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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 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.

Background Although advances in diagnosis, treatment and prognosis, coronary heart disease (CHD) is still the most prevalent cause of mortality and morbidity worldwide. Thus, there is still the need to identify both novel diagnostic and prognostic biomarkers to improve the clinical decision-making and help to stratify patients for early preventive treatment. Epigenetic-sensitive mechanisms may be related both to pathogenesis and prognosis of CHD. Among the epigenetic hallmarks, microRNAs (miRNAs), acting as flexible modulators of gene expression, could represent attractive candidate biomarkers useful in clinical practice. Purpose We prospectively investigated the expression pattern of circulating miRNAs in patients undergoing Cardiac Computed Tomography (CCT) for suspected CHD (n=95) with the aim to integrate molecular findings with morphological and clinical parameters derived by CCT. Methods CCT was performed with a third-generation dual source multidetector computed tomography scanner. Peripheral venous blood samples were collected into EDTA after a 12 h fasting in the same day of CCT, before imaging execution and the levels of 42 selected plasmatic miRNAs were analyzed by qRT-PCR. Results Let-7c-5p, miR-765, miR-483-5p, miR-31-5p and miR-206 were upregulated in CHD patients (n=66) vs healthy subjects HS (n=29) as well as let-7c-5p, miR-765, miR- 483-5p showed higher expression in obstructive CHD (n=36) compared to no obstructive CHD patients (n=66). In addition, miR-93-5p and miR-433-3p showed an upregulation in patients with critical coronary stenosis. Multivariate regression analysis demonstrated that miR-765, miR-31-5p and miR-206 were independently associated with CHD also in combination with Framingham risk score. Relevantly, miR-765, miR-93-5p and miR-433-3p were obstructive CHD predictors. ROC curve analysis also revealed a good performance for miR-765, miR-93-5p and miR-433-3p on predicting CHD severity. Circulating microRNA expression Conclusions Our study represents a combined epigenetic/imaging approach useful to support the diagnosis and prediction of CHD. Acknowledgement/Funding Italian Ministry of Health grants: “Giovani Ricercatori 2011-12” (project code GR-2011-02349436) and “Ricerca Corrente 2018”

Background How metabolic impairment leads to cardiac dysfunction in diabetics is unknown. A recent theory, derived mainly from rodent models, involves lipid over-storage to cardiomyocytes. Previous human study demonstrated cardiac steatosis on explanted hearts of diabetics with heart failure. However, this study did not provide any evidence about the effects of diabetes milieu on implanted non-diabetic heart. Purpose We evaluated intramyocyte lipid infiltration in explanted heart of type 2 diabetics with end-stage heart failure. Moreover, we studied the effects of diabetic milieu on myocyte lipid infiltration and cardiac function of non-diabetic implanted hearts in type 2 diabetics one year after heart transplantation (HTx). Methods We conducted a prospective study with a follow-up of 12 months on 88 patients over 18 years of age underwent first HTX. Patients with pre-HTx diabetes duration for at least 6 months were included in the study. Patients with endomyocardial biopsy (EMB) considered positive for rejection, according to International Society for Heart Lung Transplantation (ISHLT), and with post-HTx diabetes were excluded from the study. All patients underwent immunosuppression induction according ISHLT indications. All patients were followed applying internationally accepted patient evaluations (echocardiography and metabolic control) and EMB schedules. EBM from patients without rejection evidences were evaluated for intramyocyte lipid infiltration with oil red-O staining (Or-O). Results The patients were divided in diabetics (44%, age 51.6±7.2 y, diabetes duration 11±3 y) and no-diabetics (56%, 52.1±10.9 y). The patients were matched on the basis of eligibility for a HTx. Seven patients (3 diabetics and 4 no-diabetics) died in hospital. 5 (11%) patients developed post- HTx diabetes. No differences were seen in rejection (12% vs. 10%), infection (9% vs. 10%), renal dysfunction (9% vs. 8%) or mortality (7% vs. 8%). Therefore, the study population included 23 no-diabetics and 22 diabetics. After 1 year, we evidenced an impairment of both sx and dx ventricular function as showed by a significantly reduction of ejection fraction and TAPSE in diabetic patients (Figure-A). Although diastolic function not show significant differences among groups, the E/e' ratio showed lower reduction in diabetics. Or-O evidenced that 91% of diabetic and only 2 of no-diabetic explanted hearts (9%) showed intramyocyte lipid infiltration (Figure-B). Moreover, Or-O of EMB, for monitoring heart transplant during 1 year, evidenced a progressive intramyocyte lipid infiltration in 18 diabetics (81%), whereas none of no-diabetics showed intramyocyte lipid infiltration. Conclusions Our data show that almost all of the explanted diabetic hearts had intramyocyte lipid infiltration. More interesting, we observed that healthy heart transplanted in recipients with pretransplant diabetes were affected early by metabolic disorders leading to intramyocyte lipid infiltration.

Background Treatment with valsartan/sacubitril was found to be associated both with reduction in mortality risk for HF. Purpose To investigate the effect of sacubitril/valsartan on physical frailty (PF) in advanced HF patients in waiting list for heart transplantion (HT) in a two years follow-up study. Methods We enrolled 45 consecutive patients (status 2B UNOs). Patients tolerant to ACE-inhibitor and/or ARBs were treated with valsartan/sacubitril. Patients were required to have left ventricular ejection fraction (LVEF) of ≤35% and to be taking a stable dose of a β-blocker and an ACE inhibitor or an ARB for at least 4 weeks before enrollment. The dosage of sacubitril/valsartan was increased if tolerance was good. Frailty was assessed using an adapted version of Fried's Frailty Phenotype used in advanced HF patients in waiting list for transplantation. Patients were followed up until HT, device (TAH/LVAD) implant, or last follow-up visit. Results Mean NYHA class was 3.1±0.4, with 5.7% NYHA 2, 62.9% NYHA 3 and 31.4% NYHA 3B. LVEF was 25.1±6.4, VO2 max (ml/kg/min) was 10.3±2.3, cardiac index (L/min/m2) was 2.3±0.6, and NT-proBNP (pg/ml) was 4054.8±3977. At 3 months, 31.5% of patients received the target dose of 97/103 mg/BID, 31.6% the half dose (49/51mg BID) and 26.3% the low dose (24/26 mg BID) and 10.5% the half low dose (24/26 mg daily). The baseline mean value of physical frailty was 3.86±0.8, specifically 94.3% showed exhaustion, 91.4% physical inactivity, 88.6% weakness, 85.7% slowness and 28.6% loss of appetite. All patients had a frailty score ≥3. During follow-up there were no deaths. After treatment, NYHA class improved significantly (2.4±0.6 vs 3.1±0.4; p=0.002), with 5.7% NYHA 1, 48.6% NYHA 2, 42.9% NYHA 3 and 2.9% NYHA 3B (p Conclusions Our study shows an improvement in PH in patients with advanced HF in waiting list for HT after therapy with sacubitril/valsartan. These changes seem to appear very early after introduction of the treatment and to be maintained over time. The improvement in all physical domain was paralleled by VO2 max and 6-minute walking test increase. The pro-BNP-NT reduction was significant in the first month of treatment and remain quite stable in the follow-up.

Currently, five different statins (simvastatin,pravastatin, lovastatin, fluvastatin, and atorvastatin) are approved for treatment of hypercholesterolemia in humans and two new compounds (rosuvastatin and NK-104) are under investigation.1,2 Despite differences in their pharmacokinetic profiles, all statins have at least onecharacteristic in common: they block the conversion of HMG-CoA to mevalonic acid with consecutive attenuation of the biosynthesis of cholesterol (Fig. 1), which is associated with a reduction in serum total and low-density lipoprotein (LDL) cholesterol of as much as 20–31 and 28–42% during chronic treatment.3 Because of these properties, statins have become the most widely prescribed lipid-lowering drugs in patients with elevatedserum cholesterol levels. Several large trialsdemonstrated that statins are not only safe and well tolerated but also significantly decrease cardiovascular morbidity and mortality in hypercholesterolemic patients in both primary and secondary prevention.4–8 However, the striking benefit achieved with statin treatment in patients with a wide range of cholesterol levels, which cannot be attributed to their cholesterol lowering effect alone, has raised the question about the possible presence of additional effects of statins beyond their impact on serum cholesterol levels. Indeed, in recent years a substantial quantity of data has accumulated showing that statins exert variouseffects on multiple targets, which are independent of their plasma cholesterol lowering properties. Fig. 1 The effect of statins on the mevalonate pathway. Statins inhibit conversion of HMG-CoA to mevalonate by competitive inhibition of the rate limiting enzyme HMG-CoA reductase. Herewith, statins not only inhibit the cellular production of cholesterol but also the biosynthesis of several intermediates of the mevalonate pathway (e.g. farnesylpyrophosphate and geranylgeranylpyrophosphate). These so-called isoprenoids are essential for the posttranslational modification of several proteins involved in important intracellular signaling pathways (e.g. the small GTP-binding proteins Ras and Rho). Many of these so-called pleiotropic effects have been shown to be secondary to the …

Background Ischaemic preconditioning reduces myocardial infarct size in animal models. Clinical data suggest that episodes of angina immediately before acute myocardial infarction may be associated with smaller infarct size in man. However, it is unclear whether ischaemic episodes preceding acute myocardial infarction also affect contractile recovery in patients. Objective In this study we investigated the recovery of regional myocardial function after thrombolysis in two groups of patients at their first Q-wave acute myocardial infarction; in one group (n=42) myocardial infarction occurred unheralded, whereas patients of the second group (n=48) had experienced new-onset angina in the 48h that preceded infarction. Echocardiographic analysis of myocardial regional function in the infarct area was done at 2, 24 and 72h after thrombolysis, and at 1 week, and 1 and 3 months follow-up. Results Peak level of MB-creatine kinase was significantly lower in patients with new-onset angina (96±47 as compared with 221±108IU.l−1, P

Background and purpose: It is well established that human vascular lesions begin very early stage in life with the development of fatty streak lesions in the arteries of fetuses. Although several studies have demonstrated that maternal cholesterol may exert detrimental effects on the number and degree of vascular lesions in fetuses, however, how cholesterol affects in utero programming is still unclear. Epigenetics migh promote pathogenic mechanisms by which gene-environment interactions could occurr. The aim of this study was to verify whether maternal hypercholesterolemia during pregnancy affects epigenetic signature in fetal aortic tissues. Methods: Human fetal aortas (n=42) were analyzed by immunohistochemistry and dichotomized in their respective high and low maternal cholesterol during pregnancy. Immunohistochemistry in fetal arteries was performed with the following antibodies: Anti-trimethyl-Histone H3, (Lys4, Lys9, Lys27), and Anti-CD68 antibody. Methyl DNA immunoprecipitation was performed by MeDIP kit (DIAGENODE). Results: The data indicated a strong signal >75% of positive cells with histone trimethyl lys 27 antibody present in 27% of the biggest fetal aortic lesion area (>120mm2/section) (p>0.039). Molecular analysis of specific cholesterol regulatory circuit gene expression revealed hypermethylation in promoter and 3' UTR of SREBP2 and LDL receptor gene in aortas from hypercholesterolemic mothers compared to normocholesterolemic group (p

Background: Elevated Maternal Cholesterol during Pregnancy (MCP) enhances atherogenesis in childhood, but its impact on acute myocardial infarction (AMI) in adults is unknown. Methods: 89 AMI patients meeting narrow criteria (born after 1945, typical chest pain, transmural infarction Q-waves, elevated creatinine kinase, no cerebrovascular or terminal disease) were identified among patients admitted to coronary care unit in Naples, Italy. Patients were classified by MI severity (severe=involving 3 arteries, left ventricle ejection fraction ≤35, CK-peak >1200 mg/dl, or CK-MB >200 mg/dl). The association of MCP with AMI severity was tested by linear and multiple regression analysis that included conventional cardiovascular risk factors, gender, age, and treatment. Associations of MCP with BMI was assessed by linear correlation. Results: MCP correlated with four measures of AMI severity: number of vessels (β=0.382, p=0.001), ejection fraction (β=-0.315, p=0.003), CK (β=0.260, p=0.014) and CK-MB (β=0.334, p=0.001), as well as survival time (β=-0.252, p=0.031). In multivariate analysis of patients stratified by AMI severity, MCP predicted AMI severity independently of age, gender, and CHD risk factors (OR=1.304, 95% CI 1.107-1.559; p=0.004). Screening for point mutations ruled out that this was due to certain inherited differences in lipid metabolism. Survival was affected mainly by AMI severity. Conclusions: MCP is associated with adult BMI, atherosclerosis-related risk and severity of AMI.