the vascular remodeling in coronary heart disease as well as in cancer-associated neoangiogenesis.

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.

Background DNA methylation can play a pathogenic role in the early stages of hyperglycemia linking homeostasis imbalance and vascular damage. Material and methods We investigated DNA methylome by RRBS in CD04+ and CD08+ T cells from healthy subjects (HS) to pre-diabetics (Pre-Diab) and type 2 diabetic (T2D) patients to identify early biomarkers of glucose impairment and vascular damage. Our cross-sectional study enrolled 14 individuals from HS state to increasing hyperglycemia (pilot study, PIRAMIDE trial, NCT03792607). Results Globally, differentially methylated regions (DMRs) were mostly annotated to promoter regions. Hypermethylated DMRs were greater than hypomethylated in CD04+ T cells whereas CD08+ T showed an opposite trend. Moreover, DMRs overlapping between Pre-Diab and T2D patients were mostly hypermethylated in both T cells. Interestingly, SPARC was the most hypomethylated gene in Pre-Diab and its methylation level gradually decreased in T2D patients. Besides, SPARC showed a significant positive correlation with DBP (+0.76), HDL (+0.54), Creatinine (+0.83), LVDd (+0.98), LVSD (+0.98), LAD (+0.98), LVPWd (+0.84), AODd (+0.81), HR (+0.72), Triglycerides (+0.83), LAD (+0.69) and AODd (+0.52) whereas a negative correlation with Cholesterol (−0.52) and LDL (−0.71) in T2D. Conclusion SPARC hypomethylation in CD08+ T cells may be a useful biomarker of vascular complications in Pre-Diab with a possible role for primary prevention warranting further multicenter clinical trials to validate our findings.

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.

Network medicine can advance current medical practice by arising as response to the limitations of a reductionist approach focusing on cardiovascular (CV) diseases as a direct consequence of a single defect. This molecular-bioinformatic approach integrates heterogeneous "omics" data and artificial intelligence to identify a chain of perturbations involving key components of multiple molecular networks that are closely related in the human interactome. The clinical view of the network-based approach is greatly supported by the general law of molecular interconnection governing all biological complex systems. Recent advances in bioinformatics have culminated in numerous quantitative platforms able to identify CV disease modules underlying perturbations of the interactome. This might provide novel insights in CV disease mechanisms as well as putative biomarkers and drug targets. We describe the network-based principles and discuss their application to classifying and treating common CV diseases. We compare the strengths and weaknesses of molecular networks in comparison with the classical current reductionist approach, and remark on the necessity for a two-way approach connecting network medicine with large clinical trials to concretely translate novel insights from bench to bedside.

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.

Aims Immune endothelial inflammation, underlie 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 were considered as control subjects (Ctrls) (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 Ctrl group; 2) hypomethylation level of one specific fragment positively correlated with coronary Ca score, a relevant parameter of CCTA (p Conclusions Our results showed that reduced levels of circulating HLA-G molecules could derive from epigenetic marks inducing hypomethylation of specific regions into 5'UTR-CpG island of HLA-G gene in CHD patients with obstructive coronary stenosis vs non critical stenosis group. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Minister of Health

Background DNA methylation can play a pathogenic role in the early stages of hyperglycemia linking homeostasis imbalance and vascular damage. Material and methods We investigated DNA methylome by RRBS in CD04+ and CD08+ T cells from healthy subjects (HS) to pre-diabetics (Pre-Diab) and type 2 diabetic (T2D) patients to identify early biomarkers of glucose impairment and vascular damage. Our cross-sectional study enrolled 14 individuals from HS state to increasing hyperglycemia (pilot study, PIRAMIDE trial, NCT03792607). Results Globally, differentially methylated regions (DMRs) were mostly annotated to promoter regions. Hypermethylated DMRs were greater than hypomethylated in CD04+ T cells whereas CD08+ T showed an opposite trend. Moreover, DMRs overlapping between Pre-Diab and T2D patients were mostly hypermethylated in both T cells. Interestingly, SPARC was the most hypomethylated gene in Pre-Diab and its methylation level gradually decreased in T2D patients. Besides, SPARC showed a significant positive correlation with DBP (+0.76), HDL (+0.54), Creatinine (+0.83), LVDd (+0.98), LVSD (+0.98), LAD (+0.98), LVPWd (+0.84), AODd (+0.81), HR (+0.72), Triglycerides (+0.83), LAD (+0.69) and AODd (+0.52) whereas a negative correlation with Cholesterol (−0.52) and LDL (−0.71) in T2D. Conclusion SPARC hypomethylation in CD08+ T cells may be a useful biomarker of vascular complications in Pre-Diab with a possible role for primary prevention warranting further multicenter clinical trials to validate our findings., Highlights • We conducted the first methylome analysis by RRBS platform in circulating CD04+ and CD08+ T cells in increasing hyperglycemia. • This approach has revealed possible biomarkers for cardiovascular and kidney complications in prediabetes. • SPARC hypomethylation may underly a pro-fibrotic endophenotype to be validated in larger multicenter trials.

Background: Despite current intensive treatments, hyperglycemic patients have an unfavorable prognosis due to severe CHD and development of complications. The interplay between hyperglycemia and systemic inflammation can modify patterns of gene expression mainly affecting DNA methylation in promoter regions and, thus, endothelial damage. However, DNA methylome of CD04+ and CD08+ T cells, which play a relevant role in endothelial dysfunction has not been studied, especially during increasing hyperglycemia.Purpose: To identify differentially methylated regions (DRMs) in CD04 + and CD08 + T cells by comparing healthy subjects (HS) to Pre-Diab and type 2 (T2D) patients. This approach would investigate possible biomarkers useful to identify vascular damage already at Pre-Diab state.Methods: In this pilot study, we enrolled a subgroup of patients from our ongoing PIRAMIDE clinical trial (NCT03792607) including a total of 14 individuals classified in HS (n=2), Pre-Diab (n=6), and T2D (n=6). The DMRs were identified by using the reduced representation bisulfite sequencing platform (RRBS) which captures the majority of promoters and CpG islands. Big data analysis was performed by using the R package and ChromHMM algorithms.Results: Most of the total DMRs (30-35%) were in the promoter regions in increasing hyperglycemia vs HS. A global analysis of DMR-related genes overlapping between Pre-Diab and T2D patients showed a prevalence of DNA hypermethylation in both T cells. Interestingly, the secreted protein acidic and cysteine rich (SPARC) gene was annotated to the most hypomethylated-DMR in Pre-Diab and its methylation level gradually decreased in T2D patients.Conclusions: Preliminary data indicated that hypomethylation of the SPARC promoter may be a useful biomarker of vascular complications in Pre-Diab patients with a possible role for primary prevention. However, larger multicenter trials are needed to validate our epigenetic data in the clinical arena.

Hyperglycaemia can strongly alter the epigenetic signatures in many types of human vascular cells providing persistent perturbations of protein–protein interactions both in micro- and macro-domains. The establishment of these epigenetic changes may precede cardiovascular (CV) complications and help us to predict vascular lesions in diabetic patients. Importantly, these epigenetic marks may be transmitted across several generations (transgenerational effect) and increase the individual risk of disease. Aberrant DNA methylation and imbalance of histone modifications, mainly acetylation and methylation of H3, represent key determinants of vascular lesions and, thus, putative useful biomarkers for prevention and diagnosis of CV risk in diabetics. Moreover, a differential expression of some micro-RNAs (miRNAs), mainly miR-126, may be a useful prognostic biomarker for atherosclerosis development in asymptomatic subjects. Recently, also environmental-induced chemical perturbations in mRNA (epitranscriptome), mainly the N6-methyladenosine, have been associated with obesity and diabetes. Importantly, reversal of epigenetic changes by modulation of lifestyle and use of metformin, statins, fenofibrate, and apabetalone may offer useful therapeutic options to prevent or delay CV events in diabetics increasing the opportunity for personalized therapy. Network medicine is a promising molecular-bioinformatic approach to identify the signalling pathways underlying the pathogenesis of CV lesions in diabetic patients. Moreover, machine learning tools combined with tomography are advancing the individualized assessment of CV risk in these patients. We remark the need for combining epigenetics and advanced bioinformatic platforms to improve the prediction of vascular lesions in diabetics increasing the opportunity for CV precision medicine.

Background Endothelial cells arise from endothelial progenitor cells (EPCs), which reside in bone marrow vascular niches, and are classified according to specific functional differences. Oxidative stress and inflammation lead to endothelial dysfunction that is a critical event in the initiation and progression of atherosclerotic plaques. Patients with chronic renal failure frequently show endothelial dysfunction and are at a greatly increased risk of developing atherosclerosis. Owing to their ability to partially restore vascular damage, both the number and functional changes in EPCs may be considered as useful prognostic biomarkers of cardiovascular events in hemodialysis (HD) patients. Purpose This study investigated EPC features and several biomarkers of systemic vascular inflammation and carotid atherogenesis in hemodialysis (HD) patients. Methods We studied 104 HD patients (males=55 and females 49, mean age: 51±12 years) and 40 healthy controls (males=20/females=20; mean age: 52±11 years). Isolated EPCs were cultured in the fibronectin-covered culture dishes and counted. EPC markers were studied by flow cytometry (FACS) by using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), IL-6, TNF-α, and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also evaluated. Results In our experimental conditions, EPC number was decreased in HD patients when compared to controls (p Conclusions Our study shows that EPC number was decreased in HD patients and it was associated with systemic inflammation. TNF-α could activate inhibitory actions on EPC in HD patients. A significant relationship was present between ICAM/VCAM and carotid atherosclerosis, while this was not evident with EPC number. These pathogenic mechanisms can contribute to the high incidence of cardiovascular diseases in HD patients. However, further larger studies should investigate this working hypothesis.

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.

Cardiovascular diseases (CVDs) are the first cause of death in the World. Mediator (MED) is an evolutionarily conserved protein complex, which mediates distinct protein-protein interactions. Pathogenic events in MED subunit have been associated with human diseases. Novel increasing evidence showed that missense mutations in MED13L gene are associated with transposition of great arteries while MED12, MED13, MED15, and MED30, have been correlated with heart development. Moreover, MED23 and MED25 have been associated with heart malformations in humans. Relevantly, MED1, MED13, MED14, MED15, MED23, MED25, and CDK8, were found modify glucose and/or lipid metabolism. Indeed, MED1, MED15, MED25, and CDK8 interact in the PPAR- and SREBP-mediated signaling pathways. MED1, MED14 and MED23 are involved in adipocyte differentiation, whereas MED23 mediates smooth muscle cell differentiation. MED12, MED19, MED23, and MED30 regulate endothelial differentiation by alternative splicing mechanism. Thus, MEDs have a central role in early pathogenic events involved in CVDs representing novel targets for clinical prevention and therapeutic approaches.

The regenerative potential of tissues and organs could promote survival, extended lifespan and healthy life in multicellular organisms. Niches of adult stemness are widely distributed and lead to the anatomical and functional regeneration of the damaged organ. Conversely, muscular regeneration in mammals, and humans in particular, is very limited and not a single piece of muscle can fully regrow after a severe injury. Therefore, muscle repair after myocardial infarction is still a chimera. Recently, it has been recognized that epigenetics could play a role in tissue regrowth since it guarantees the maintenance of cellular identity in differentiated cells and, therefore, the stability of organs and tissues. The removal of these locks can shift a specific cell identity back to the stem-like one. Given the gradual loss of tissue renewal potential in the course of evolution, in the last few years many different attempts to retrieve such potential by means of cell therapy approaches have been performed in experimental models. Here we review pathways and mechanisms involved in the in vivo repair of cardiovascular muscle tissues in humans. Moreover, we address the ongoing research on mammalian cardiac muscle repair based on adult stem cell transplantation and pro-regenerative factor delivery. This latter issue, involving genetic manipulations of adult cells, paves the way for developing possible therapeutic strategies in the field of cardiovascular muscle repair.

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

Circulating biomarkers available in clinical practice do not allow to stratify patients with coronary heart disease (CHD) prior the onset of a clinically relevant event. We evaluated the methylation status of specific genomic segments and gene expression in peripheral blood of patients undergoing Cardiac Computed Tomography (CCT) for CHD (n = 95). We choose to investigate cholesterol metabolism. Methylation and gene expression of low density lipoprotein receptor (LDLR), sterol regulatory element-binding factor 2 (SREBF2) and ATP-binding cassette transporter 1 (ABCA1) were evaluated by qRT-PCR. Calcium score (CACS), stenosis degree, total plaque volume (TPV), calcified plaque volume (CPV), non-calcified plaque volume (NCPV) and plaque burden (PB) were assessed in all CHD patients (n = 65). The percentage of methylation at the specific analyzed segment of LDLR promoter was higher in CHD patients vs healthy subjects (HS) (n = 30) (p = 0.001). LDLR, SREBF2 and ABCA1 mRNAs were up-regulated in CHD patients vs HS (p = 0.02; p = 0.019; p = 0.008). SREBF2 was overexpressed in patients with coronary stenosis 50% vs subjects with stenosis

INTRODUCTION: Blood transfusion is a lifesaving procedure for patients affected by hematological diseases or hemorrhage risk. AIM: This retrospective study was aimed to evaluate clinical safety of pediatric transfusions by comparing the frequency of adverse events caused by apheretic blood components vs whole blood. METHODS: From 2011 to 2015, 214 patients (blood malignancy patients, n = 144 and thalassemic patients, n = 70) received 12 531 units of blood components. The adverse acute reactions occurred during patient hospitalization were reported to the Hemovigilance system and assessed by fitting a logistic mixed-effect model. RESULTS: A total of 33 (0.3%) adverse acute events occurred. Odds ratio (OR) of adverse events from apheresis vs whole blood transfusion adjusted by patient classification was not statistically significant (OR [95% CI], 0.75 [0.23-2.47]). CONCLUSION: Our findings showed no significant differences in the prevalence of adverse acute events between blood component collected by apheresis vs whole blood in our study center.

Cardiovascular disease is still the major cause of death worldwide despite the remarkable progress in its prevention and treatment. Endothelial progenitor cells (EPCs) have recently emerged as key players of vascular repair and regenerative medicine applied to cardiovascular disease. A large amount of effort has been put into discovering the factors that could aid or impair the number and function of EPCs, and also into characterizing these cells at the molecular level in order to facilitate their therapeutic applications in vascular disease. Interestingly, the major cardiovascular risk factors have been associated with reduced number and function of EPCs. The bacterial contribution to cardiovascular disease represents a long-standing controversy. The discovery that Bartonella henselae can infect and damage EPCs revitalizes the enduring debate about the microbiological contribution to atherosclerosis, thus allowing the hypothesis that this infection could impair the cardiovascular regenerative potential and increase the risk for cardiovascular disease. In this review, we summarize the rationale suggesting that Bartonella henselae could favour atherogenesis by infecting and damaging EPCs, thus reducing their vascular repair potential. These mechanisms suggest a novel link between communicable and non-communicable human diseases, and put forward the possibility that Bartonella henselae could enhance the susceptibility and worsen the prognosis in cardiovascular disease.

Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test – the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre.

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.

Background Patients with end-stage liver disease (ESLD) have a compromised nutritional status because of the liver crucial role in regulating metabolic homeostasis and energy balance. Data sources A systematic review of literature based on extensive relevant articles published from 2001 to 2017 in English in PubMed database was performed by searching keywords such as liver disease, non-alcoholic liver disease, alcoholic liver disease, malnutrition, epigenetics, gut microbiota, and probiotics. Results Liver transplantation would be one eligible therapy for ESLD patients, even if, the clinical outcome is negatively influenced by malnutrition and/or infections. The malnutrition is a condition of nutrient imbalance with a high incidence in ESLD patients. An accurate evaluation of nutritional status could be fundamental for reducing complications and prolonging the survival of ESLD patients including those undergoing liver transplantation. In addition, the interaction among nutrients, diet and genes via epigenetics has emerged as a potential target to reduce the morbidity and mortality in ESLD patients. The malnutrition induces changes in gut microbiota causing dysbiosis with a probable translocation of bacteria and/or pathogen-derived factors from the intestine to the liver. Gut microbiota contribute to the progression of chronic liver diseases as well as hepatocellular carcinoma. The administration of probiotics modulating gut microbiota could improve all chronic liver diseases. Conclusions This review provides an update on malnutrition status linked to epigenetics and the potential benefit of some probiotics on the management of ESLD patients. In support of this view and to reveal the constant and growing interest in this field, some clinical trials are reported.

The major issue in coronary heart disease (CHD) diagnosis and management is that symptoms onset in an advanced state of disease. Despite the availability of several clinical risk scores, the prediction of cardiovascular events is lacking, and many patients at risk are not well stratified according to the canonical risk factors alone. Therefore, adequate risk assessment remains the most challenging issue. Recently, the integration of imaging data with biochemical markers in a radiogenomic framework has been proposed in many fields of medicine as well as in cardiology. Multimodal imaging and advanced processing techniques can provide both direct (e.g., remodeling index, calcium score, total plaque volume, plaque burden) and indirect (e.g., myocardial perfusion index, coronary flow reserve) imaging features of CHD. Furthermore, the identification of novel non-invasive biochemical markers, mainly focused on plasma and/or serum samples, has increased the specificity of findings, reflecting several pathophysiological pathways of atherosclerosis, the principal actor in CHD. In this context, a multifaced approach, derived from the strengths of all these modalities, appears promising for finer risk stratification and treatment strategies, facilitating the decision-making and clinical management of patients. This review underlines the role of different imaging modalities in the quantification of coronary atherosclerosis and describes novel blood-based markers that could improve diagnosis and have a better predictive value in CHD.

From the 1980s, extracorporeal photochemotherapy (ECP) has been shown to be effective in a variety of pathological conditions such as erythrodermic cutaneous T-cell lymphoma, autoimmune diseases, solid organ allograft rejection and graft versus host disease. To date, ECP represents a non-aggressive immune modulatory therapy with a low spectrum of toxicity. ECP reduces the alloreactivity promoting the immune tolerance to self. At the same time, it allows the maintenance of immune response integrity of both naive and memory T-cells. However, the molecular mechanisms of action by which ECP exerts its therapeutic activity are still under investigation. Here, we review molecular mechanisms and clinical applications involved in ECP. The outcome of ECP is difficult due to the lack of reliable predictor factors for the selection of patients and their adequate follow-up. Since the study of such predictors is important, we also describe some biological markers that enable us to investigate the clinical management of the patients considered for the use of ECP.

Background: The main challenge in type 2 diabetes (T2D) is to detect the regulators of pathogenic events during early stages of disease, as well as prevention and progression follow-up of cardiovascular (CV) complications. DNA methylation and micro-RNAs (miRNAs) are major components of the epigenome, which are involved in the diabetic interactome. This study protocol may contribute to advance our knowledge on molecular basis underlying T2D and its CV complications, as well as provide putative useful prognostic biomarkers.Methods: The perturbation of interactome through micro-RNA and methylome analysis in diabetes endophenotypes: the PIRAMIDE pathogenic clinical study protocol is a cross-sectional research program planned to combine big data and network-based analysis aimed to investigate whether DNA methylation and miRNAs may act as simultaneous regulators of the interactome in T2D patients. Clinical datasets will be aggregate to large-scale DNA methylation, mRNA-Seq, and miRNA-Seq analysis performed both on purified CD4+ and CD8+ T cells isolated from 35 T2D patients and 35 sex and age-matched controls. DNA methylome data will be used as input for the weighted human DNA methylation PPI network (WMPN) algorithm. RNA sequencing data will be used as input data for the TargetScan algorithm. The primary endpoint will be to integrate both DNA methylation and miRNA networks to potentially capture which genes are simultaneously modulate by interactions between epigenetic changes. Then, statistical analysis will be performed to correlate these molecular modifications with development of T2D-related CV complications.Conclusions: PIRAMIDE pathogenic clinical study protocol will test the hypothesis that simultaneous interactions between DNA methylation and miRNAs may hit T2D-associated candidate genes and predict the development of T2D-related CV complications.Trial Registration: The ongoing PIRAMIDE pathogenic clinical study protocol has been registered on NIH website (NCT03792607).

Intravenous immunoglobulin (IVIG) products are known to have beneficial immunomodulatory effects on several inflammatory and autoimmune disorders. These effects could be attributed to a different inhibitory action on complement factors, but other mechanisms could be implicated, e.g., immunocomplexes development and/or anti-idiotypic antibodies. Positive results on the reduction of anti-Human Leukocyte Antigens (HLA) antibodies in highly sensitized patients have also been found. The present study focuses on the effect of IVIG on the reduction of Panel Reactive Antibody level and crossmatch positivity in sensitized patients awaiting kidney transplantation.The study was performed adapting an in vitro assay on sensitized patients' sera in waiting list for kidney transplantation. Sera of twelve highly sensitized patients were evaluated for the cytotoxicity inhibition after 10% IVIG treatment.A reduction of anti- HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12) resulted unresponsiveness. Particularly, our data showed a significantly higher Panel Reactive Antibody reduction for T lymphocytes (p0.010) than B lymphocytes (p0.032).In this study, we have used an in vitro assay to investigate susceptibility to desensitization with IVIG treatment of sensitized patient sera. These findings reveal that the variable effect of IVIG on reducing Panel Reactive Antibody in our immunized patients could be attributed to a different inhibitory action on complement, likely due to the type and the titre of anti-HLA antibodies.

In this chapter we will describe the use of the tubular network assay as a useful tool in the evaluation of cancer neoangiogenesis (as reported by Arnaoutova et al. [Angiogenesis 12:267–274, 2009]). Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. A detailed working protocol applicable to the study of any type of endothelial cells is provided below and in addition, details on data interpretation analysis and troubleshooting.

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P

The CD146 cell membrane adhesion molecule is highly expressed on the cell surface of several tumours. The level of its expression has been found to correlate directly with tumour progression and metastatic potential, thus establishing CD146 as an important candidate of tumour growth and metastasis. In order to characterize its expression in human osteosarcoma (OS) cell lines, we have examined the CD146 expression at protein and RNA levels in both normal and tumour osteoblast-like cell lines by several methods. Our results indicate that CD146 protein is expressed at low levels in normal osteoblast cells whereas it is highly expressed in all OS cell lines analysed, (SaOS, MG-63, U-2OS). Moreover, CD146 overexpression was partially reduced in shYY1 cells, where the Yin Yang 1 transcription factor, also found over-expressed in human OS cells, has been silenced.

The growing improvements of computed tomography have made this technique more and more available for cardiac evaluation. Coronary artery anomalies (CAAs) are often incidental findings in subjects with suspected coronary artery disease (CAD) undergoing coronary angiography or computed tomography coronary angiography (CTCA). In some cases, CAAs can be clinically relevant so their identification could change radically patient management and treatment. We report the case of a 68-year-old male patient with known CAD and associated anomalous origination of the left coronary artery from the opposite sinus.

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

Circulating endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischaemic tissues and in re-endothelization of injured blood vessels. Identification of compounds able to enhance EPC levels and improve their functional activity, noticeably compromised by risk factors for coronary heart disease, is of clinical interest. This study evaluates the effects of red wine on EPCs. After being isolated from total peripheral blood mononuclear cells, EPC phenotype was confirmed by the presence of double positive cells for DiLDL uptake and lectin binding and by expression of CD34, CD133 and VE-cadherin cell surface markers. Long-term culture in the presence of red wine (1 microl/ml), containing resveratrol (Resv) at physiological concentration (nM), determined a time-dependent amelioration of cell number (P < 0.05). The presence of red wine prevented the TNF-alpha-induced reduction of EPC number (P < 0.05) and this effect was accompanied by reduced p38-phosphorylation expression levels (P < 0.05) and increased NOx levels (P < 0.05) Indeed, pure Resv alone significantly improved the TNF-alpha reduced EPC number (P < 0.05). This evidence indicates novel beneficial effects of red wine and Resv in the positive modulation of EPCs levels.

PALB2 gene is mutated in about 1–2 % of familial breast cancer as well as in 3–4 % of familial pancreatic cancer cases. Few studies have reported mutations in Italian patients with breast or pancreatic cancer. We evaluate the occurrence of PALB2 mutations in Italian patients affected with hereditary breast and ovarian cancers and define the pathological significance of the putative allelic variants. We recruited 98 patients (F = 93, M = 5) affected with breast and/or ovarian cancer, negative for mutations in BRCA1 and BRCA2 (BRCAX). Genomic DNA was isolated from peripheral blood lymphocytes, PALB2 coding regions and adjacent intronic were sequenced; in silico predictions were carried out using prediction programs. Mutational analysis of PALB2 gene revealed the novel mutation c.1919C>A (p.S640X) in a 29 years old woman with breast cancer. The c.1919C>A (p.S640X) mutation causes the lack of C-terminus region inducing alteration of MORF4L1–PALB2 association and the lack of interaction of PALB2 with RAD51 and BRCA2. In addition, we identified two novel PALB2 variants, c.3047T>C (p.F1016S) and c.*146A>G. In silico analysis conducted for c.*146A>G indicates that this variant does not affect the splicing while c.3047T>C (p.F1016S) was predicted as damaging in three classifier algorithms. The proband carrier of c.3047T>C (p.F1016S) showed two breast cancer cases, two ovarian cancer cases and one pancreatic cancer in mother’s family. c.3047T>C (p.F1016S) and c.*146A>G should be considered PALB2 UVs even though the genotype–phenotype correlation for these variants remains still unclear. Our findings indicate that the presence of PALB2 mutation should be routinely investigated in hereditary breast and ovarian cancers families since it could be of clinical relevance for clinical management.

Background: The use of cardiac resynchronization therapy (CRT) and implantable cardioverter- defibrillator (ICD) for advanced heart failure (HF) is increasing. Renal dysfunction is a common condition in HF which is associated with a worse survival. The study aims at identifying in patients with advanced HF treated with CRT the effect of baseline glomerular filtration rate (GFR), GFR improvement and left ventricular ejection fraction (LVEF) change, after 6-months of CRT implant, on survival. Methods: The study population consisted of 375 advanced HF patients who received a CRT between 1999 and 2009, of these 277 received also an ICD implant. Clinical characteristics (New York Heart Association [NYHA] functional class, ischemic vs. non-ischemic etiology, atrial fibrillation, diabetes, hypertension, LVEF, QRS duration and GFR were recorded. The use of common used drugs was evaluated. Cox proportional hazards analysis was calculated in order to evaluate variables associated to mortality. Results: During a median follow-up of 43.0 months, 93 (24.8%) patients died. Patients deceased during the study had at baseline higher NYHA class and lower LVEF and GFR. In Cox regression analysis, GFR predicts long-term mortality (hazard ratio [HR] 0.983; 95% confidence interval [CI] 0.969–0.998; p = 0.023) independently from the effect of others covariates. In addition, a positive GFR improvement 6 months after CRT implant is significantly associated with a lower hazard of mortality (for each 10 mL/min of GFR improvement HR 0.86; 95% CI 0.75–0.99; p = 0.038). Conclusions: GFR is a significant predictor of mortality in advanced HF patients who received CRT. A GFR improvement 6 months after CRT implant is significantly associated with a lower hazard of mortality.

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.

From the 1980s, extracorporeal photochemotherapy (ECP) has been shown to be effective in a variety of pathological conditions such as erythrodermic cutaneous T-cell lymphoma, autoimmune diseases, solid organ allograft rejection and graft versus host disease. To date, ECP represents a non-aggressive immune modulatory therapy with a low spectrum of toxicity. ECP reduces the alloreactivity promoting the immune tolerance to self. At the same time, it allows the maintenance of immune response integrity of both naive and memory T-cells. However, the molecular mechanisms of action by which ECP exerts its therapeutic activity are still under investigation. Here, we review molecular mechanisms and clinical applications involved in ECP. The outcome of ECP is difficult due to the lack of reliable predictor factors for the selection of patients and their adequate follow-up. Since the study of such predictors is important, we also describe some biological markers that enable us to investigate the clinical management of the patients considered for the use of ECP.