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Acute heart failure (AHF) affects millions of people worldwide, and it is a potentially life-threatening condition for which the cardiologist is more often brought into play. It is crucial to rapidly identify, among patients presenting with dyspnoea, those with AHF and to accurately stratify their risk, in order to define the appropriate setting of care, especially nowadays due to the coronavirus disease 2019 (COVID-19) outbreak. Furthermore, with physical examination being limited by personal protective equipment, the use of new alternative diagnostic and prognostic tools could be of extreme importance. In this regard, usage of biomarkers, especially when combined (a multimarker approach) is beneficial for establishment of an accurate diagnosis, risk stratification and post-discharge monitoring. This review highlights the use of both traditional biomarkers such as natriuretic peptides (NP) and troponin, and emerging biomarkers such as soluble suppression of tumourigenicity (sST2) and galectin-3 (Gal-3), from patients' emergency admission to discharge and follow-up, to improve risk stratification and outcomes in terms of mortality and rehospitalization., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

In pre-hospital care, an accurate and quick diagnosis of ST-segment elevation myocardial infarction (STEMI) is imperative to promptly kick-off the STEMI network with a direct transfer to the cardiac catheterization laboratory (cath lab) in order to reduce myocardial infarction size and mortality. Aa atherosclerotic plaque rupture is the main mechanism responsible for STEMI. However, in a small percentage of patients, emergency coronarography does not reveal any significant coronary stenosis. The fluoropyrimidine agents such as 5-Fluorouracil (5-FU) and capecitabine, widely used to treat gastrointestinal, breast, head and neck cancers, either as a single agent or in combination with other chemotherapies, can cause potentially lethal cardiac side effects. Here, we present the case of a patient with 5-FU cardiotoxicity resulting in an acute coronary syndrome (ACS) with recurrent episodes of chest pain and ST-segment elevation.. Our case report highlights the importance of widening the knowledge among cardiologists of the side effects of chemotherapeutic drugs, especially considering the rising number of cancer patients around the world and that fluoropyrimidines are the main treatment for many types of cancer, both in adjuvant and advanced settings.

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.

Heart failure (HF) is one of the major causes of morbidity and mortality worldwide and represents an escalating problem for healthcare systems. The identification of asymptomatic patients with underlying cardiac subclinical disease would create an opportunity for early intervention and prevention of symptomatic HF. Traditional biomarkers are very useful as diagnostic and prognostic tools in the cardiovascular field; however, their application is usually limited to overt cardiac disease. On the other hand, a growing number of studies is investigating the diagnostic and prognostic potential of new biomarkers, such as micro-RNAs (miRNA), long non-coding RNAs, and exosome cargo, because of their involvement in the early phases of cardiac dysfunction. Unfortunately, their use in asymptomatic phases remains a distant goal. The aim of this review is to gather the current knowledge of old and novel biomarkers in the early diagnosis of cardiac dysfunction in asymptomatic individuals.

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of Coronavirus Disease-2019 (COVID-19) in humans. ACE-2 is a type I transmembrane metallocarboxypeptidase expressed in vascular endothelial cells, alveolar type 2 lung epithelial cells, renal tubular epithelium, Leydig cells in testes and gastrointestinal tract. ACE2 mediates the interaction between host cells and SARS-CoV-2 spike (S) protein. However, ACE2 is not only a SARS-CoV-2 receptor, but it has also an important homeostatic function regulating renin-angiotensin system (RAS), which is pivotal for both the cardiovascular and immune systems. Therefore, ACE2 is the key link between SARS-CoV-2 infection, cardiovascular diseases (CVDs) and immune response. Susceptibility to SARS-CoV-2 seems to be tightly associated with ACE2 availability, which in turn is determined by genetics, age, gender and comorbidities. Severe COVID-19 is due to an uncontrolled and excessive immune response, which leads to acute respiratory distress syndrome (ARDS) and multi-organ failure. In spite of a lower ACE2 expression on cells surface, patients with CVDs have a higher COVID-19 mortality rate, which is likely driven by the imbalance between ADAM metallopeptidase domain 17 (ADAM17) protein (which is required for cleavage of ACE-2 ectodomain resulting in increased ACE2 shedding), and TMPRSS2 (which is required for spike glycoprotein priming). To date, ACE inhibitors and Angiotensin II Receptor Blockers (ARBs) treatment interruption in patients with chronic comorbidities appears unjustified. The rollout of COVID-19 vaccines provides opportunities to study the effects of different COVID-19 vaccines on ACE2 in patients on treatment with ACEi/ARB.

The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically., (Copyright © 2020. Published by Elsevier Ltd.)

Ischemic heart disease (IHD) is among the leading causes of death in developed countries. Its pathological origin is traced back to coronary atherosclerosis, a lipid-driven immuno-inflammatory disease of the arteries that leads to multifocal plaque development. The primary clinical manifestation of IHD is acute myocardial infarction (AMI),) whose prognosis is ameliorated with optimal timing of revascularization. Paradoxically, myocardium re-perfusion can be detrimental because of ischemia-reperfusion injury (IRI), an oxidative-driven process that damages other organs. Amyloid-β (Aβ) plays a physiological role in the central nervous system (CNS). Alterations in its synthesis, concentration and clearance have been connected to several pathologies, such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Aβ has been suggested to play a role in the pathogenesis of IHD and cerebral IRI. The purpose of this review is to summarize what is known about the pathological role of Aβ in the CNS; starting from this evidence, we will illustrate the role played by Aβ in the development of coronary atherosclerosis and its possible implications in the pathophysiology of IHD and myocardial IRI. Better elucidation of Aβ's contribution to the molecular pathways underlying IHD and IRI could be of great help in developing new therapeutic strategies.

(1)Background: Chronic heart failure (CHF) contributes to the overall burden of cardiovascular disease. Early identification of at-risk individuals may facilitate the targeting of precision therapies. Plasma microRNAs are promising circulating biomarkers for their implications with cardiac pathologies. In this pilot study, we investigate the possible exploitability of circulating micro-RNAs (miRNAs) to track chronic heart failure (CHF) occurrence, and progression from NYHA class I to IV. (2)Methods: We screened 367 microRNAs using TaqMan microRNA Arrays in plasma samples from healthy controls (HC) and CHF NYHA-class I-to-IV patients (5/group). Validation was performed by singleplex assays on 10 HC and 61 CHF subjects. Differences in the expression of validated microRNAs were evaluated through analysis of covariance (ANCOVA). Associations between N-terminal pro-BNP (NT-proBNP), left ventricular end-diastolic volume (LVEDV) or peak oxygen uptake (VO2 peak) and plasma microRNA were assessed by multivariable linear regression analysis. (3)Results: Twelve microRNAs showed higher expression in CHF patients vs. HC. Seven microRNAs were associated with NT-proBNP concentration; of these, miR-423-5p was also an independent predictor of LVEDV. Moreover, miR-499-5p was a predictor of the VO2 peak. Finally, a cluster of 5 miRNAs discriminated New York Heart Association (NYHA) class-I from HC subjects. (4)Conclusions: Our data suggest that circulating miRNAs have the potential to serve as pathophysiology-based markers of HF status and progression, and as indicators of pre-symptomatic individuals.

Low-grade gliomas (LGG) are infiltrative primary brain tumors that in 70% of the cases undergo anaplastic transformation, deeply affecting prognosis. However, the timing of progression is heterogeneous. Recently, the tumor microenvironment (TME) has gained much attention either as prognostic factor or therapeutic target. Through the release of extracellular vesicles, the TME contributes to tumor progression by transferring bioactive molecules such as microRNA. The aim of the study was to take advantage of glioma-associated stem cells (GASC), an in vitro model of the glioma microenvironment endowed with a prognostic significance, and their released exosomes, to investigate the possible role of exosome miRNAs in favoring the anaplastic transformation of LGG. Therefore, by deep sequencing, we analyzed and compared the miRNA profile of GASC and exosomes obtained from LGG patients characterized by different prognosis. Results showed that exosomes presented a different signature, when compared to their cellular counterpart and that, although sharing several miRNAs, exosomes of patients with a bad prognosis, selectively expressed some miRNAs possibly responsible for the more aggressive phenotype. These findings get insights into the value of TME and exosomes as potential biomarkers for precision medicine approaches aimed at improving LGG prognostic stratification and therapeutic strategies.

Background: The aim of this study was to match clinical outcomes of heart transplantation (HTx) against histopathologic and ultrastructural characteristics of marginal grafts preserved by cold storage (CS) or ex vivo normothermic perfusion., Methods: Since 2011, 100 patients had undergone HTx at our institution by using marginal donors (aged ≥55 years, expected ischemic time of >4 hours, left ventricular ejection fraction of ≤50%, interventricular septum thickness of ≥14 mm, drug abuse history, episodes of cardiac arrest, and presence of mild coronary artery disease). CS was utilized in 79 cases (Group 1, 79%), and ex vivo perfusion was utilized in 21 (Group 2, 21%). Pre-operative data, survival, and complications in the first 5 years after HTx were analyzed. Myocardial biopsies were collected at graft harvesting, just before implantation, and immediately after aortic declamping., Results: Pre-operative demographics were similar in the 2 groups. Graft utilization rate with ex vivo perfusion was 81%. Ischemic, cardiopulmonary bypass, and surgical times were shorter in Group 2 patients, who showed a lower incidence of overall complications (33% vs 13%, p = 0.04) and better 5-year survival (log-rank, p = 0.04). Moreover, restoration of hypertrophy-related sarcomere changes and mitigation of reperfusion-dependent myocardium injuries were more frequently observed in Group 2 hearts., Conclusions: Ex vivo perfusion allows for continuous evaluation of marginal donor hearts, favoring exclusion of unsuitable grafts, reduction of complications, and optimal survival of up to 5 years. Such results, supported by consistent histopathologic and ultrastructural findings, suggest better myocardial preservation., (Copyright © 2020 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Renin-angiotensin system (RAS) blockers are extensively used worldwide to treat many cardiovascular disorders, where they are effective in reducing both mortality and morbidity. These drugs are known to induce an increased expression of angiotensin-converting enzyme 2 (ACE2). ACE2 acts as receptor for the novel SARS coronavirus-2 (SARS-CoV-2) which raising the important issue of possible detrimental effects that RAS blockers could exert on the natural history and pathogenesis of the coronavirus disease-19 (COVID-19) and associated excessive inflammation, myocarditis and cardiac arrhythmias. We review the current knowledge on the interaction between SARS-CoV-2 infection and RAS blockers and suggest a scientific rationale for continuing RAS blockers therapy in patients with COVID-19 infection., (© 2020 The Association for the Publication of the Journal of Internal Medicine.)

Aims: Homozygosity for a four-missense single-nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this longevity-associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy., Methods and Results: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild-type-BPIFB4 exerted no benefit. Interestingly, LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and up-regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 up-regulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor., Conclusions: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease., (© 2020 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Survivors after a myocardial infarction (MI), especially those with diabetes mellitus (DM),remain at high risk of further events. Identifying and treating factors that may influence survivalmay open new therapeutic strategies. We assessed the impact on prognosis of DM andhypovitaminosis D (hypovitD), alone or combined. In this prospective, observational study, 1081patients were enrolled surviving an MI and divided into four groups according to their diabetic andVitD status. The primary end-point was composite of all-cause mortality, angina/MI and heartfailure (HF). Secondary outcomes were mortality, HF and angina/MI. During a follow-up of 26.1months (IQR 6.6-64.5), 391 subjects experienced the primary end-point. Patients with DM orhypovitD had similar rate of the composite end-point. Patients with only hypovitD or DM did notdiffer regarding components of composite end-point (angina p = 0.97, HF p = 0.29, mortality p = 0.62).DM and VitD deficiency had similarly adjusted risks for primary end-point (HR 1.3, 95%CI 1.05-1.61; HR 1.3, 95% CI 1.04-1.64). The adjusted HR for primary composite end-point for patients withhypovitD and DM was 1.69 (95%CI 1.25-2.29, p = 0.001) in comparison to patients with neitherhypoD nor DM. In conclusion, DM and hypovitD, individually and synergistically, are associatedwith a worse outcome after MI.

High neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte ratio (MLR) are respectively associated with systemic inflammation and immune suppression and have been associated with a poor outcome. Plasmatic exosomes are extracellular vesicles involved in the intercellular communication system that can exert an immunosuppressive function. Aim of this study was to investigate the interplay between the immune system and circulating exosomes in metastatic breast cancer (MBC). A threshold capable to classify patients according to MLR, NLR and PLR, was computed through a receiving operator curve analysis after propensity score matching with a series of female blood donors. Exosomes were isolated from plasma by ExoQuick solution and characterized by flow-cytometry. NLR, MLR, PLR and exosomal subpopulations potentially involved in the pre-metastatic niche were significantly different in MBC patients with respect to controls. MLR was significantly associated with number of sites at the onset of metastatic disease, while high levels of MLR and NLR were found to be associated with poor prognosis. Furthermore, exosomal subpopulations varied according to NLR, MLR, PLR and both were associated with different breast cancer subtypes and sites of distant involvement. This study highlights the nuanced role of immunity in MBC spread, progression and outcome. Moreover, they suggest potential interaction mechanisms between immunity, MBC and the metastatic niche.

Background: Incidentally discovered diffuse low-grade gliomas (iLGG) are poorly documented in the literature. They are diagnosed by chance during radiological examinations., Objective: To review a cohort of patients with iLGG surgically treated in our institution, analyzing clinical, molecular, and surgical aspects., Methods: Clinical, radiological, and treatment data of iLGG were retrieved and compared with those of symptomatic diffuse LGGs (sLGG). Histological and molecular review was carried out as well. The extent of resection was evaluated on preoperative and postoperative T2-weighted magnetic resonance imaging., Results: Thirty-four iLGG cases were identified within a monoinstitutional cohort of 332 patients operated for low-grade gliomas from 2000 to 2017. Clinically, patients with iLGG had higher preoperative karnofsky performance scale (KPS) (P = .003), smaller tumor volume (P = .0001), lower frequency of eloquent areas involvement (P = .0001), and higher rate of complete resection (P = .0001) compared to those with sLGG. No differences in the molecular profile and O6-methylguanine-DNA-methyltransferase promoter methylation were detected between iLGG and sLGG. Importantly, patients with iLGG had longer overall survival than those with sLGG (P = .0001), even when a complete surgical resection was achieved (P = .001)., Conclusion: Although the therapeutic strategy of iLGG is still a matter of debate, our data support the safety and the effectiveness of early surgical resection. The favorable prognosis of iLGG may be due to the higher practicability of extensive resection, noneloquent tumor location, and smaller tumor volume., (Copyright © 2019 by the Congress of Neurological Surgeons.)

Although the lack of a robust cardiomyocyte proliferative response has been considered to be a crucial determinant of cardiac pathology and Heart Failure in adult mammalians, the emerging picture is that myocardial regeneration is a complex phenotype involving many actors, including acute cellular senescence and inflammation. However, three major and interconnected events occur in response to tissue injury: loss of protein homeostasis, accumulation of dysfunctional mitochondria and chronic inflammation. These events blunt the reparative response of the heart, are associated with the accumulation of chronically senescent cells and progressively lead to cardiac dysfunction. Therefore, it is crucial to understand which are the pivotal players of this process, in order to devise strategies aimed at reducing the occurrence of chronic cell senescence in the heart in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

(1) Background: Recently, it has been shown that the extent of resection (EOR) and molecular classification of low-grade gliomas (LGGs) are endowed with prognostic significance. However, a prognostic stratification of patients able to give specific weight to the single parameters able to predict prognosis is still missing. Here, we adopt classic statistics and an artificial intelligence algorithm to define a multiparametric prognostic stratification of grade II glioma patients. (2) Methods: 241 adults who underwent surgery for a supratentorial LGG were included. Clinical, neuroradiological, surgical, histopathological and molecular data were assessed for their ability to predict overall survival (OS), progression-free survival (PFS), and malignant progression-free survival (MPFS). Finally, a decision-tree algorithm was employed to stratify patients. (3) Results: Classic statistics confirmed EOR, pre-operative- and post-operative tumor volumes, Ki67, and the molecular classification as independent predictors of OS, PFS, and MPFS. The decision tree approach provided an algorithm capable of identifying prognostic factors and defining both the cut-off levels and the hierarchy to be used in order to delineate specific prognostic classes with high positive predictive value. Key results were the superior role of EOR on that of molecular class, the importance of second surgery, and the role of different prognostic factors within the three molecular classes. (4) Conclusions: This study proposes a stratification of LGG patients based on the different combinations of clinical, molecular, and imaging data, adopting a supervised non-parametric learning method. If validated in independent case studies, the clinical utility of this innovative stratification approach might be proved.

Background: The clinical outcome of patients affected by dilated cardiomyopathy (DCM) is heterogeneous, since its pathophysiology is only partially understood. Interleukin 1β levels could predict the mortality and necessity of cardiac transplantation of DCM patients., Objective: To investigate mechanisms triggering sterile inflammation in dilated cardiomyopathy (DCM)., Methods: Hearts explanted from 62 DCM patients were compared with 30 controls, employing immunohistochemistry, cellular and molecular biology, as well as metabolomics studies., Results: Although misfolded protein accumulation and aggresome formation characterize DCM hearts, aggresomes failed to trigger the autophagy lysosomal pathway (ALP), with consequent accumulation of both p62 SQSTM1 and dysfunctional mitochondria. In line, DCM hearts are characterized by accumulation of lipoperoxidation products and activation of both redox responsive pathways and inflammasome. Consistently with the fact that mTOR signaling may impair ALP, we observed, an increase in DCM activation, together with a reduction in the nuclear localization of Transcription Factor EB -TFEB- (a master regulator of lysosomal biogenesis). These alterations were coupled with metabolomic alterations, including accumulation of branched chain amino acids (BCAAs), known mTOR activators. Consistently, reduced levels of PP2Cm, a phosphatase that regulates the key catabolic step of BCAAs, coupled with increased levels of miR-22, a regulator of PP2Cm levels that triggers senescence, characterize DCM hearts. The same molecular defects were present in clinically relevant cells isolated from DCM hearts, but they could be reverted by downregulating miR-22., Conclusion: We identified, in human DCM, a complex series of events whose key players are miR-22, PP2Cm, BCAA, mTOR, and ALP, linking loss of proteostasis with inflammasome activation. These potential therapeutic targets deserve to be further investigated.

Introduction: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS., Methods: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice., Findings: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis., Interpretation: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. FUND: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: "Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities". M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Ghrelin may exert positive effects on cardiac structure and function in heart failure (HF) patients., Methods: We assessed ghrelin levels in 266 dilated cardiomyopathy (DCM) patients and in 200 age, gender and body mass index (BMI) matched controls. Further, we evaluated the expression of ghrelin and growth hormone secretagogue-receptor (GHSR) in the myocardium of 41 DCM patients and in 11 controls., Results: DCM patients had significantly lower levels of total, acylated and unacylated ghrelin when compared to controls ( p < 0.05 for all). In controls, we observed a negative correlation of ghrelin with age, male gender and BMI. These correlations were lost in the DCM group, except for male gender. Total ghrelin was higher in patients with more recent diagnosis when compared to patients with longer duration of the DCM ( p = 0.033). Further, total ghrelin was higher in patients with lower left ventricular systolic function (<40% LVEF, vs. 40% ≤ LVEF < 49% vs. LVEF ≥ 50%: 480.8, vs. 429.7, vs. 329.5 pg/mL, respectively, p = 0.05). Ghrelin prepropeptide was expressed more in DCM patients than in controls (p = 0.0293) while GHSR was expressed less in DCM patients (p < 0.001). Furthermore, ghrelin showed an inverse correlation with its receptor (= -0.406, p = 0.009), and this receptor showed a significant inverse correlation with Interleukin-1 (= -0.422, p = 0.0103)., Conclusion: DCM duration and severity are accompanied by alterations in the ghrelin-GHSR system., Competing Interests: The authors declare no conflict of interest.

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.

Exosomes are one of the most important mediators of the cross talk occurring between glioma stem cells (GSCs) and the surrounding microenvironment. We have previously shown that exosomes released by patient-derived glioma-associated stem cells (GASC) are able to increase, in vitro, the aggressiveness of both GSC and glioblastoma cell lines. To understand which molecules are responsible for this tumour-supporting function, we performed a descriptive proteomic analysis of GASC-exosomes and identified, among the others, Semaphorin7A (SEMA7A). SEMA7A was described as a promigratory cue in physiological and pathological conditions, and we hypothesised that it could modulate GSC migratory properties. Here, we described that SEMA7A is exposed on GASC-exosomes' surface and signals to GSC through Integrin β1. This interaction activates focal adhesion kinase into GSC and increases their motility, in our patient-based in vitro model. Our findings suggest SEMA7A-β1-integrin as a new target to disrupt the communication between GSCs and the supporting microenvironment., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Background: Acute myocardial infarction (AMI) survivors are at risk of major adverse cardiac events and their risk stratification is a prerequisite to tailored therapeutic approaches. Biomarkers could be of great utility in this setting., Methods: We sought to evaluate the utility of the combined assessment of Galectin 3 (Gal-3) and Galectin 3 binding protein (Gal-3bp) for post-AMI risk stratification in a large, consecutive population of AMI patients. The primary outcomes were: Recurrent angina/AMI and all-cause mortality at 12 months after the index event., Results: In total, 469 patients were included. The median Gal-3bp was 9.1 μg/mL (IQR 5.8-13.5 μg/mL), while median Gal-3 was 9.8 ng/mL (IQR 7.8-12.8 ng/mL). During the 12 month follow-up, 34 patients died and 41 had angina pectoris/reinfarction. Gal-3 was associated with all-cause mortality, while Gal-3bp correlated with the risk of angina/myocardial infarction even when corrected for other significant covariates. The final multivariable model for mortality prediction included patients' age, left ventricular ejection fraction (LVEF), Gal-3, and renal function. The ROC curve estimated for this model has an area under the curve (AUC) of 0.84 (95%CI 0.78-0.9), which was similar to the area under the ROC curve obtained using the GRACE score 1-year mortality., Conclusions: The integrated assessment of Gal-3 and Gal-3bp could be helpful in risk stratification after AMI.

Soluble ST2 (sST2) has recently emerged as a promising biomarker in the field of acute cardiovascular diseases. Several clinical studies have demonstrated a significant link between sST2 values and patients' outcome. Further, it has been found that higher levels of sST2 are associated with an increased risk of adverse left ventricular remodeling. Therefore, sST2 could represent a useful tool that could help the risk stratification and diagnostic and therapeutic work-up of patients admitted to an emergency department. With this review, based on recent literature, we have built sST2-assisted flowcharts applicable to three very common clinical scenarios of the emergency department: Acute heart failure, type 1, and type 2 acute myocardial infarction. In particular, we combined sST2 levels together with clinical and instrumental evaluation in order to offer a practical tool for emergency medicine physicians.

Aging is by far the dominant risk factor for the development of cardiovascular diseases, whose prevalence dramatically increases with increasing age reaching epidemic proportions. In the elderly, pathologic cellular and molecular changes in cardiac tissue homeostasis and response to injury result in progressive deteriorations in the structure and function of the heart. Although the phenotypes of cardiac aging have been the subject of intense study, the recent discovery that cardiac homeostasis during mammalian lifespan is maintained and regulated by regenerative events associated with endogenous cardiac stem cell (CSC) activation has produced a crucial reconsideration of the biology of the adult and aged mammalian myocardium. The classical notion of the adult heart as a static organ, in terms of cell turnover and renewal, has now been replaced by a dynamic model in which cardiac cells continuously die and are then replaced by CSC progeny differentiation. However, CSCs are not immortal. They undergo cellular senescence characterized by increased ROS production and oxidative stress and loss of telomere/telomerase integrity in response to a variety of physiological and pathological demands with aging. Nevertheless, the old myocardium preserves an endogenous functionally competent CSC cohort which appears to be resistant to the senescent phenotype occurring with aging. The latter envisions the phenomenon of CSC ageing as a result of a stochastic and therefore reversible cell autonomous process. However, CSC aging could be a programmed cell cycle-dependent process, which affects all or most of the endogenous CSC population. The latter would infer that the loss of CSC regenerative capacity with aging is an inevitable phenomenon that cannot be rescued by stimulating their growth, which would only speed their progressive exhaustion. The resolution of these two biological views will be crucial to design and develop effective CSC-based interventions to counteract cardiac aging not only improving health span of the elderly but also extending lifespan by delaying cardiovascular disease-related deaths.

Extracellular vesicles (EV) are at the center of an intense activity of investigation, both for their possible employment as biomarkers of ongoing pathologic processes and for their broad range of biological activities. EV can promote tissue repair in very different pathologic settings, including hindlimb and myocardial ischemia. Importantly, the exact mode of action of EV is still partly understood, since they may act by modulating growth factors and cytokines, signaling pathways, and by transferring non-coding RNAs to target cells. However, the term EV identifies cell derived, enveloped particles very heterogeneous in size, composition, and biogenesis. Therefore, part of the controversies on the biological effects exerted by EV is a consequence of differences in methods of separation that result in the enrichment of different entities. Since technical challenges still hamper the highly specific sorting of different EV subpopulations, up to now only few investigators have tried to verify differences in the biological effects of specific EV subtypes. This review summarizes the current state of the art on the comprehension of mechanisms involved in EV biogenesis and release, which is a prerequisite for understanding and investigating the impact that pathology and drug therapy may exert on the secretion and composition of EV. Finally, we described both the mechanism involved in the modulation of EV secretion by drugs commonly used in patients affected by heart failure, and how pathophysiological mechanisms involved in heart disease modify EV secretion.

Background: While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment., Methods: By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs., Results: The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs., Conclusion: This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Purpose of Review: The purpose of this study was to retrospectively evaluate the use of carmustine wafers (CWs) in the management of high-grade gliomas (HGGs). The data from our monoinstitutional series was compared with studies reported in the literature. Special emphasis was placed on the evaluation of side effects and the analysis of extent of resection and molecular profile as risk factors., Recent Findings: The implantation of CWs into the resection cavity during HGG treatment to deliver localized chemotherapy, followed by the Stupp protocol, remains debated in a clinical setting, largely due to the lack of appropriate phase III studies. Given the high expense and poorly characterized side effects associated with CW treatment, identification of patients most likely to benefit from this therapy could be clinically relevant. CWs may represent an effective and safe first-line treatment for patients with HGG that exhibit complete tumor resection and harboring a methylated MGMT promoter. Our investigation showed a much larger group of patients exhibiting long-term survival (> = 36 months), strongly supporting a potential survival benefit conferred via CW treatment. The pre-surgical definition of the MGMT promoter status could be of clinical use in identifying "good responders" to CW implantation.

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results.

A side effect of the medical improvements of the last centuries is the progressive aging of the world population, which is estimated to reach the impressive number of 2 billion people with more than 65 years by 2050. As a consequence, age-related diseases, such as heart failure, will affect more and more patients in the next years. To understand the biological bases of these diseases will be a crucial task in order to find better treatments, and possibly slow age-related morbidity and mortality. Cardiac stem cells have been at the center of a heated debate and their potential involvement in cardiac homeostasis has been questioned. In this review, we summarize evidence obtained by independent groups, on different animal models and humans, that strongly support the important role played by immature, cardiac resident cells in the cardioprotection against heart failure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clinical data and basic research indicate that the structural and functional alterations that characterize the evolution of cardiac disease towards heart failure may be, at least in part, reversed. This paradigm shift is due to the accumulation of evidence indicating that, in peculiar settings, cardiomyocytes may be replenished. Moving from the consideration that cardiomyocytes are rapidly withdrawn from the cell cycle after birth, independent laboratories have tested the hypothesis that cardiac resident stem/progenitor cells resided in mammalian hearts and were important for myocardial repair. After almost two decades of intensive investigation, several (but partially overlapping) cardiac resident stem/progenitor cell populations have been identified. These primitive cells are characterized by mesenchymal features, unique properties that distinguish them from mesodermal progenitors residing in other tissues, and heterogeneous embryological origins (that include the neural crest and the epicardium). A further layer of complexity is related to the nature, in vivo localization and properties of mesodermal progenitors residing in adult tissues. Intriguingly, these latter, whose possible perivascular pericyte/mural cell origin has been shown, have been identified in human hearts too. However, their exact anatomical localization, pathophysiological role, and their relationship with cardiac stem/progenitor cells are emerging only recently. Therefore, aim of this review is to discuss the different origin, the distinct nature, and the complementary effect of cardiac stem cells and pericytes supporting regenerative strategies based on the combined use of both myogenic and angiogenic factors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. EVs contain cell type-specific signatures and have been proposed as biomarkers in a variety of diseases. Their small size (<1 μm) and biological and physical functions make them obvious candidates for therapeutic agents in immune therapy, vaccination, regenerative medicine and drug delivery. However, due to the complexity and heterogeneity of their origin and composition, the actual mechanism through which these vesicles exert their functions is still unknown and represents a great biomedical challenge. Moreover, because of their small dimensions, the quantification, size distribution and biophysical characterization of these particles are challenging and still subject to controversy. Here, we address the advantage of atomic force microscopy (AFM), for the characterization of isolated EVs. We review AFM imaging of EVs immobilized on different substrates (mica, glass) to identify the influence of isolation and deposition methods on the size distribution, morphology and mechanical properties of EVs.

This report describes the case of undiagnosed posttraumatic coronary artery dissection in a young multiorgan donor. Ex vivo preservation with the Organ Care System (TransMedics, Inc, Andover, MA) revealed the presence of coronary disease and avoided transplantation of an organ at high risk for failure., (Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Aging is characterized by a progressive loss of the ability of the organism to cope with stressors and to repair tissue damage. As a result, chronic diseases, including cardiovascular disease, increase their prevalence with aging, underlining the existence of common mechanisms that lead to frailty and age-related diseases. In this frame, the progressive decline of the homeostatic and reparative function of primitive cells has been hypothesized to play a major role in the evolution of cardiac pathology to heart failure. Although initially it was believed that reactive oxygen species (ROS) were produced in an unregulated manner as a byproduct of cellular metabolism, causing macromolecular damage and aging, accumulating evidence indicate the major role played by redox signaling in physiology. Aim of this review is to critically revise evidence linking ROS to cell senescence and aging and to provide evidence of the primary role played by redox signaling, with a particular emphasis on the multifunctional protein APE1/Ref in stem cell biology. Finally, we will discuss evidence supporting the role of redox signaling in cardiovascular cells.

Aims: The prognostic stratification of patients with Idiopathic Dilated Cardiomyopathy (iDCM) is a difficult task. Here, we assessed the additive value of the evaluation of biomarkers of inflammasome activation and systemic inflammation for the long-term risk stratification of iDCM patients., Methods and Results: We studied 156 ambulatory iDCM patients (mean age 58 years, 77% men, 79% in NYHA class 1-2, median Left Ventricular Ejection Fraction (LVEF) 35%, mean sodium 139 mEq/L, median BNP 189 pg/mL, median IL-1 beta (IL-1β) 1.08 pg/mL, median IL-6 1.7 pg/mL and median IL-10 2.7 pg/mL).During the follow-up period of 89.6 months, 35 patients (22%) died/underwent heart transplantation. Patients who died/underwent heart transplantation were more likely to be male, to be in NYHA class III, to have atrial fibrillation, to have lower LVEF and higher BNP levels. IL-1β, IL-6 and IL-10 levels did not differ significantly between the groups of patients with good or bad prognosis. IL-1β levels did not vary significantly among either the different NYHA classes or the quartiles of LVEF. In a multivariable model, however, IL-1β was a strong and independent predictor of all-cause mortality (HR 1.193, 95% CI 1.056 - 1.349, p=0.005 for log squared transformed values). Other factors associated with the outcome were: male gender, presence of atrial fibrillation and sodium concentration. The estimated time-dependent ROC curve of the multivariable model showed an AUC 0.74 (95% CI 0.65-0.86)., Conclusions: Serum levels of IL-1β could be useful to predict the long-term outcome of patients with iDCM.

The ability of exosomes to elicit specific cellular responses suggests that they may be increasingly used as therapeutics. Their vesicular nature makes them suitable as potential nanocarriers for drugs or nucleic acids delivery. Here we address the question whether the method of preparation of enriched exosomal fractions can affect their uptake by cells and their ability to trigger a response. We compared ultracentrifugation and polymer-based precipitation methods on supernatants of glioma-associated stem cells isolated from a high-grade glioma patient. We determined particle size distributions after purification and their correlation with uptake, proliferation and migration in glioblastoma cell cultures. Our findings indicate that polymer-based precipitation leads to smaller particle size distributions, faster uptake by target cells and increased cellular motility. The different effect that isolation method-dependent populations of particles have on cell motility suggests their size distribution could also profoundly affect exosomes therapeutic potential., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Improved protocols/devices for in vitro culture of 3D cell spheroids may provide essential cues for proper growth and differentiation of stem/progenitor cells (S/PCs) in their niche, allowing preservation of specific features, such as multi-lineage potential and paracrine activity. Several platforms have been employed to replicate these conditions and to generate S/PC spheroids for therapeutic applications. However, they incompletely reproduce the niche environment, with partial loss of its highly regulated network, with additional hurdles in the field of cardiac biology, due to debated resident S/PCs therapeutic potential and clinical translation. In this contribution, the essential niche conditions (metabolic, geometric, mechanical) that allow S/PCs maintenance/commitment will be discussed. In particular, we will focus on both existing bioreactor-based platforms for the culture of S/PC as spheroids, and on possible criteria for the scaling-up of niche-like spheroids, which could be envisaged as promising tools for personalized cardiac regenerative medicine, as well as for high-throughput drug screening.

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression., Competing Interests: The authors have declared that no competing interests exist.

The life-long story of the heart starts concomitantly with primary differentiation events occurring in multipotent progenitors located in the so-called heart tube. This initially tubular structure starts a looping process, which leads to formation of the final four-chambered heart with a primary contribution of geometric and position-associated cell sensing. While this establishes the correct patterning of the final cardiac structure, it also provides feedbacks to fundamental cellular machineries controlling proliferation and differentiation, thus ensuring a coordinated restriction of cell growth and a myocyte terminal differentiation. Novel evidences provided by embryological and cell engineering studies have clarified the relevance of mechanics-supported position sensing for the correct recognition of cell fate inside developing embryos and multicellular aggregates. One of the main components of this pathway, the Hippo-dependent signal transduction machinery, is responsible for cell mechanics intracellular transduction with important consequences for gene transcription and cell growth control. Being the Hippo pathway also directly connected to stress responses and altered metabolism, it is tempting to speculate that permanent alterations of mechanosensing may account for modifying self-renewal control in tissue homeostasis. In the present contribution, we translate these concepts to the aging process and the failing of the human heart, two pathophysiologic conditions that are strongly affected by stress responses and altered metabolism.

Hemoglobin (Hb) is the major protein in erythrocytes and carries oxygen (O 2 ) throughout the body. Recently, Hb has been found synthesized in atypical sites, including the brain. Hb is highly expressed in A9 dopaminergic (DA) neurons of the substantia nigra (SN), whose selective degeneration leads to Parkinson's disease (PD). Here we show that Hb confers DA cells' susceptibility to 1-methyl-4-phenylpyridinium (MPP + ) and rotenone, neurochemical cellular models of PD. The toxic property of Hb does not depend on O 2 binding and is associated with insoluble aggregate formation in the nucleolus. Neurochemical stress induces epigenetic modifications, nucleolar alterations and autophagy inhibition that depend on Hb expression. When adeno-associated viruses carrying α- and β-chains of Hb are stereotaxically injected into mouse SN, Hb forms aggregates and causes motor learning impairment. These results position Hb as a potential player in DA cells' homeostasis and dysfunction in PD.

Background: Previous studies in the setting of patients with acute myocardial infarction (AMI) have demonstrated that hypovitaminosis D is associated with increased mortality risk during a follow-up whose median did not exceed two years., Objective: To evaluate the impact of vitamin D levels on long-term mortality in patients with AMI., Results: In our study 477 patients with AMI were included. During a median follow-up period of 57 (IQR 53-64) months, 93 patients (20%) died. A non-linear U-shaped relationship between 25(OH)D levels and long-term mortality was observed; patients with vitamin D<10ng/mL and >30ng/mL had higher mortality rate than those with intermediate values. After adjustment for differences in baseline features and treatment, it was confirmed that extreme values of vitamin D (<10 or >30ng/mL) are independent predictors of mortality with HR of 3.02 (95% CI 1.78-5.11). Other independent predictors of outcome were age, NYHA class at discharge, treatment with ACE inhibitors and statins. The estimated time-dependent ROC curve of the multivariable model including vitamin D showed an AUC significantly higher than the model without vitamin D: AUC 0.82 (95% CI 0.76-0.87) vs. 0.77 (95% CI 0.71-0.83), p=0.005. Addition of vitamin D to the model that included all significant factors for mortality improved the prognostic accuracy as showed by the metrics of reclassification (NRI 0.34 (95% CI 0.14-0.48), p=0.003 and IDI 0.06 (95% CI 0.01-0.12, p=0.005 p=0.03)., Conclusions: We report a U-shaped relationship between vitamin D levels and long-term outcome of patients surviving AMI., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24-39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.

Although the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM⁺/CK⁺) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a "surrogate" of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The aryl hydrocarbon receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: (i) examined the cytokine expression profile; (ii) counted AhR-expressing MCs; (iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; (iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); (v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and (vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors released by MCs on AhR activation. These results suggest a new mechanistic role of MCs in the pathogenesis of endometriosis., Competing Interests: The authors declare no conflict of interest.

Unlabelled: The in vivo reparative potential of Cardiac Stem Cells (CSC), cultured from explanted failing hearts (E-), is impaired by cellular senescence. Moreover, E-CSC are characterized, with respect to CSC obtained from healthy donors (D-), by an arrest in the autophagic degradation. Although the lysosome plays a pivotal role in cellular homeostasis and defects of this organelle may be associated with aging and heart failure, the lysosomal function of CSC has never been investigated. The aim of this work was to focus on the Lysosomal Compartment (LC) of E-CSC, evaluating elements that could jeopardize lysosome functionality., Methods and Results: Bioinformatics analysis conducted on genes differentially expressed between D- and E-CSC identified lysosomal-related gene sets as significantly enriched. Moreover, 29 differentially expressed genes were part of CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, by which Transcription Factor EB (TFEB) regulates cellular clearance. Consistently, live cell imaging and flow cytometry analyses showed that the lysosomes of E-CSC are less acidic than the D-CSC ones. Furthermore, confocal microscopy showed in E-CSC: an accumulation of intralysosomal lipofuscins, a reduction of cathepsin B activity, evidence of lysosome membrane permeabilization, and the reduction of the nuclear active TFEB. The use of Rapamycin (TORC1 inhibitor) was able on one hand to increase TFEB activation and, on the other hand, to reduce lipofuscin mass, potentiating the lysosomal functionality., Conclusions: This study demonstrated for the first time that E-CSC are characterized by a blunted activation of TFEB and an altered proteostasis. TORC1 hyperactivation plays a central role in this phenomenon., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Long noncoding RNAs (lncRNAs) are non-protein coding transcripts regulating a variety of physiological and pathological functions. However, their implication in heart failure is still largely unknown. The aim of this study is to identify and characterize lncRNAs deregulated in patients affected by ischemic heart failure., Methods: LncRNAs were profiled and validated in left ventricle biopsies of 18 patients affected by non end-stage dilated ischemic cardiomyopathy and 17 matched controls. Further validations were performed in left ventricle samples derived from explanted hearts of end-stage heart failure patients and in a mouse model of cardiac hypertrophy, obtained by transverse aortic constriction. Peripheral blood mononuclear cells of heart failure patients were also analyzed. LncRNA distribution in the heart was assessed by in situ hybridization. Function of the deregulated lncRNA was explored analyzing the expression of the neighbor mRNAs and by gene ontology analysis of the correlating coding transcripts., Results: Fourteen lncRNAs were significantly modulated in non end-stage heart failure patients, identifying a heart failure lncRNA signature. Nine of these lncRNAs (CDKN2B-AS1/ANRIL, EGOT, H19, HOTAIR, LOC285194/TUSC7, RMRP, RNY5, SOX2-OT and SRA1) were also confirmed in end-stage failing hearts. Intriguingly, among the conserved lncRNAs, h19, rmrp and hotair were also induced in a mouse model of heart hypertrophy. CDKN2B-AS1/ANRIL, HOTAIR and LOC285194/TUSC7 showed similar modulation in peripheral blood mononuclear cells and heart tissue, suggesting a potential role as disease biomarkers. Interestingly, RMRP displayed a ubiquitous nuclear distribution, while H19 RNA was more abundant in blood vessels and was both cytoplasmic and nuclear. Gene ontology analysis of the mRNAs displaying a significant correlation in expression with heart failure lncRNAs identified numerous pathways and functions involved in heart failure progression., Conclusions: These data strongly suggest lncRNA implication in the molecular mechanisms underpinning HF.

Background: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data., Methods: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively., Results: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment., Conclusion: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.