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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.

Background and purpose Some studies have shown that air pollution, often assessed by thin particulate matter with diameter below 2.5 µg/m3 (PM2.5), may contribute to severe COVID‐19 courses, as well as play a role in the onset and evolution of multiple sclerosis (MS). However, the impact of air pollution on COVID‐19 has never been explored specifically amongst patients with MS (PwMS). This retrospective observational study aims to explore associations between PM2.5 and COVID‐19 severity amongst PwMS. Methods Data were retrieved from an Italian web‐based platform (MuSC‐19) which includes PwMS with COVID‐19. PM2.5 2016–2018 average concentrations were provided by the Copernicus Atmospheric Monitoring Service. Italian patients inserted in the platform from 15 January 2020 to 9 April 2021 with a COVID‐19 positive test were included. Ordered logistic regression models were used to study associations between PM2.5 and COVID‐19 severity. Results In all, 1087 patients, of whom 13% required hospitalization and 2% were admitted to an intensive care unit or died, were included. Based on the multivariate analysis, higher concentrations of PM2.5 increased the risk of worse COVID‐19 course (odds ratio 1.90; p = 0.009). Conclusions Even if several other factors explain the unfavourable course of COVID‐19 in PwMS, the role of air pollutants must be considered and further investigated., Air pollution, often assessed by particulate matter with diameter below 2.5 µg/m3, may contribute to severe COVID‐19 courses. 1087 patients were included, of whom 13% required hospitalization and 2% were admitted to an intensive care unit or died. Even if several other factors explain the unfavourable course of COVID‐19 in patients with multiple sclerosis, the role of air pollutants must be considered and further investigated.

Nitric oxide (NO) is a powerful mediator with biological activities such as vasodilation and prevention of vascular smooth muscle cell proliferation as well as functional regulation of cardiac cells. Thus, impaired production or reduced bioavailability of NO predisposes to the onset of different cardiovascular (CV) diseases. Alterations in the redox balance associated with excitation–contraction coupling have been identified in heart failure (HF), thus contributing to contractile abnormalities and arrhythmias. For its ability to influence cell proliferation and angiogenesis, NO may be considered a therapeutic option for the management of several CV diseases. Several clinical studies and trials investigated therapeutic NO strategies for systemic hypertension, atherosclerosis, and/or prevention of in stent restenosis, coronary heart disease (CHD), pulmonary arterial hypertension (PAH), and HF, although with mixed results in long-term treatment and effective dose administered in selected groups of patients. Tadalafil, sildenafil, and cinaguat were evaluated for the treatment of PAH, whereas vericiguat was investigated in the treatment of HF patients with reduced ejection fraction. Furthermore, supplementation with hydrogen sulfide, tetrahydrobiopterin, and nitrite/nitrate has shown beneficial effects at the vascular level.

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.

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.

Background: The MuSC-19 project is an Italian cohort study open to international partners that collects data on multiple sclerosis (MS) patients with COVID-19. During the second wave of the pandemic, serological tests became routinely available. Objective: To evaluate the seroprevalence of anti-SARS-CoV-2 antibodies according to the use of disease-modifying therapy (DMT) in a subset of patients included in the MuSC-19 data set who had undergone a serological test. Methods: We evaluated the association between positive serological test results and time elapsed since infection onset, age, sex, Expanded Disability Status Scale score, comorbidities and DMT exposure using a multivariable logistic model. Results: Data were collected from 423 patients (345 from Italy, 61 from Turkey and 17 from Brazil) with a serological test performed during follow-up. Overall, 325 out of 423 tested patients (76.8%) had a positive serological test. At multivariate analysis, therapy with anti-CD20 was significantly associated with a reduced probability of developing antibodies after COVID-19 (odds ratio (OR) = 0.20, p = 0.002). Conclusion: Patients with MS maintain the capacity to develop humoral immune response against SARS-COV-2, although to a lesser extent when treated with anti-CD20 drugs. Overall, our results are reassuring with respect to the possibility to achieve sufficient immunization with vaccination.

Background: It is unclear how multiple sclerosis (MS) affects the severity of Covid-19. Methods: Hospitalization, Intensive Care Unit (ICU) admission and death after Covid-19 diagnosis of 1362 MS patients were compared to the age and sex-matched Italian population in a retrospective observational case-cohort study with population-based control. The observed vs the expected events were compared in the whole MS cohort and in different subgroups (higher-risk: EDSS>3 or at least one comorbidity, lower-risk: EDSS

espanolMediante la Sentencia de 1 de octubre de 2018, la Corte Internacional de Justicia se ha pronunciado sobre la demanda del Estado Plurinacional de Bolivia acerca de una pretendida obligacion a cargo de Chile, para otorgarle acceso soberano al mar. La Corte Internacional de Justicia ha decidido que, de la conducta de las partes y del Derecho internacional general, no se derivaba la existencia de tal obligacion. El caso planteaba la pregunta acerca de donde y como se habia generado esa obligacion, si lo habia sido por la conducta de la parte demandada, o por acuerdo de las partes, y que papel cumplia el Derecho internacional general. El caso ha permitido evocar tambien el debate teorico segun el cual pueden presentarse obligaciones dignas de calificarse como propias de un pactum de negotiando, o de un pactum de contrahendo. La respuesta de la Corte a los distintos elementos expuestos fue que ella no pudo llegar a la conclusion que Chile posee «la obligacion de negociar con Bolivia a fin de alcanzar un acuerdo que otorgue a Bolivia un acceso soberano al oceano Pacifico», como lo expusiese Bolivia. Entre otros elementos, la Corte ha afirmado que el art. 2.3 de la Carta de las Naciones Unidas establece un deber general de solucionar las controversias a fin de preservar la paz y seguridad internacionales, y la justicia. Pero aquel no indica que las partes estan obligadas a recurrir a un metodo especifico de arreglo, como es la negociacion, termino que tampoco menciona. En consecuencia, la Corte no acepta las demas peticiones de Bolivia, cuya premisa era la supuesta obligacion de negociar. EnglishBy judgment of October 1, 2018, the International Court of Justice has decided on the submission of the Plurinational State of Bolivia regarding an alleged existence of an obligation under Chile, to negotiate an agreement granting Bolivia sovereign access to the sea. The Court has ruled that neither general international law nor the conduct of the parties resulted in the existence of such an obligation. The case raised the question of where and how that obligation had been generated, if it had been through the conduct of the respondent, or by agreement of the parties, as well as about the role of general international law. The case has recalled the theoretical debate according related to obligations characterized as part of a pactum of negotiando or of a pactum of contrahendo. The response of the Court to these various elements has been that it could not reach the conclusion that Chile had «the obligation to negotiate with Bolivia in order to reach an agreement granting Bolivia a fully sovereign access to the Pacific Ocean», as stated by Bolivia in its submissions. Among other elements, the Court also affirms that art. 2.3 of the Charter of the United Nations establishes a general duty to settle disputes in order to preserve international peace and security, and justice. And, it does not indicate that the parties are obliged to resort to a specific method of settlement, such as negotiations, a term which is not mentioned in the said provision. Accordingly, the Court could not accept the other final submissions presented by Bolivia, which were premised on the existence of such an obligation.

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

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

The early identification of pathogenic mechanisms is essential to predict the incidence and progression of cardiomyopathies and to plan appropriate preventive interventions. Noninvasive cardiac imaging such as cardiac computed tomography, cardiac magnetic resonance, and nuclear imaging plays an important role in diagnosis and management of cardiomyopathies and provides useful prognostic information.Most molecular factors exert their functions by interacting with other cellular components, thus many diseases reflect perturbations of intracellular networks. Indeed, complex diseases and traits such as cardiomyopathies are caused by perturbations of biological networks. The network medicine approach, by integrating systems biology, aims to identify pathological interacting genes and proteins, revolutionizing the way to know cardiomyopathies and shifting the understanding of their pathogenic phenomena from a reductionist to a holistic approach.In addition, artificial intelligence tools, applied to morphological and functional imaging, could allow imaging scans to be automatically analyzed to extract new parameters and features for cardiomyopathy evaluation. The aim of this review is to discuss the tools of network medicine in cardiomyopathies that could reveal new candidate genes and artificial intelligence imaging-based features with the aim to translate into clinical practice as diagnostic, prognostic, and predictive biomarkers and shed new light on the clinical setting of cardiomyopathies. The integration and elaboration of clinical habits, molecular big data, and imaging into machine learning models could provide better disease phenotyping, outcome prediction, and novel drug targets, thus opening a new scenario for the implementation of precision medicine for cardiomyopathies.

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.

Coronary artery fistulas (CAFs) are often incidental findings in patients undergoing conventional coronary angiography (CAG). CAFs represent a congenital or acquired abnormal vascular communication of coronary arteries with cardiac chambers or any segment of the systemic or pulmonary circulation bypassing capillaries within myocardium (1).

La jurisdiccion de la Corte Internacional de Justicia conforme al Pacto de Bogota forma parte de un sistema de normas que atribuyen competencia a dicho tribunal, asi como a otros medios de solucion. El sistema establece principios y excepciones generales que se aplican a todos los medios de solucion previstos en tal instrumento. Este trabajo pasa revista a casos especificos que muestran como ha operado el Pacto, asi como tambien a los principales debates relativos a su eficacia y reforma. Mientras que el primer tema se relaciona con materias de derecho internacional general sobre la solucion de controversias y la interpretacion de los tratados, el segundo ambito se vincula a las dinamicas del Sistema Interamericano, sus componentes institucionales y las perspectivas de una reforma. Finalmente, el Pacto no ha sido ajeno a la politica y diplomacia hemisfericas.

Early identification of coronary atherosclerotic pathogenic mechanisms is useful for predicting the risk of coronary heart disease (CHD) and future cardiac events. Epigenome changes may clarify a significant fraction of this “missing hereditability”, thus offering novel potential biomarkers for prevention and care of CHD. The rap-idly growing disciplines of systems biology and network science are now poised to meet the fields of precision medicine and personalized therapy. Network medicine integrates standard clinical recording and non-invasive, advanced cardiac imaging tools with epigenetics into deep learning for in-depth CHD molecular phenotyping. This approach could potentially explore developing novel drugs from natural compounds (i.e. polyphenols, folic acid) and repurposing current drugs, such as statins and metformin. Several clinical trials have exploited epigenetic tags and epigenetic sensitive drugs both in primary and secondary prevention. Due to their stability in plasma and easiness of detection, many ongoing clinical trials are focused on the evaluation of circulating miRNAs (e.g. miR-8059 and miR-320a) in blood, in association with imaging parameters such as coronary calcifications and stenosis degree detected by coronary computed tomography angiography (CCTA), or functional parameters pro-vided by FFR/CT and PET/CT. Although epigenetic modifications have also been prioritized through network based approaches, the whole set of molecular interactions (interactome) in CHD is still under investigation for primary prevention strategies.

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”

La Sociedad Chilena de Derecho Internacional rinde homenaje al reconocido jurista y académico Francisco Orrego Vicuña, recientemente fallecido.

Objectives Nitric oxide (NO) represents the most powerful endogenous molecule with vasodilator action mainly produced by endothelial nitric oxide synthase (eNOS) enzyme. Polymorphisms and epigenetic-sensitive mechanisms can modulate the expression of eNOS gene, leading to the endothelial dysfunction. This review updates on the mechanistic role of NO in the regulation of platelet activation, as well as the impact of eNOS genetic and epigenetic modifications on arterial thrombosis onset. Design A systematic search was addressed to examination of PubMed databases with the following terms: nitric oxide; arterial thrombosis; endothelial dysfunction; DNA variations; epigenetic modifications; personalized therapy; network medicine. Results G894T, -786T/C, and 4b/4a variable number tandem repeat (VNTR), are the main classes of polymorphisms harbored in eNOS gene associated to increased arterial thrombosis risk. DNA methylation, histone/non-histone modifications, and microRNA (miRNAs) can modulate eNOS gene expression. Investigators largely focused on the role of miRNAs in modulating NO production in arterial thrombosis development. In detail, miR-195, and miR-582 are inversely correlated both to eNOS and NO levels, thus suggesting novel biomarkers. Conclusion We are far from incorporating omics pathogenic data from bench to arterial thrombosis bedside. Network medicine is an emerging paradigm that ideally overcomes the current shortcomings of the reductionist approach. Despite several clinical limitations, the network-based analysis of the interactome might reveal the key nodes underlying the perturbations of the arterial thrombosis, thus advancing personalized therapy.

China and Russia stated in 2016 that “It is crucial for the maintenance of international legal order that all dispute settlement means and mechanisms are based on consent and used in good faith and in the spirit of cooperation, and their purposes shall not be undermined by abusive practices.” Does this sentence reflect their shared view of the signing states or are these ideas endorsed by other international players?

Moderate to severe spasticity is commonly reported in Multiple Sclerosis (MS) and its management is still a challenge. Cannabinoids were recently suggested as add-on therapy for the treatment of spasticity and chronic pain in MS but there is no conclusive scientific evidence on their safety, especially on cognition and over long periods. The aim of this prospective pilot study was to assess the long-term effects of a tetrahydrocannabinol-cannabidiol (THC/CBD) oromucosal spray (Sativex®) on cognition, mood and anxiety.An extensive and specific battery of neuropsychological tests (Symbol Digit Modalities Test-SDMT, California Verbal Learning Test-CVLT, Brief Visuospatial Memory Test-BVMT; PASAT-3 and 2; Free and Cued Selective Remind Test-FCSRT, Index of Sensitivity of Cueing-ISC) was applied to longitudinally investigate different domains of cognition in 20 consecutive MS patients receiving Sativex for spasticity. The primary endpoint was to assess any variation in cognitive performance. Secondary outcomes regarding mood and anxiety were investigated by means of Beck Depression Inventory (BDI) and Hamilton Anxiety Rating Scale (HAM-A). Any change in patients' spasticity was evaluated using the 0-10 Numerical Rating Scale (NRS).Twenty per protocol patients were followed up and evaluated at baseline, 6 and 12 months. Domains involving processing speed and auditory verbal memory significantly improved within the first 6 months of therapy (SDMT: p 0.001; CVLT: p = 0.0001). Mood and anxiety did not show any significant variation. Additionally, the NRS score significantly improved since the beginning (p 0.0001).These results are encouraging in supporting possible long-term benefits of Sativex on cognition and a wider role than symptom alleviator. Further studies on larger groups of patients would be necessary in order to test this intriguing possibility.

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

Epigenetic-sensitive mechanisms may be correlated both to pathogenesis and prognosis of coronary heart disease (CHD). We prospectively investigated some plasma circulating microRNA levels in patients undergoing cardiac computed tomography for suspected CHD (n = 95). We show that let-7c-5p, miR-765, miR-483-5p, miR-31-5p, and miR-206 were upregulated in CHD patients (n = 66) versus healthy subjects HS (n = 29); moreover, let-7c-5p, miR-765, miR- 483-5p showed higher expression in obstructive CHD (n = 36) compared to no obstructive CHD patients (n = 66). Remarkably, miR-765, 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 while circulating levels of miR-765 (p = 0.035), miR-433-3p (p = 0.043), and miR-93-5p (p = 0.041) were significantly higher in critical stenosis patients. Receiver operating characteristic curve analysis revealed a good performance for miR-765, miR-93-5p, and miR-433-3p on predicting CHD severity. In conclusion, our study represents a combined epigenetic/imaging approach useful to support the diagnosis and prediction of CHD.

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

Although venous thromboembolism (VTE) shows a polygenic nature, the crossroad between genome and environment is not fully understood. Genetics explains only a part of VTE hereditability and not defined molecular causes are found in approximately 50% of thrombotic patients. Thus, a major understanding of molecular mechanisms may clarify the missing hereditability. Concerning epigenetics, a particular histone modification (citrullination) plays a key role in increasing the rate of venous occlusive events by inducing neutrophil apoptosis and expulsion of neutrophil extra-cellular traps (NETs), which may be useful biomarkers of active disease. Moreover, an over-expression of miR-320a/b, miR-582, miR-195, miR-424-5p, and miR-532, or a down-regulation of miR495, miR-136-5p and miR-26a may improve the accuracy of VTE diagnosis. No clinical studies have focused on DNA methylation in VTE. Nowadays, no validated epigenetics biomarkers are routinely used for diagnosis and prevention of VTE. In the era of personalized therapy, several clinical trials are investigating the putative role of statins, a class of lipid-lowering epigenetic-based drugs, as additional therapeutic agents in VTE. Furthermore, single nucleotide polymorphisms (SNPs) in CYP2C9, VKORC1, and MIR133 genes can help physicians to predict individual warfarin dose requirement. Consequently, a comprehensive understanding of the mechanisms involved in the control of blood clot development is crucial to design novel therapeutic strategies. This review summarizes the current clinical concepts both in genetic and epigenetic VTE framework. Furthermore, we discuss the contribution of the innovative network medicine paradigm into advancing our knowledge about molecular underpinnings needed to support novel VTE diagnostic and therapeutic options.

Purpose:To compare image quality and radiation dose among different protocols in patients who underwent a 128-slice dual source computed tomography coronary angiography (DSCT-CTCA).Methods:Ninety patients were retrospectively grouped according to heart rate (HR): 26 patients (group A) with stable HR ≤60 bpm were acquired using high pitch spiral mode (FLASH); 48 patients (group B) with irregular HR ≤60 bpm or stable HR between 60 and 70 bpm using step and shoot mode; and 16 patients (group C) with irregular HR >60 bpm or stable HR ≥70 bpm by retrospective electrocardiogram pulsing acquisition. Signal to noise ratio (SNR) and contrast to noise ratio (CNR) were measured for the main vascular structures. Moreover, the dose-length product and the effective dose were assessed.Results:Both SNR and CNR were higher in group A compared to group C (18.27 ± 0.32 vs 11.22 ± 0.50 and 16.75 ± 0.32 vs 10.17 ± 0.50; P = .001). The effective dose was lower in groups A and B (2.09 ± 1.27 mSv and 4.60 ± 2.78 mSv, respectively) compared to group C (9.61 ± 5.95 mSv) P < .0001.Conclusion:The correct selection of a low-dose, HR-matched CTCA scan protocol with a DSCT scanner provides substantial reduction of radiation exposure and better SNR and CNR.

Cardiac Magnetic Resonance (CMR), thanks to high spatial resolution and absence of ionizing radiation, has been widely used in myocardial infarction (MI) assessment to evaluate cardiac structure, function, perfusion and viability. Nevertheless, it suffers from limitations in tissue and assessment of myocardial pathophysiological changes subsequent to MI. In this issue, nanoparticle-based contrast agents offer the possibility to track biological processes at cellular and molecular level underlying the various phases of MI, infarct healing and tissue repair. In this paper, first we examine the conventional CMR protocol and its findings in MI patients. Next, we looked at how nanoparticles can help in the imaging of MI and give an overview of the major approaches currently explored. Based on the presentation of successful nanoparticle applications as contrast agents (CAs) in preclinical and clinical models, we discuss promises and outstanding challenges facing the field of CMR in MI, their translational potential and clinical application.