At its earliest appearance, atrial fibrillation (AF) is often unnoticed, asymptomatic, and/or merely device-detected. Widespread use of heart-rate monitoring technologies has facilitated such “nascent atrial fibrillation (nAF)” recognition. Consequently, clinicians face a growing number of patients affected by new-onset AF in the absence of a definite indication for anticoagulation due to several counterarguments: (1) a CHA2DS2-VA score ≤ 1 in otherwise apparently healthy subjects; (2) an uncertain embolic/hemorrhagic benefit/risk ratio with anticoagulation; (3) EKG demonstration and confirmation of AF; and (4) existence of a pathogenic mechanism other than atrial hypercoagulability. In this frustrating limitation of pharmacological options, cardiologists may miss a complete comprehension of drugs with proven anti-ictal potential, whose administration may serve both as a bridge strategy toward future anticoagulation and as a consolidative strategy paralleling anticoagulation. This review aims to summarize and elucidate such therapeutic strategies and their preventative mechanisms.

Objective: Atherosclerosis is a chronic cardiovascular disease. High plasma concentration of LDL is the main risk factor but the role of inflammation in all the stages become recently evident. Atherosclerosis and lipid metabolism change according to gender differences. Several possible markers of endothelial dysfunction and vascular stiffness have been identified, including clinical-instrumental and biochemical markers of inflammation and/or fibrosis (i.e., TNF-alpha, prostaglandin, L-Arginine/Asymmetric dimethylarginine - ADMA). A common mechanism identified in the pathogenesis of numerous CVD is endothelial dysfunction that correlates with circulating endothelial progenitor cells (EPCs). These cells are recruited from the bone marrow in response to vascular damage or tissue ischemia. In the peripheral blood, EPCs contribute to reendothelialization and neovascularization, acting as positive regulators of hemostasis and vascular integrity. A reduction in their number or function has been correlated to progression of CVD and MACE. Our project aims to assess the role of EPCs and L-ARG/ADMA in arterial stiffness and atherosclerosis. Design and method: Our study is a prospective experimental investigation, enrolling all patients with cardiovascular disease. Patients will undergo routine hematological and biochemical tests as well as instrumental examinations in accordance with good clinical practice. The enrolled patients will undergo a series of clinical and instrumental examinations (ABI,PWV). In in vitro experiments, EPCs will be isolated from PBMCs and functional assays will be performed. Also, Proteome Profiler Target Analytes study and ELISA assay (L-Arg/ADMA) will be performed. Results: There was a direct correlation between SCORE2/2-OP and PWV (r=0.70; p<0.0001). Preliminary data indicated a correlation between L-Arg/ADMA to SCORE2/2OP (r=0.36, p=0.04), Total Cholesterol (r=0.75, p=0.033) and LDL (r=0.75, p=0.031), as well as EPCs and their migration correlates to a change in folate cycle (P<0.007 Conclusions: preliminary data suggests the importance of EPCs as specific read-out of cell activation and tissue damage in response to cardiovascular risk factors.

The pathophysiology of atrial fibrillation (AF) may involve atrial fibrosis/remodeling and dysfunctional endothelial activities. Despite the currently available treatment approaches, the progression of AF, its recurrence rate, and the high mortality risk of related complications underlay the need for more advanced prognostic and therapeutic strategies. There is increasing attention on the molecular mechanisms controlling AF onset and progression points to the complex cell to cell interplay that triggers fibroblasts, immune cells and myofibroblasts, enhancing atrial fibrosis. In this scenario, endothelial cell dysfunction (ED) might play an unexpected but significant role. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level. In the cardiovascular compartment, both free circulating and exosomal miRNAs entail the control of plaque formation, lipid metabolism, inflammation and angiogenesis, cardiomyocyte growth and contractility, and even the maintenance of cardiac rhythm. Abnormal miRNAs levels may indicate the activation state of circulating cells, and thus represent a specific read-out of cardiac tissue changes. Although several unresolved questions still limit their clinical use, the ease of accessibility in biofluids and their prognostic and diagnostic properties make them novel and attractive biomarker candidates in AF. This article summarizes the most recent features of AF associated with miRNAs and relates them to potentially underlying mechanisms.

Multiple myeloma (MM) is a plasma cell (PC) malignancy whose development flourishes in the bone marrow microenvironment (BMME). The BMME components’ immunoediting may foster MM progression by favoring initial immunotolerance and subsequent tumor cell escape from immune surveillance. In this dynamic process, immune effector cells are silenced and become progressively anergic, thus contributing to explaining the mechanisms of drug resistance in unresponsive and relapsed MM patients. Besides traditional treatments, several new strategies seek to re-establish the immunological balance in the BMME, especially in already-treated MM patients, by targeting key components of the immunoediting process. Immune checkpoints, such as CXCR4, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), PD-1, and CTLA-4, have been identified as common immunotolerance steps for immunotherapy. B-cell maturation antigen (BCMA), expressed on MMPCs, is a target for CAR-T cell therapy, antibody-(Ab) drug conjugates (ADCs), and bispecific mAbs. Approved anti-CD38 (daratumumab, isatuximab), anti-VLA4 (natalizumab), and anti-SLAMF7 (elotuzumab) mAbs interfere with immunoediting pathways. New experimental drugs currently being evaluated (CD137 blockers, MSC-derived microvesicle blockers, CSF-1/CSF-1R system blockers, and Th17/IL-17/IL-17R blockers) or already approved (denosumab and bisphosphonates) may help slow down immune escape and disease progression. Thus, the identification of deregulated mechanisms may identify novel immunotherapeutic approaches to improve MM patients’ outcomes.

Abstract Funding Acknowledgements Type of funding sources: Private hospital(s). Main funding source(s): dedicated cardiovascular research foud Background Recent advances support the concept that pre-persistent Atrial Fibrillation (AF) does not explain the wholeness of embolic strokes, suggesting the recently postulated hypothesis of a broad Atrial Cardiomyopathy (AC). In contrast to its worldwide distribution and its very inclusive definition, pathogenic mechanisms underlying AC are still largely unknown. Folate cycle disorders (FCD) are a yet underrated dysmetabolism only partly explained by methylene tetrahydrofolate reductase (MTHFR)-inherited defects. On a translational basis, FCD could hinder both endothelial and circulating endothelial progenitor cell (EPCs) functioning, therefore providing one-shot explanation to both atrial stasis (increasing atrial fibrosis and generating atrial hypocontractility) and endothelial dysfunction (ED). If such cardiac-bone marrow networking would be verified, a fundamental pathogenic mechanism of AC and subsequent AF would be unraveled. Purpose This study aims to enquire for the hypothesis that: 1) atrial fibrosis (AFib) would relate to FCD (intended as both: a)MTHFR C677T inherited mutations and b)bone-marrow function disorders, here referring to erythropoiesis diversions) and 2) AF patients would show dysfunctional EPCs. Methods We studied 59 consecutive patients admitted to the Cardiology Unit of the General Hospital "F.Miulli", with preserved EF, subjected to AF ablation. AFib was quantified by relative % of low-voltage (<0,5 mV) bipolar peak-to-peak points, with respect to the wholeness of the endocavitary mapping. Blood count cell was evaluated. MTHFR C677T genotypes were elucidated by RT-PCR. Folate were measured by a commercial laboratory test. EPCs isolation and characterization were performed by Ficoll-Hypaque gradient and flow cytometry analysis for cell surface antigens: CD45, CD34, CD133, VEGFR2 and KDR. EPCs functional wound healing assay was performed. Results Baseline characteristics did not differ between Sample and Control groups (Fig. 1 – Left Table). % of Afib significantly differs between C677T MTHFR homozigosis patients (n=15) with respect to non-C677T MTHFR homozygosis patients (n=44) (Fig.1 – Right graph. - p < 0,02). Once univariate analysis was performed, subsequent multivariate analysis highlights highest fit once merged RBC, RDW-SD and folates values were inputed: Goodness of fit was proper, modelling good (Fig.2 – superior graph. - R2=0,39; p=0,0001). Either RBC, RDW-SD and folates coefficient reached significance (p < 0,0001; p < 0,01; p < 0,05 respectively). Number of EPCs significantly differs between AF patients and matched controls (Fig 2 – inferior graph. – p < 0,001). Conclusions Our findings support the hypothesis that genetically determined folates dysmetabolism (MTHFR dysfunction) promotes AFib via a complex cardiac-bone marrow networking involving circulating EPCs and unraveled by erythropoiesis diversions. Such results suggest a pathogenic role of folate cycle disorders in the AC development.

A growing number of clinical and epidemiological studies support the hypothesis of a tight correlation between type 2 diabetes mellitus (T2DM) and the development risk of Alzheimer’s disease (AD). Indeed, the proposed definition of Alzheimer’s disease as type 3 diabetes (T3D) underlines the key role played by deranged insulin signaling to accumulation of aggregated amyloid beta (Aβ) peptides in the senile plaques of the brain. Metabolic disturbances such as hyperglycemia, peripheral hyperinsulinemia, dysregulated lipid metabolism, and chronic inflammation associated with T2DM are responsible for an inefficient transport of insulin to the brain, producing a neuronal insulin resistance that triggers an enhanced production and deposition of Aβ and concomitantly contributes to impairment in the micro-tubule-associated protein Tau, leading to neural degeneration and cognitive decline. Furthermore, the reduced antioxidant capacity observed in T2DM patients, together with the impairment of cerebral glucose metabolism and the decreased performance of mitochondrial activity, suggests the existence of a relationship between oxidative damage, mitochondrial impairment, and cognitive dysfunction that could further reinforce the common pathophysiology of T2DM and AD. In this review, we discuss the molecular mechanisms by which insulin-signaling dysregulation in T2DM can contribute to the pathogenesis and progression of AD, deepening the analysis of complex mechanisms involved in reactive oxygen species (ROS) production under oxidative stress and their possible influence in AD and T2DM. In addition, the role of current therapies as tools for prevention or treatment of damage induced by oxidative stress in T2DM and AD will be debated.

We explored the significance of the L-Arginine/asymmetric dimethylarginine (L-Arg/ADMA) ratio as a biomarker of endothelial dysfunction in stroke patients. To this aim, we evaluated the correlation, in terms of severity, between the degree of endothelial dysfunction (by L-Arg/ADMA ratio), the methylene tetrahydrofolate reductase (MTHFR) genotype, and the interatrial septum (IAS) phenotype in subject with a history of stroke. Methods and Results: L-Arg, ADMA, and MTHFR genotypes were evaluated; the IAS phenotype was assessed by transesophageal echocardiography. Patients were grouped according to the severity of IAS defects and the residual enzymatic activity of MTHFR-mutated variants, and values of L-Arg/ADMA ratio were measured in each subgroup. Of 57 patients, 10 had a septum integrum (SI), 38 a patent foramen ovale (PFO), and 9 an ostium secundum (OS). The L-Arg/ADMA ratio differed across septum phenotypes (p ≤ 0.01), and was higher in SI than in PFO or OS patients (p ≤ 0.05, p ≤ 0.01, respectively). In the PFO subgroup a negative correlation was found between the L-Arg/ADMA ratio and PFO tunnel length/height ratio (p ≤ 0.05; r = − 0.37; R2 = 0.14). Interestingly, the L-Arg/ADMA ratio varied across MTHFR genotypes (p ≤ 0.0001) and was lower in subgroups carrying the most impaired enzyme with respect to patients carrying the conservative MTHFR (p ≤ 0.0001, p ≤ 0.05, respectively). Consistently, OS patients carried the most dysfunctional MTHFR genotypes, whereas SI patients the least ones. Conclusions: A low L-Arg/ADMA ratio correlates with impaired activity of MTHFR and with the jeopardized IAS phenotype along a severity spectrum encompassing OS, PFO with long/tight tunnel, PFO with short/large tunnel, and SI. This infers that genetic MTHFR defects may underlie endothelial dysfunction-related IAS abnormalities, and predispose to a cryptogenic stroke. Our findings emphasize the role of the L-Arg/ADMA ratio as a reliable marker of stroke susceptibility in carriers of IAS abnormalities, and suggest its potential use both as a diagnostic tool and as a decision aid for therapy.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder clinically characterized by impairment of cognitive functions and memory loss. The two core neuropathological hallmarks of AD are deposits of beta-amyloid (A beta) fibrils in senile plaques and accumulation of hyperphosphorylated tau protein filaments in neurofibrillary tangles. An increasing number of murine models of AD have been generated. Animal models have been valuable tools for reproducing aspects of the neuropathological characteristics of AD strongly supporting the amyloid cascade hypothesis. However, we need to learn much more about the link between the pathological cascade of AD and the emergence of clinical symptoms to identify the factors which best predict the risk of progression from normal cognition to mild cognitive impairment and AD dementia. The few therapeutic agents currently available, such as galantamine, memantine, rivastigmine and donepezil are useful for symptomatic management and none of them is able to modify the disease progression. Studies with transgenic mouse models and data from clinical trials suggest that A beta-modifying therapies have no or limited effect after neuronal degeneration has begun, and stress the need of validated biomarkers for stratification of patients more responsive to immunotherapeutic agents. It is hoped that further efforts will move toward defining the preclinical stage of AD when intervention with anti-A beta immunotherapy may be most efficacious. The present paper reports a brief overview of AD and some murine models focusing on what we have learned from AD modeling.

Cancer cells express unusual antigens which activate signal transduction pathways resulting in unregulated tumor growth.Hence, an effective immune response can be elicited via antibodies targeting the tumoral neoantigens.In particular, modulation of immunological pathways involved in the adaptive immune response is currently considered an innovative approach to fight against aggressive malignancies.Antigenspecific immune responses require critical interaction between antigen presenting cells (APC), T-lymphocytes, and target cells.Upon T-cell activation, the programmed cell death protein-1 (PD-1) blocks the early TCR/CD28 signalling pathway, thus hindering cytokine production and cell cycle progression.Furthermore, cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibits the activated T-cell by binding with high affinity to the B7 molecules and hampering the CD28-mediated signalling.The characterization of these negative immune regulators led to the successful development of fully human monoclonal antibodies (mAbs), specifically designed to promote T-cell activation and increase the anti-tumor immune response.This article provides a brief overview of immune modulation of T-cell function by anti-CTLA-4 and anti-PD-1 antibodies, known as checkpoint inhibitors, and summarizes clinical benefits and limitations of their use in cancer management.

In both developing and industrialized Countries, the growing prevalence of Type 2 Diabetes Mellitus (T2DM) and the severity of its related complications make T2DM one of the most challenging metabolic diseases worldwide. The close relationship between genetic and environmental factors suggests that eating habits and unhealthy lifestyles may significantly affect metabolic pathways, resulting in dynamic modifications of chromatin-associated proteins and homeostatic transcriptional responses involved in the progression of T2DM. Epigenetic mechanisms may be implicated in the complex processes linking environmental factors to genetic predisposition to metabolic disturbances, leading to obesity and type 2 diabetes mellitus (T2DM). Endothelial dysfunction represents an earlier marker and an important player in the development of this disease. Dysregulation of the endothelial ability to produce and release vasoactive mediators is recognized as the initial feature of impaired vascular activity under obesity and other insulin resistance conditions and undoubtedly concurs to the accelerated progression of atherosclerotic lesions and overall cardiovascular risk in T2DM patients. This review aims to summarize the most current knowledge regarding the involvement of epigenetic changes associated with endothelial dysfunction in T2DM, in order to identify potential targets that might contribute to pursuing "precision medicine" in the context of diabetic illness.

The association of obesity and diabetes, termed "diabesity", defines a combination of primarily metabolic disorders with insulin resistance as the underlying common pathophysiology. Cardiovascular disorders associated with diabesity represent the leading cause of morbidity and mortality in the Western world. This makes diabesity, with its rising impacts on both health and economics, one of the most challenging biomedical and social threats of present century. The emerging comprehension of the genes whose alteration confers inter-individual differences on risk factors for diabetes or obesity, together with the potential role of genetically determined variants on mechanisms controlling responsiveness, effectiveness and safety of anti-diabetic therapy underlines the need of additional knowledge on molecular mechanisms involved in the pathophysiology of diabesity. Endothelial cell dysfunction, resulting from the unbalanced production of endothelial-derived vascular mediators, is known to be present at the earliest stages of insulin resistance and obesity, and may precede the clinical diagnosis of diabetes by several years. Once considered as a mere consequence of metabolic abnormalities, it is now clear that endothelial dysfunctional activity may play a pivotal role in the progression of diabesity. In the vicious circle where vascular defects and metabolic disturbances worsen and reinforce each other, a low-grade, chronic, and 'cold' inflammation (metaflammation) has been suggested to serve as the pathophysiological link that binds endothelial and metabolic dysfunctions. In this paradigm, it is important to consider how traditional antidiabetic treatments (specifically addressing metabolic dysregulation) may directly impact on inflammatory processes or cardiovascular function. Indeed, not all drugs currently available to treat diabetes possess the same anti-inflammatory potential, or target endothelial cell function equally. Perspective strategies pointing at reducing metaflammation or directly addressing endothelial dysfunction may disclose beneficial consequences on metabolic regulation. This review focuses on existing and potential new approaches ameliorating endothelial dysfunction and vascular inflammation in the context of diabesity.

Background: Green tea catechins seem to contribute toward reducing body weight and fat. Objective: We aimed to investigate whether chronic administration of (–)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, reduces weight gain in spontaneously hypertensive rats (SHR), an animal model of metabolic syndrome, by increasing motor activity and/or by altering gastrointestinal motility. Design: Nine-week-old SHR were randomly assigned to two groups and treated by gavage for 3 weeks with vehicle dimethyl sulfoxide or EGCG (200 mg/kg/day). Age-matched Wistar-Kyoto (WKY) control rats were treated with vehicle alone. The effect of chronic administration of EGCG was evaluated on open-field motor activity and on ex vivo colonic and duodenal motility. Moreover, in vitro acute effect of 20-min incubation with EGCG (100 µM) or vehicle was evaluated in colonic and duodenal specimens from untreated WKY rats and SHR. Results: Vehicle-treated SHR were normoglycemic and hyperinsulinemic, and showed a reduction of plasma adiponectin when compared to vehicle-treated WKY rats. In addition, consistent with fasting glucose and insulin values, vehicle-treated SHR were more insulin resistant than age-matched vehicle-treated WKY rats. Chronic treatment for 3 weeks with EGCG improved insulin sensitivity, raised plasma adiponectin levels, and reduced food intake and weight gain in SHR. Vehicle-treated SHR showed increased open-field motor activity (both crossings and rearings) when tested after each week of treatment. The overall hyperactivity of vehicle-treated SHR was significantly reduced to the levels of vehicle-treated WKY rats after 2 and 3 weeks of EGCG treatment. Colonic and duodenal preparations obtained from SHR chronically treated in vivo with EGCG showed reduced responses to carbachol (0.05–5 µM) and increased inhibitory response to electrical field stimulation (EFS, 1–10 Hz, 13 V, 1 msec, 10-sec train duration), respectively. In vitro acute EGCG incubation (100 µM, 20 min) of colonic and duodenum strips obtained from untreated SHR and WKY rats showed a reduced contractile colonic response to a fixed dose of carbachol (1.5 µM) only in SHR with respect to its own vehicle, whereas the inhibitory duodenal response to a fixed EFS frequency (5 Hz) was significantly reduced in both WKY rats and SHR groups with respect to their own vehicle. Conclusions: These data suggest that EGCG affects body weight gain in rats and this effect seems to be due to the altered intestinal motility and not to increased motor activity.

Increased TNFα-mediated JNK signaling in the perivascular adipose tissue (PVAT) may contribute to the pathogenesis of vascular complications in T1DM by reducing adiponectin (Ad) synthesis and therefore impairing Ad-mediated activity in the contiguous blood vessel system. We evaluated whether in vivo treatment with the TNFα blocking antibody infliximab normalized expression of Ad and Ad receptors in various fat depots, and whether this effect correlated with improved endothelial activity and vasodilator function in streptozotocin (STZ)-induced diabetic mice. STZ mice were studied at 1 and 2weeks after diabetes onset, and compared to age-matched infliximab-treated diabetic (I-STZ) and control animals (CTRL) (n=10 each group). In STZ mice, activation of pro-inflammatory JNK signaling was faster in PVAT (P<0.01) than in visceral (VAT), epididymal (EAT) and subcutaneous (SAT) adipose depots, and associated with decreased Ad synthesis and dysregulated AdipoR1/R2 levels. In parallel, activation of JNK in aortic endothelial cells and mesenteric arteries was associated with decreased expression/phosphorylation of eNOS and impaired ACh-mediated vasodilation (P<0.05 vs. CTRL). Treatment with infliximab abrogated JNK activation, ameliorated Ad protein expression, and normalized expression of both AdipoR1 and AdipoR2 in PVAT, concomitantly improving eNOS expression and vessel relaxation in mesenteric arteries from I-STZ mice (P<0.01 vs. STZ). These observations underline the early susceptibility of PVAT to activation of pro-inflammatory JNK signaling, and highlight its potential importance in early vascular changes of T1DM. Further elucidation of the role of PVAT in cardiovascular complications may allow for the design of novel therapeutic strategies directly addressing PVAT pathophysiology.

AIM:To investigate the mechanisms underlying the potential contribution of the heme oxygenase/carbon monoxide (HO/CO) pathway in the constipating effects of granisetron.METHODS:For in vivo studies, gastrointestinal motility was evaluated in male rats acutely treated with granisetron [25, 50, 75 μg/kg/subcutaneous (sc)], zinc protoporphyrin IX [ZnPPIX, 50 μg/kg/intraperitoneal (ip)] and hemin (50 μmol/L/kg/ip), alone or in combination. For in vitro studies, the contractile neurogenic response to electrical field stimulation (EFS, 3, 5, 10 Hz, 14 V, 1 ms, pulse trains lasting 10 s), as well as the contractile myogenic response to acetylcholine (ACh, 0.1-100 μmol/L) were evaluated on colon specimens incubated with granisetron (3 μmol/L, 15 min), ZnPPIX (10 μmol/L, 60 min) or CO-releasing molecule-3 (CORM-3, 100, 200, 400 μmol/L) alone or in combination. These experiments were performed under co-treatment with or without atropine (3 μmol/L, a muscarinic receptor antagonist) or NG-nitro-L-Arginine (L-NNA, 100 μmol/L, a nitric oxide synthase inhibitor).RESULTS:Administration of granisetron (50, 75 μg/kg) in vivo significantly increased the time to first defecation (P = 0.045 vs vehicle-treated rats), clearly suggesting a constipating effect of this drug. Although administration of ZnPPIX or hemin alone had no effect on this gastrointestinal motility parameter, ZnPPIX co-administered with granisetron abolished the granisetron-induced constipation. On the other hand, co-administration of hemin and granisetron did not modify the increased constipation observed under granisetron alone. When administered in vitro, granisetron alone (3 μmol/L) did not significantly modify the colon's contractile response to either EFS or ACh. Incubation with ZnPPIX alone (10 μmol/L) significantly reduced the colon's contractile response to EFS (P = 0.016) but had no effect on contractile response to ACh. Co-administration of ZnPPIX and atropine (3 μmol/L) abolished the ZnPPIX-mediated decrease in contractile response to EFS. Conversely, incubation with CORM-3 (400 μmol/L) alone increased both the contractile response to EFS at 10 Hz (10 Hz: 71.02 ± 19.16 vs 116.25 ± 53.70, P = 0.01) and the contractile response to ACh (100 μmol/L) (P = 0.012). Co-administration of atropine abolished the CORM-3-mediated effects on the EFS-mediated response. When granisetron was co-incubated in vitro with ZnPPIX, the ZnPPIX-mediated decrease in colon contractile response to EFS was lost. On the other hand, co-incubation of granisetron and CORM-3 (400 μmol/L) further increased the colon's contractile response to EFS (at 5 Hz: P = 0.007; at 10 Hz: P = 0.001) and to ACh (ACh 10 μmol/L: P = 0.001; ACh 100 μmol/L: P = 0.001) elicited by CORM-3 alone. L-NNA co-administered with granisetron and CORM-3 abolished the potentiating effect of CORM-3 on granisetron on both the EFS-induced and ACh-induced contractile response.CONCLUSION:Taken together, findings from in vivo and in vitro studies suggest that the HO/CO pathway is involved in the constipating effects of granisetron.