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Objective To explore the longitudinal association of quantitative infrapatellar fat pad (IPFP) signal intensity alteration with OA-related biomarkers. Methods Eighteen OA-related biochemical biomarkers of 600 knee OA participants in the Foundation for the National Institutes of Health OA Biomarkers Consortium (FNIH) study were extracted. The quantitative IPFP signal intensity measures were acquired based on magnetic resonance imaging, including mean value [Mean (IPFP)] and standard deviation [sDev (IPFP)] of the whole IPFP signal intensity, median value [Median (H)] and upper quartile value [UQ (H)] of high signal intensity, the ratio of volume of high signal intensity to volume of whole IPFP signal intensity [Percentage (H)] and Clustering factor (H). The linear mixed-effect model was applied to determine the longitudinal associations between IPFP signal intensity alteration and biochemical biomarkers over 2 years. Results All IPFP measures except for Clustering factor (H) were positively associated with urine collagenase-cleaved type II collagen neoepitope (uC2C), urine C-terminal cross-linked telopeptide of type II collagen (uCTX-II), urine C-terminal cross-linked telopeptide of type I collagen-α (uCTX-Iα) and urine N-terminal cross-linked telopeptide of type I collagen (uNTX-I). Mean (IPFP), Median (H) and Percentage (H) were positively associated with the nitrated form of an epitope located in the triple helix of type II collagen (Coll2-1 NO2). Mean (IPFP), Median (H) and UQ (H) were positively associated with sCTX-I and uCTX-Iβ. Positive associations between sDev (IPFP), Percentage (H) and serum hyaluronic acid (sHA) were found. Conclusion Our results suggest a role of IPFP signal intensity alteration in joint tissue remodelling on a molecular level.

Objectives This study assessed whether circulating levels of adiponectin and leptin are associated with higher mortality in patients with RA. Methods Participants were adults from the Veterans Affairs RA Registry. Adipokines and inflammatory cytokines were measured as part of a multi-analyte panel on banked serum at enrolment. Dates and causes of death were derived from the Corporate Data Warehouse and the National Death Index. Covariates were derived from medical record, biorepository and registry databases. Multivariable Cox proportional hazard models evaluated associations between biomarkers and all-cause and cause-specific mortality. Results A total of 2583 participants were included. Higher adiponectin levels were associated with older age, male sex, white race, lower BMI, autoantibody seropositivity, radiographic damage, longer disease duration, prednisone use and osteoporosis. Higher adiponectin concentrations were also associated with higher levels of inflammatory cytokines but not higher disease activity at enrolment. Leptin was primarily associated with greater BMI and comorbidity. The highest quartile of adiponectin (vs lowest quartile) was associated with higher all-cause mortality [hazard ratio (HR): 1.46 (95% CI: 1.11, 1.93), P = 0.009] and higher cardiovascular mortality [HR: 1.85 (95% CI: 1.24, 2.75), P = 0.003], after accounting for covariates. Higher leptin levels were also associated with greater all-cause and cancer mortality. Conclusions Elevations in adipokines are associated with age, BMI, comorbidity and severe disease features in RA and independently predict early death. Associations between adiponectin and inflammatory cytokines support the hypothesis that chronic subclinical inflammation promotes metabolic changes that drive elevations in adipokines and yield adverse health outcomes.

Objectives The Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial compared hematopoietic stem cell transplant to CYC treatment in patients with early SSc with progressive skin and lung or kidney involvement. Here we describe lymphocyte phenotype abnormalities at study entry and the relation to prior DMARD therapy. Methods Lymphocyte subsets (n = 26) measured by flow cytometry were compared in 123 heathy controls and 71 SCOT participants, including those given (n = 57) or not given (n = 14) DMARDs within 12 months of randomization. Results Compared with healthy controls, individuals with SSc showed significant reductions in central memory CD8 T cells, activated total and CD4 T cells, γ/δ T cells, memory B cells, myeloid and plasmacytoid dendritic cells and FOXP3+CD25+ Treg cells and increases in naïve CD4 T cells, effector memory CD4 T cells and effector CD8 T cells. A greater bias towards a IL-4+ Th2/T cytotoxic 2 (Tc2) phenotype based on the Th2:Th1 CD4 ratio and Tc2:Tc1 CD8 T cells was also found. Notably, no difference in any lymphocyte subset was observed between those given or not given prior DMARDs. Conclusions In patients with early, severe SSc, significant lymphocyte subset abnormalities were observed. Prior treatment with immunosuppressive therapy did not impact the immunophenotype, suggesting that lymphocyte disturbances in scleroderma appeared to be due to the disease itself. Trial registration ClinicalTrials.gov (https://clinicaltrials.gov), NCT00114530.

Objective This study was initiated to evaluate mammalian target of rapamycin (mTOR) activation in renal tissue of LN patients. Methods This retrospective study included 187 LN patients, 20 diabetic nephropathy (DN) patients, 10 minimal change disease (MCD) patients and 10 normal controls (NCs). Seven of 187 LN patients had repeated renal biopsies. mTORC1/2 activation was evaluated by immunohistochemistry and multiplexed immunofluorescence. The association of mTORC1/2 activation with the clinicopathologic indices and prognostic outcomes was analysed among 187 LN patients. Proteomics was performed in renal biopsies of 20 LN patients. Proteomics was employed to comprehensively evaluate the impact of mTOR activation on intrarenal gene expression. Results mTORC1/2 was significantly activated in podocytes, mesangial cells, endothelial cells and tubular epithelial cells of LN patients as compared with those with MCD or NC. The glomerular mTORC1 activation was higher in LN patients compared with DN patients. mTORC1, but not mTORC2, activation strongly correlated with serum albumin, complement C3, proteinuria and the following pathological biomarkers of LN: crescent formation, interstitial inflammation and fibrosis. Moreover, mTORC1 activation was identified as a prognostic marker in LN patients. Bioinformatic analyses of proteomics and immunohistochemical data unveiled increased complement activation, antigen presentation and phagocytosis in LN patients with mTORC1 activation. Conclusion Renal mTORC1 activation could be a biomarker to reveal disease activity and predict clinical prognosis in LN patients.

Introduction Electronic cigarettes (e-cigs) have increased in popularity as a potentially less harmful alternative to tobacco smoking; however, the literature suggests “vaping” can cause endothelial damage, which can adversely affect erectile function. While there is a known link between smoking cigarettes and erectile dysfunction (ED), the effect of e-cigs on erectile function has been understudied. Objectives To review the evidence for e-cig use causing endothelial dysfunction, to explore endothelial dysfunction as a potential mechanism for ED, and to determine if there is literature to support e-cigs as a cause of ED. Methods A literature review was performed to identify publications pertaining to e-cig consumption and ED. Publications regarding e-cig consumption and vascular or endothelial damage were also included. The search was conducted through PubMed, MEDLINE database, the Cochrane Library Central Search, Web of Science, and Google Scholar. We identified 40+ publications for review, including 6 clinical trials and 3 basic science studies. Results The literature suggests that e-cig use induces oxidative stress, including the direct formation of reactive oxygen species and hydroxyl radicals, leading to endothelial damage. Endothelial dysfunction is a potential mechanism for ED, but there is a paucity of studies evaluating the relationship between e-cig use and ED. Conclusion E-cig use may cause ED in men, but more studies, specifically clinical trials, are needed to establish a relationship between e-cigs and ED. E-cig-induced endothelial dysfunction is a potential mechanism for ED that is worth exploring further. J Pincus, V Sandoval, B Dick, et al. E-Cigarette-Associated Endothelial Damage: A Potential Mechanism for Erectile Dysfunction. Sex Med Rev 2021;XX:XXX–XXX.

Aim Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. Methods and results We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2–5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and β-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2–5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. Conclusions Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or β-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Objectives RA is a chronic autoimmune disease characterized by joint inflammation and tissue destruction. Immune responses mediated by T cells and autoantibodies are known to play critical roles in RA. Collagen type II (CII)-induced arthritis (CIA) is a commonly used animal model of human RA. We have previously reported the identification of a new T cell inhibitory molecule CD300c. Here we investigate the ability of recombinant CD300c-IgG2a Fc (CD300c-Ig) fusion protein to prevent and treat CIA. Methods Mice were induced to develop CIA by CII and injected with CD300c-Ig or control Ig protein before or after CIA symptoms occur. The mice were examined for CIA clinical and pathological scores, and analysed for the expression of proinflammatory cytokines, the percentage and activation of CD4 T cells and regulatory T cells, CII-specific T cell proliferation and cytokine production, and CII-specific autoantibody production. Results In a prevention model, CD300c-Ig significantly decreases CIA incidence, and reduces clinical and pathological arthritis scores. In the treatment model, CD300c-Ig ameliorates established CIA. The beneficial effects of CD300c-Ig are related to decreased expansion and activation of T cells in the spleen and reduced expression of proinflammatory cytokines in the joints. CD300c-Ig also inhibits CII-specific T cell proliferation and Th1 and Th17 cytokine production. In addition, CD300c-Ig treatment reduced the production of CII autoantibodies in the serum. Furthermore, CD300c-Ig inhibits the proliferation and activation of T cells from RA patients in vitro. Conclusion CD300c-Ig protein has the potential to be used in the treatment of patients with RA.

Aims Coronavirus disease of 2019 (COVID-19) has rapidly become a worldwide pandemic. Many clinical trials have been initiated to fight the disease. Among those, hydroxychloroquine and azithromycin had initially been suggested to improve clinical outcomes. Despite any demonstrated beneficial effects, they are still in use in some countries but have been reported to prolong the QT interval and induce life-threatening arrhythmia. Since a significant proportion of the world population may be treated with such COVID-19 therapies, evaluation of the arrhythmogenic risk of any candidate drug is needed. Methods and results Using the O'Hara-Rudy computer model of human ventricular wedge, we evaluate the arrhythmogenic potential of clinical factors that can further alter repolarization in COVID-19 patients in addition to hydroxychloroquine (HCQ) and azithromycin (AZM) such as tachycardia, hypokalaemia, and subclinical to mild long QT syndrome. Hydroxychloroquine and AZM drugs have little impact on QT duration and do not induce any substrate prone to arrhythmia in COVID-19 patients with normal cardiac repolarization reserve. Nevertheless, in every tested condition in which this reserve is reduced, the model predicts larger electrocardiogram impairments, as with dofetilide. In subclinical conditions, the model suggests that mexiletine limits the deleterious effects of AZM and HCQ. Conclusion By studying the HCQ and AZM co-administration case, we show that the easy-to-use O'Hara-Rudy model can be applied to assess the QT-prolongation potential of off-label drugs, beyond HCQ and AZM, in different conditions representative of COVID-19 patients and to evaluate the potential impact of additional drug used to limit the arrhythmogenic risk.

Objective Cartilage and bone damage in RA are associated with elevated IL-1β. The effects of IL-1β can be reduced by biological therapies that target IL-1β or TNF-α. However, the mechanisms responsible for increased IL-1β and the effect of anti-TNF-α have not been fully elucidated. Recently, sterile-α and armadillo motif containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1β secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR-induced IL-1β secretion in RA peripheral blood monocytes and in patients commencing anti-TNF-α treatment. Methods Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1β secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM1, IL-1β and the components of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. Results TLR1/2 activation induced elevated IL-1β in RA monocytes compared with healthy controls (P = 0.0009), which negatively correlated with SARM1 expression (P = 0.0086). Lower SARM expression also correlated with higher disease activity (P = 0.0246). Additionally, patients responding to anti-TNF-α treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. Conclusion Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1β associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNF-α blockade can modify IL-1β secretion.

Aims Genetically altered mice are powerful models to investigate mechanisms of atrial arrhythmias, but normal ranges for murine atrial electrophysiology have not been robustly characterized. Methods and results We analyzed results from 221 electrophysiological (EP) studies in isolated, Langendorff-perfused hearts of wildtype mice (114 female, 107 male) from 2.5 to 17.7 months (mean 7 months) with different genetic backgrounds (C57BL/6, FVB/N, MF1, 129/Sv, Swiss agouti). Left atrial monophasic action potential duration (LA-APD), interatrial activation time (IA-AT), and atrial effective refractory period (ERP) were summarized at different pacing cycle lengths (PCLs). Factors influencing atrial electrophysiology including genetic background, sex, and age were determined. LA-APD70 was 18 ± 0.5 ms, atrial ERP was 27 ± 0.8 ms, and IA-AT was 17 ± 0.5 ms at 100 ms PCL. LA-APD was longer with longer PCL (+17% from 80 to 120 ms PCL for APD70), while IA-AT decreased (−7% from 80 to 120 ms PCL). Female sex was associated with longer ERP (+14% vs. males). Genetic background influenced atrial electrophysiology: LA-APD70 (−20% vs. average) and atrial ERP (−25% vs. average) were shorter in Swiss agouti background compared to others. LA-APD70 (+25% vs. average) and IA-AT (+44% vs. average) were longer in 129/Sv mice. Atrial ERP was longer in FVB/N (+34% vs. average) and in younger experimental groups below 6 months of age. Conclusion This work defines normal ranges for murine atrial EP parameters. Genetic background has a profound effect on these parameters, at least of the magnitude as those of sex and age. These results can inform the experimental design and interpretation of murine atrial electrophysiology.

Aims Irreversible electroporation (IRE) ablation is a non-thermal ablation method based on the application of direct current between a multi-electrode catheter and skin electrode. The delivery of current through blood leads to electrolysis. Some studies suggest that gaseous (micro)emboli might be associated with myocardial damage and/or (a)symptomatic cerebral ischaemic events. The aim of this study was to compare the amount of gas generated during IRE ablation and during radiofrequency (RF) ablation. Methods and results In six 60–75 kg pigs, an extracorporeal femoral shunt was outfitted with a bubble-counter to detect the size and total volume of gas bubbles. Anodal and cathodal 200 J IRE applications were delivered in the left atrium (LA) using a 14-electrode circular catheter. The 30 and 60 s 40 W RF point-by-point ablations were performed. Using transoesophageal echocardiography (TOE), gas formation was visualized. Average gas volumes were 0.6 ± 0.6 and 56.9 ± 19.1 μL (P Conclusion Anodal IRE applications result in significantly less gas formation than both cathodal IRE applications and RF applications. This finding is supported by TOE observations.

Graphical abstract Establishment of an autoreactive B cell memory after myocardial infarction: a working hypothesis. The demise of cardiac cells leads to the release of cryptoantigens that induce a humoral immune response that leads to accumulation of immunoglobulins in plaques and eventually amplifies atherogenesis at remote sites.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive.Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs.ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

Aims Adipocyte fatty acid-binding protein (A-FABP) is an adipokine implicating in various metabolic diseases. Elevated circulating levels of A-FABP correlate positively with poor prognosis in ischaemic stroke (IS) patients. No information is available concerning the role of A-FABP in the pathogenesis of IS. Experiments were designed to determine whether or not A-FABP mediates blood–brain barrier (BBB) disruption, and if so, to explore the molecular mechanisms underlying this deleterious effects. Methods and results Circulating A-FABP and its cerebral expression were increased in mice after middle cerebral artery occlusion. Genetic deletion and pharmacological inhibition of A-FABP alleviated cerebral ischaemia injury with reduced infarction volume, cerebral oedema, neurological deficits, and neuronal apoptosis; BBB disruption was attenuated and accompanied by reduced degradation of tight junction proteins and induction of matrix metalloproteinases-9 (MMP-9). In patients with acute IS, elevated circulating A-FABP levels positively correlated with those of MMP-9 and cerebral infarct volume. Mechanistically, ischaemia-induced elevation of A-FABP selectively in peripheral blood monocyte-derived macrophages and cerebral resident microglia promoted MMP-9 transactivation by potentiating JNK/c-Jun signalling, enhancing degradation of tight junction proteins and BBB leakage. The detrimental effects of A-FABP were prevented by pharmacological inhibition of MMP-9. Conclusion A-FABP is a key mediator of cerebral ischaemia injury promoting MMP-9-mediated BBB disruption. Inhibition of A-FABP is a potential strategy to improve IS outcome.

Introduction The regulatory role of microRNA (miRNA) in several conditions has been studied, but their function in tendon healing remains elusive. This review summarizes how miRNAs are related to the pathogenesis of tendon injuries and highlights their clinical potential, focusing on the issues related to their delivery for clinical purposes. Sources of data We searched multiple databases to perform a systematic review on miRNA in relation to tendon injuries. We included in the present work a total of 15 articles. Areas of agreement The mechanism of repair of tendon injuries is probably mediated by resident tenocytes. These maintain a fine equilibrium between anabolic and catabolic events of the extracellular matrix. Specific miRNAs regulate cytokine expression and orchestrate proliferation and differentiation of stromal cell lines involved in the composition of the extracellular matrix. Areas of controversy The lack of effective delivery systems poses serious obstacles to the clinical translation of these basic science findings. Growing point In vivo studies should be planned to better explore the relationship between miRNA and tendon injuries and evaluate the most suitable delivery system for these molecules. Areas timely for developing research Investigations ex vivo suggest therapeutic opportunities of miRNA for the management of tendon injuries. Given the poor pharmacokinetic properties of miRNAs, these must be delivered by an adequate adjuvant transport system.

Aims Pulsed field ablation (PFA) is a novel, non-thermal modality that selectively ablates myocardium with ultra-short electrical impulses while sparing collateral tissues. In a proof-of-concept study, the safety and feasibility of ventricular PFA were assessed using a prototype steerable, endocardial catheter. Methods and results Under general anaesthesia, the left and right ventricles of four healthy swine were ablated using the 12-Fr deflectable PFA catheter and a deflectable sheath guided by electroanatomic mapping. Using the study catheter, electrograms were recorded for each site and pre-ablation and post-ablation pacing thresholds (at 2.0 ms pulse width) were recorded in two of four animals. After euthanasia at 35.5 days, the hearts were submitted for histology. The PFA applications (n = 39) resulted in significant electrogram reduction without ventricular arrhythmias. In ablation sites where it was measured, the pacing thresholds increased by >16.8 mA in the right ventricle (3 sites) and >16.1 mA in the left ventricle (7 sites), with non-capture at maximum amplitude (20 mA) observable in 8 of 10 sites. Gross measurements, available for 28 of 30 ablation sites, revealed average lesion dimensions to be 6.5 ± 1.7 mm deep by 22.6 ± 4.1 mm wide, with a maximum depth and width of 9.4 mm and 28.6 mm, respectively. In the PFA lesions, fibrous tissue homogeneously replaced myocytes with a narrow zone of surrounding myocytolysis and no overlying thrombus. When present, nerve fascicles and vasculature were preserved within surrounding fibrosis. Conclusion We demonstrate that endocardial PFA can be focally delivered using this prototype catheter to create homogeneous, myocardium-specific lesions., Graphical Abstract Graphical Abstract

Aims Electronic (e)-cigarettes have been marketed as a ‘healthy’ alternative to traditional combustible cigarettes and as an effective method of smoking cessation. There are, however, a paucity of data to support these claims. In fact, e-cigarettes are implicated in endothelial dysfunction and oxidative stress in the vasculature and the lungs. The mechanisms underlying these side effects remain unclear. Here, we investigated the effects of e-cigarette vapour on vascular function in smokers and experimental animals to determine the underlying mechanisms. Methods and results Acute e-cigarette smoking produced a marked impairment of endothelial function in chronic smokers determined by flow-mediated dilation. In mice, e-cigarette vapour without nicotine had more detrimental effects on endothelial function, markers of oxidative stress, inflammation, and lipid peroxidation than vapour containing nicotine. These effects of e-cigarette vapour were largely absent in mice lacking phagocytic NADPH oxidase (NOX-2) or upon treatment with the endothelin receptor blocker macitentan or the FOXO3 activator bepridil. We also established that the e-cigarette product acrolein, a reactive aldehyde, recapitulated many of the NOX-2-dependent effects of e-cigarette vapour using in vitro blood vessel incubation. Conclusions E-cigarette vapour exposure increases vascular, cerebral, and pulmonary oxidative stress via a NOX-2-dependent mechanism. Our study identifies the toxic aldehyde acrolein as a key mediator of the observed adverse vascular consequences. Thus, e-cigarettes have the potential to induce marked adverse cardiovascular, pulmonary, and cerebrovascular consequences. Since e-cigarette use is increasing, particularly amongst youth, our data suggest that aggressive steps are warranted to limit their health risks.

Introduction Female sexual dysfunction (FSD) is a common endocrine disease that impairs the quality of life for many women. The existing therapy strategies still have many disadvantages. It is necessary to explore new pharmacologic treatments that are effective and safe. Aim The aim of this study was to explore the effects of soy isoflavone (SI) on FSD in mice and the underlying mechanisms. Methods and Main Outcome Measures Laser Doppler flowmetry was used to determine vaginal blood flow. Serum hormone levels and histologic changes of the vagina were analyzed by enzyme-linked immunosorbent assay (ELISA) and by hematoxylin and eosin (H&E) and Masson’s trichome staining. The mRNA and protein expression of endothelial nitric oxide synthase (eNOS) was then evaluated by quantitative real-time polymerase chain reaction and western blot assays. Results Vaginal blood flow was found to be remarkably lower in adult mice, and SI was shown to increase vaginal blood flow in a dose-dependent manner (P < .05). The results of ELISA and H&E and Masson’s trichome staining suggest that SI had a positive effect on FSD, as evidenced by the levels of hormones in serum and histologic changes of the vagina, which changed consistently. In addition, the level of eNOS was positively correlated with the concentration of SI, and eNOS inhibitor was able to reverse the improvement in sexual function induced by SI. Conclusion Our study demonstrated that SI could improve sexual function by upregulating the eNOS pathway. Therefore, SI might serve as a promising candidate for the treatment of sexual dysfunction.

AIMS: Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. METHODS AND RESULTS: By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. CONCLUSION: The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Aims Genetic disposition and lifestyle factors are understood as independent components underlying the risk of multiple diseases. In this study, we aim to investigate the interplay between genetics, educational attainment—an important denominator of lifestyle—and coronary artery disease (CAD) risk. Methods and results Based on the effect sizes of 74 genetic variants associated with educational attainment, we calculated a ‘genetic education score’ in 13 080 cases and 14 471 controls and observed an inverse correlation between the score and risk of CAD [P = 1.52 × 10−8; odds ratio (OR) 0.79, 95% confidence interval (CI) 0.73–0.85 for the higher compared with the lowest score quintile]. We replicated in 146 514 individuals from UK Biobank (P = 1.85 × 10−6) and also found strong associations between the ‘genetic education score’ with ‘modifiable’ risk factors including smoking (P = 5.36 × 10−23), body mass index (BMI) (P = 1.66 × 10−30), and hypertension (P = 3.86 × 10−8). Interestingly, these associations were only modestly attenuated by adjustment for years spent in school. In contrast, a model adjusting for BMI and smoking abolished the association signal between the ‘genetic education score’ and CAD risk suggesting an intermediary role of these two risk factors. Mendelian randomization analyses performed with summary statistics from large genome-wide meta-analyses and sensitivity analysis using 1271 variants affecting educational attainment (OR 0.68 for the higher compared with the lowest score quintile; 95% CI 0.63–0.74; P = 3.99 × 10−21) further strengthened these findings. Conclusion Genetic variants known to affect educational attainment may have implications for a health-conscious lifestyle later in life and subsequently affect the risk of CAD.

Objective This is a comprehensive, structured review synthesizing and summarizing the current experimental data and knowledge about the mechanisms of action (MOA) underlying spinal cord stimulation with the burst waveform (as defined by De Ridder) in chronic pain treatment. Methods Multiple database queries and article back-searches were conducted to identify the relevant literature and experimental findings for results integration and interpretation. Data from recent peer-reviewed conference presentations were also included for completeness and to ensure that the most up-to-date scientific information was incorporated. Both human and animal data were targeted in the search to provide a translational approach in understanding the clinical relevance of the basic science findings. Results/Conclusions Burst spinal cord stimulation likely provides pain relief via multiple mechanisms at the level of both the spinal cord and the brain. The specific waveforms and temporal patterns of stimulation both play a role in the responses observed. Differential modulation of neurons in the dorsal horn and dorsal column nuclei are the spinal underpinnings of paresthesia-free analgesia. The burst stimulation pattern also produces different patterns of activation within the brain when compared with tonic stimulation. The latter may have implications for not only the somatic components of chronic pain but also the lateral and affective pathway dimensions as well.

Background Healthy longevity is the result of the interaction between favourable environment and unique genetic makeup. We showed that horizontal transfer of a longevity-associated gene variant (LAV-BPIFB4) improves endothelial function and accelerates the recovery from ischemia. Purpose To determine if the benefit of LAV-BPIFB4 gene therapy can be extended to diabetic cardiomyopathy. Methods and results We confirmed that human diabetic patients with heart failure (n=13) show a decreased cardiac expression of BPIFB4 compared with healthy subjects (n=10). Obese db/db mice received a systemic injection of adeno-associated viral vector (AAV9)-LAV-BPIFB4, AAV9-wild type (WT)-BPIFB4 (both 100 μL at 1×1012 GC/mL) or vehicle before the onset of cardiomyopathy, and were euthanised four weeks later for histological, metabolic and transcriptional analyses. Echocardiographic evaluation (n=8/group), performed at baseline and after gene therapy, showed that LAV-BPIFB4 treatment, despite not resolving hyperglycaemia, improved left ventricular function compared with the other groups. Histological analyses of the hearts (n=5 to 10/group) revealed that LAV-BPIFB4 reduced myocardial fibrosis and increased angiogenesis compared with vehicle and WT-hearts; moreover, LAV increased the expression of the alpha-isoform of the cardiac myosin heavy chain, which is associated with a superior cardiomyocyte contractility. Interestingly, LAV-BPIFB4 treatment induced an increase in cardiac SDF1 expression compared with WT and vehicle, despite the mechanism linking the two events is still unknown. The oral administration of the CXCR4 antagonist AMD-070, given at 2 mg/kg/day for four weeks, abolished several of the beneficial effects exerted by the LAV-BPIFB4 therapy in the obese diabetic mice, as assessed by echocardiography and histology (n=7/group). At the molecular level, next-generation RNA sequencing (n=3 to 4 /group) showed 8 genes were differentially expressed by LAV-BPIFB4-hearts compared with vehicle-hearts. These genes are associated with mitochondrial and metabolic functions. Among them, changes in the UCP3, HMGCS2, CS, ATPB and TOMM20 expression were also validated at the protein level by western blotting. Lipidomics using ultrahigh-performance liquid chromatography-mass spectrometry (n=6 or 7/group) showed 63 metabolites differentially expressed by LAV-BPIFB4- compared with vehicle-hearts, with only 3 (two cardiolipins and one glycerophospholipid) returning close to the non-diabetic phenotype following LAV-BPIFB4 treatment. Conclusions This study newly shows the possibility of transferring the benefit of salutary polymorphic gene variants to protect the cardiovascular system from metabolic pressure. Rather than combating pathogenic mechanisms, the strategy activates alternative pathways overriding disease risk factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation project grant “Longevity-associated BPIFB4 gene therapy for treatment of ischemic disease”

BackgroundPericytes are attracting much attention as potential candidates for successful cell therapy of myocardial ischaemia. Intramyocardially delivered adventitial pericytes (APCs) secrete paracrine factors which stimulate angiogenesis and recruitment of cardiac stromal cells, reduce fibrosis and promote cardiomyocyte proliferation and viability. However, factors responsible for these biological effects have not been elucidated yet.PurposeTo exploit the components of APC secretome exerting a biological effect on cardiac cells with the aim to discover new druggable targets with potential therapeutic activity.Methods and resultsAPCs were derived from saphenous veins of adult patients (n=13, 68±11 yrs, all with coronary artery disease - CAD). The APC-conditioned medium (CM) stimulated the proliferation of human iPS-derived cardiomyocytes compared with unconditioned medium (UCM) (EdU incorporation, 1.3-fold increases, P=0.004). Stimulation with APC-CM increased the number of mitotic figures in cardiomyocytes (Aurora B, 1.5-fold increases compared to UCM, P=0.002). Furthermore, APC-CM abrogated the hypoxia-induced apoptosis in cardiomyocytes (2-fold increase in Caspase 3/7 activity in hypoxic cells exposed to UCM compared to normoxic cells, P=0.002). We also found that APC-CM stimulates the migration of human cardiac stromal cells (CSCs) obtained from healthy donors (n=6, 54±11 yrs) in both a transwell and scratch migration assays (n=6, P30KDa. The pro-migratory fractions of the APC-CM obtained from size exclusion chromatography underwent mass spectrometry analysis (n=3 APCs). This identified 14 proteins uniquely present in the pro-migratory fractions. The two most relevant candidates were SPARC and TGFBI, both confirmed by ELISA. Intriguingly, the recombinant SPARC and TGFBI failed to reproduce the biological effect of APC-CM on CSC migration, suggesting that the secreted proteins may carry unique post-translational modifications not found in synthetic peptides. Further analyses are being carried out to reveal the biological properties of the endogenous SPARC and TGFBI.ConclusionsThis study suggests a fascinating approach based on the use of the active component of the APC-CM as a surrogate of APC therapy. If the biological properties of the cellular proteins will be successfully reproduced in synthetic peptides in vitro, this innovative approach may extend the benefits of APC therapy to all those patients with CAD for whom cell therapy is not an available option.

Introduction Despite recent promising clinical results, the underlying mechanism of action of low-intensity extracorporeal shockwave therapy (Li-ESWT) for erectile dysfunction (ED) is mostly unclear and currently under investigation. Aim To systematically identify and evaluate evidence regarding the basic science behind Li-ESWT for ED, discuss and propose a putative mechanism of action, address the limitations, and imply insights for further investigation in the field. Methods Using Cochrane’s methodologic recommendations on scoping studies and systematic reviews, we conducted a systematic scoping review of the literature on experimental research regarding Li-ESWT for ED and other pathologic conditions. The initial systematic search was carried between January and November 2017, with 2 additional searches in April and August 2018. All studies that applied shockwave treatment at an energy flux density >0.25 mJ/mm2 were excluded from the final analysis. Main Outcome Measure We primarily aimed to clarify the biological responses in erectile tissue after Li-ESWT that could lead to improvement in erectile function. Results 59 publications were selected for inclusion in this study. 15 experimental research articles were identified on Li-ESWT for ED and 44 on Li-ESWT for other pathologic conditions. Li-ESWT for ED seems to improve erectile function possibly through stimulation of mechanosensors, inducing the activation of neoangiogenesis processes, recruitment and activation of progenitor cells, improving microcirculation, nerve regeneration, remodeling of erectile tissue, and reducing inflammatory and cellular stress responses. Clinical Implications Improving our understanding of the mechanism of action of Li-ESWT for ED can help us improve our study designs, as well as suggest new avenues of investigation. Strengths & Limitations A common limitation in all these studies is the heterogeneity of the shockwave treatment application and protocol. Conclusion Li-ESWT for ED, based on current experimental studies, seems to improve erectile function by inducing angiogenesis and reversing pathologic processes in erectile tissue. These studies provide preliminary insights, but no definitive answers, and many questions remain unanswered regarding the mechanism of action, as well as the ideal treatment protocol.

Introduction Although basic science and clinical research indicate that the vascular physiopathology of male and female sexual dysfunction (FSD) is similar, to date the association between FSD and cardiovascular (CV) diseases has been only marginally explored. Aim To discuss the potential reasons for differences in the role of CV diseases and risk factors in sexual function in women vs men in the 2nd part of a 2-part review. Methods A thorough literature search of peer-reviewed publications on the topic was performed using the PubMed database. Main Outcome Measures We present a review of the main factors that could account for this gap: (i) actual physiologic discrepancies and (ii) factors related to the inadequacy of the methodologic approach used to investigate CV risk in patients with FSD. A summary of the available methods to assess female sexual response, focusing on genital vascularization, is reported. Results The microanatomy and biochemistry of the male and female peripheral arousal response are similar; in contrast, there are differences in the interplay between the metabolic profile and sex steroid milieu, in the relative weighting of cardiometabolic risk factors in the pathogenesis of CV disease, and their clinical presentation and management. CV diseases in women are under-recognized, leading to less aggressive treatment strategies and poorer outcomes. Moreover, evaluation of hemodynamic events that regulate the female sexual response has thus far been plagued by methodologic problems. Conclusion To clarify whether sexuality can be a mirror for CV health in women, the female genital vascular district should be objectively assessed with standardized and validated methods. Studies designed to establish normative values and longitudinal intervention trials on the effect of the treatment of CV risk factors on FSD are urgently needed. Maseroli E, Scavello I, Vignozzi L. Cardiometabolic Risk and Female Sexuality—Part II. Understanding (and Overcoming) Gender Differences: The Key Role of an Adequate Methodological Approach. Sex Med Rev 2018;6:525–534.

Aims: We aimed to study the differences in biventricular scar characterization using bipolar voltage mapping compared with state-of-the-art in vivo delayed gadolinium-enhanced cardiac magnetic resonance (LGE-CMR) imaging and ex vivo T1 mapping. Methods and results: Ten pigs with established myocardial infarction (MI) underwent in vivo scar characterization using LGE-CMR imaging and high-density voltage mapping of both ventricles using a 3.5-mm tip catheter. Ex vivo post-contrast T1 mapping provided a high-resolution reference. Voltage maps were registered onto the left and right ventricular (LV and RV) endocardium, and epicardium of CMR-based geometries to compare voltage-derived scars with surface-projected 3D scars. Voltage-derived scar tissue of the LV endocardium and the epicardium resembled surface projections of 3D in vivo and ex vivo CMR-derived scars using 1-mm of surface projection distance. The thinner wall of the RV was especially sensitive to lower resolution in vivo LGE-CMR images, in which differences between normalized low bipolar voltage areas and CMR-derived scar areas did not decrease below a median of 8.84% [interquartile range (IQR) (3.58, 12.70%)]. Overall, voltage-derived scars and surface scar projections from in vivo LGE-CMR sequences showed larger normalized scar areas than high-resolution ex vivo images [12.87% (4.59, 27.15%), 18.51% (11.25, 24.61%), and 9.30% (3.84, 19.59%), respectively], despite having used optimized surface projection distances. Importantly, 43.02% (36.54, 48.72%) of voltage-derived scar areas from the LV endocardium were classified as non-enhanced healthy myocardium using ex vivo CMR imaging. Conclusion: In vivo LGE-CMR sequences and high-density voltage mapping using a conventional linear catheter fail to provide accurate characterization of post-MI scar, limiting the specificity of voltage-based strategies and imaging-guided procedures. The CNIC (Madrid, Spain) is supported by the Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation; The CNIC and the BSC (Barcelona, Spain) are Severo Ochoa Centers of Excellence (SEV-2015-0505 and SEV-2011-0067, respectively); Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional (RD12/0042/0036, CB16/11/00458), Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2016-80324-R, PI16/02110, and DTS17/00136), and by the European Commission [ERA-CVD Joint Call (JTC2016/APCIN-ISCIII-2016), Grant no. AC16/00021]; Fundacion Interhospitalaria para la Investigacion Cardiovascular (FIC, Madrid, Spain) and the heart rhyhtm section of the Spanish Society of Cardiology (DFR), in part; R01 Grant HL122352 from the National Heart Lung and Blood Institute, USA, National Institutes of Health to J.J.; CompBioMed project, H2020-EU.1.4.1.3 European Union's Horizon 2020 research and innovation program, (Grant no. 675451 to J.A.-S.); D.G.L. has received financial support through the 'la Caixa' Fellowship Grant for Doctoral Studies, 'la Caixa' Banking Foundation, Barcelona, Spain. Sí

Extracellular vesicles (EVs) have gained increasing attention as underexplored intercellular communication mechanisms in basic science and as potential diagnostic tools in translational studies, particularly those related to cancers and neurological disorders. This article summarizes accumulated findings in the basic biology of EVs, EV research methodology, and the roles of EVs in brain cell function and dysfunction, as well as emerging EV studies in human brain disorders. Further research on EVs in neurobiology and psychiatry may open the door to a better understanding of intercellular communications in healthy and diseased brains, and the discovery of novel biomarkers and new therapeutic strategies in psychiatric disorders.

Background Inflatable penile prosthetic (IPP) infections are unusual but carry high patient morbidity and healthcare costs. Aim To increase the bactericidal effect of IPP tubing material to prevent future bacterial infections and to determine whether this effect is time-dependent. Methods A modified disk diffusion assay was developed to measure the zones of inhibition against Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Staphylococcus epidermidis when tubing was immersed in gentamycin, ampicillin, tetracycline, kanamycin, erythromycin, or ciprofloxacin. To further assess the efficacy of this approach, IPP tubing was exposed to ampicillin or ciprofloxacin for 30 seconds, 2 minutes, 10 minutes, or 60 minutes. Outcomes Bacterial zones of inhibition against IPP tubing material exposed to various treatments. Results IPP tubing was more effective against Gram-positive bacteria (S aureus and S epidermidis) then Gram-negative bacteria (E coli and P mirabilis). Immersing IPP tubing material in ampicillin or ciprofloxacin increased bactericidal effect of tubing material against Gram-positive and Gram-negative bacteria, respectively. The observed inhibitory effect was time dependent. Clinical Translation Exposing IPP to a specific antimicrobial directly before implantation increases the bactericidal properties of the material, potentially decreasing the likelihood of infection. Strengths & Limitations This study is limited in that it is in vitro experimentation observing the effect of a single strain of each bacterium. Although the strains used were clinically relevant, further analysis is required to determine whether these results were strain specific. Conclusion Immersing IPP material into an antibiotic solution, such as ampicillin or ciprofloxacin, increases the bactericidal properties and may aid in the prevention of infection.

Background Autologous fat transfer, also known as lipofilling, is a minimally invasive technique that uses the patient's own fat to correct disfiguring sequelae after breast cancer surgery. Despite its obvious clinical benefits, experimental research has demonstrated that autologous fat transfer inherently stimulates angiogenesis and tissue regeneration, which is feared to increase the risk of locoregional recurrence of breast cancer. This meta-analysis is founded on recently completed large cohort studies on this highly relevant topic. Methods A literature search was performed in PubMed, Embase and the Cochrane Library on 1 September 2017, adhering to the PRISMA guidelines, to identify all relevant studies of patients with breast cancer exposed to autologous fat transfer. The difference in incidence rate of locoregional recurrence between patients who had autologous fat transfer and controls was the primary outcome in the meta-analysis. Results Fifty-nine studies and a total of 4292 patients were included. These consisted of seven matched cohorts, 12 cohorts and 40 case series. Mean follow-up was 5·7 years from the date of primary cancer surgery and 2·7 years after autologous fat transfer. Meta-analysis of matched cohorts revealed an incidence rate difference of –0·15 (95 per cent c.i. –0·36 to 0·07) per cent per year, which was not statistically significant (P = 0·419). This finding was confirmed in the pooled results of the remaining cohorts and case series. Conclusion This meta-analysis of all oncological data from the published literature demonstrated that autologous fat transfer did not result in an increased rate of locoregional recurrence in patients with breast cancer. Autologous fat transfer can therefore be performed safely in breast reconstruction after breast cancer.

Non-coding RNAs are increasingly recognized not only as regulators of various biological functions but also as targets for a new generation of RNA therapeutics and biomarkers. We hereby review recent insights relating to non-coding RNAs including microRNAs (e.g. miR-126, miR-146a), long non-coding RNAs (e.g. MIR503HG, GATA6-AS, SMILR), and circular RNAs (e.g. cZNF292) and their role in vascular diseases. This includes identification and therapeutic use of hypoxia-regulated non-coding RNAs and endogenous non-coding RNAs that regulate intrinsic smooth muscle cell signalling, age-related non-coding RNAs, and non-coding RNAs involved in the regulation of mitochondrial biology and metabolic control. Finally, we discuss non-coding RNA species with biomarker potential. This article is part of the Mini Review Series from the Varenna 2017 meeting of the Working Group of Myocardial Function of the European Society of Cardiology.

Aims Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. Methods and results Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-β1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-β1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-β1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFβ-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. Conclusion These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFβ/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signalling leads to chronic fibrosis., National Institutes of Health (Grant R01HL097357 to D.L.); Ministerio de Economía y Competitividad (MINECO) SAF 2012-31338, CSD 2007-00020, Comunidad de Madrid ‘Fibroteam’ S2010/BMD-2321 to S.L.

AIMS: Obesity is a risk factor of abdominal aortic aneurysm (AAA). Inflammatory cytokine interleukin-18 (IL18) has two receptors: IL18 receptor (IL18r) and Na-Cl co-transporter (NCC). In human and mouse AAA lesions, IL18 colocalizes to its receptors at regions rich in adipocytes, suggesting a role of adipocytes in promoting IL18 actions in AAA development. METHODS AND RESULTS: We localized both IL18r and NCC in human and mouse AAA lesions. Murine AAA development required both receptors. In mouse AAA lesions, IL18 binding to these receptors increased at regions enriched in adipocytes or adjacent to perivascular adipose tissue. 3T3-L1 adipocytes enhanced IL18 binding to macrophages, aortic smooth muscle cells (SMCs), and endothelial cells by inducing the expression of both IL18 receptors on these cells. Adipocytes also enhanced IL18r and IL18 expression from T cells and macrophages, AAA-pertinent protease expression from macrophages, and SMC apoptosis. Perivascular implantation of adipose tissue from either diet-induced obese mice or lean mice but not that from leptin-deficient ob/ob mice exacerbated AAA development in recipient mice. Further experiments established an essential role of adipocyte leptin and fatty acid-binding protein 4 (FABP4) in promoting IL18 binding to macrophages and possibly other inflammatory and vascular cells by inducing their expression of IL18, IL18r, and NCC. CONCLUSION: Interleukin-18 uses both IL18r and NCC to promote AAA formation. Lesion adipocyte and perivascular adipose tissue contribute to AAA pathogenesis by releasing leptin and FABP4 that induce IL18, IL18r, and NCC expression and promote IL18 actions.

Aims Radiotherapy-induced cardiovascular disease is an emerging problem in a growing population of cancer survivors where traditional treatments, such as anti-platelet and lipid-lowering drugs, have limited benefits. The aim of the study was to investigate vascular inflammatory patterns in human cancer survivors, replicate the findings in an animal model, and evaluate whether interleukin-1 (IL-1) inhibition could be a potential treatment. Methods and results Irradiated human arterial biopsies were collected during microvascular autologous free tissue transfer for cancer reconstruction and compared with non-irradiated arteries from the same patient. A mouse model was used to study the effects of the IL-1 receptor antagonist, anakinra, on localized radiation-induced vascular inflammation. We observed significant induction of genes associated with inflammasome biology in whole transcriptome analysis of irradiated arteries, a finding supported by elevated protein levels in irradiated arteries of both, pro-caspase and caspase-1. mRNA levels of inflammasome associated chemokines CCL2, CCL5 together with the adhesion molecule VCAM1, were elevated in human irradiated arteries as was the number of infiltrating macrophages. A similar pattern was reproduced in Apoe−/− mouse 10 weeks after localized chest irradiation with 14 Gy. Treatment with anakinra in irradiated mice significantly reduced Ccl2 and Ccl5 mRNA levels and expression of I-Ab. Conclusion Anakinra, administered directly after radiation exposure for 2 weeks, ameliorated radiation induced sustained expression of inflammatory mediators in mice. Further studies are needed to evaluate IL-1 blockade as a treatment of radiotherapy-induced vascular disease in a clinical setting.

Background Brain metastases (BMs) are reported in 20–40% patients with METex14 skipping NSCLC. Tepotinib, a highly selective MET inhibitor, has demonstrated an objective response rate (ORR) of 45% and median duration of response (mDOR) of 11.1 months, in METex14 skipping NSCLC patients in Cohort A of the Phase II VISION study. Here, we report the intracranial activity of tepotinib in Cohort A. Methods Patients received oral tepotinib 500 mg QD. Study eligibility allowed for patients with BM (asymptomatic and symptomatic/stable). Primary endpoint: systemic objective response (RECIST v1.1); subgroup analysis in patients with BM (RECIST v1.1) was predefined. An ad hoc retrospective analysis of brain lesions (by CT/MRI) was conducted by an IRC using Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteria. Responses were determined in patients with ≥1 evaluable post-baseline tumor assessment. For non-measurable lesions (enhancing and non-enhancing non-target lesions [NTL]), disease control in the brain was defined as non-complete response/non-progressive disease. Data cut-off: July 1, 2020. Results Twenty-three patients had baseline BM. Systemic efficacy in patients with BM (ORR 47.8% [95% CI: 26.8, 69.4]; mDOR 9.5 months [95% CI: 5.5, not estimable]) was consistent with the overall population. Fifteen patients were evaluable by RANO-BM; 12 received prior radiotherapy for BM (median 6.4 weeks before treatment). Systemic best objective responses (BORs) were partial response (PR, n=9), stable disease (SD, n=3), and progressive disease (PD; n=3). Of seven patients with measurable CNS disease (all of whom received prior radiotherapy), intracranial BORs were PR (n=5), SD (n=1), and PD (n=1). For patients with NTL only (n=8), one had PD, and seven achieved intracranial disease control with three patients achieving CR of the enhancing NTL. Conclusions Tepotinib demonstrated intracranial activity in patients with METex14 skipping NSCLC with BM. Prospective evaluation of intracranial activity in VISION Cohort C is ongoing.

Objective MuT Homolog1 (MTH1) is an enzyme involved in DNA repair in normal cells and is often up- regulated in cancer cells. The enzyme catalyses the hydrolysis of oxidised dNTPs, to prevent their incorporation into DNA or RNA, resulting in mutations or cell damage/death. Cancer cells can have a high concentration of ROS, due to defective redox regulation. This results in the damage of DNA and oxidises free dNTPS, which in turn leads to mutations in DNA replication or cell death. Identifying MTH1 in brain metastases could present a target for treatment with MTH1-Inhibitors. Methods The quantification of MTH1 expression was shown using Western Blot analysis. 16 tumours were obtained during neurosurgery, 8 metastatic and 8 recurrent tumours of the same patient, and immediately frozen using liquid nitrogen. The proteins were extracted using RIPA lysis buffer. Western Blot was performed and detection followed via peroxidase linked secondary antibodies. Results MTH1 expression was shown to be up-regulated in brain metastases (1,442+/-0,6374) versus normal brain tissue (0,4133+/-0,277). However, in the recurrent tumour of the brain metastases, MTH1 was not expressed in a significantly higher amount compared the controll tissue and less than in the brain metastases (0,6941+/-0,4146). Conclusion The high expression of MTH1 in cerebral metastases is not uncommon for many cancers and thus presents a therapeutic target for MTH1-inhibitors, provided these are able to cross the blood brain barrier. Comparison to the primary melanoma tumour would be useful in showing significant differences of the metastases. Lower levels of MTH1 in recurrent brain metastases may suggest dedifferentiation from the original metastases. It may present a target for specific treatment of brain metastases of melanoma.

Cytokines signal through specific cell surface receptors to broadly regulate immune development, differentiation, proliferation, and survival, thereby influencing cellular fate and function. Accordingly, cytokines are potential therapeutic targets for modulation of immune responses. Indeed, over the last several decades, an increasing number of cytokine-based clinical trials have been performed, collectively using either recombinant forms of cytokines or blocking agents that modulate the actions of cytokines. The pleiotropic actions of cytokines, including their abilities to mediate both inflammatory and protective immune responses, indicate that using or targeting cytokines can have desired but also potentially undesirable effects. Here, we focus on the immunomodulatory cytokine, IL-2. We review the underlying basic science related to IL-2 and its biologic actions, discuss the current state of IL-2-based immunotherapy, and focus on emerging concepts of modulating the activities of this cytokine, including the generation of novel partial cytokine agonists as new potential therapeutics.

Aims We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P

Introduction Vacuum erectile device (VED) therapy is commonly used for penile rehabilitation after radical prostatectomy, however, the underlying mechanism of this effect is not fully understood. Aim To evaluate the presence of label-retaining cells (LRCs), cells with long-term retention of 5-ethynyl-2-deoxyuridine (EdU) labeling and recognized as adult stem cells or progenitor-like cells, in cavernous tissue after VED treatment using a BCNC rat model. Methods Postnatal pups (1 day old) of Sprague Dawley (SD) rats were intraperitoneally injected with EdU (50 ug/g, BID for 3 days) and BCNC surgery was conducted at 6 weeks old (designated as natal-labeled rats). Adult SD rats underwent BCNC surgery and EdU injection (50 ug/g, once) after surgery (designated as adult-labeled rats). One week after surgery, both natal- and adult-labeled rats received daily VED treatment for 4 weeks. Intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured for all rats and then the penile tissue was harvested. The ratio of ICP/MAP was calculated to represent erectile function. Penile tissue was examined by immunofluorescence staining to detect EdU positive cells. Main Outcome Measures The ratio of Intracavernous pressure (ICP) /MAP and the percentage of EdU positive cells were measured. Results The erectile function was impaired after BCNC and partially restored after VED treatment in both natal- and adult-labeled rats (P < .05). There was no difference in the percentage of EdU positive cells in natal-labeled rat cavernous tissue in BCNC group compared with VED group. Among the adult-labeled rats, the percentage of EdU positive cells increased in BCNC group (P < .05) but didn't change significantly after VED treatment (P = .35). Conclusion LRCs may play a limited role in the restoration of erectile dysfunction through VED treatment after BCNC.

Aims In patients with acute coronary syndrome (ACS), the higher activity of effector T-cells suggests that mechanisms involving adaptive immunity dysregulation might play a role in coronary instability. The shedding of the functional CD31 domain 1–5 leads to uncontrolled lymphocyte activation. In experimental models, matrix metalloproteinase-9 (MMP-9) has been implicated in endothelial CD31 cleavage. Interestingly, higher serum levels of MMP-9 have been observed in ACS. We aim to investigate the mechanisms underlying CD31 dysregulation in ACS. Methods and results To assess CD31 cleavage on CD4+ T-cells, we analysed by flow cytometry CD4+ T-cells of 30 ACS, 25 stable angina (SA) patients, and 28 controls (CTRL) using two different CD31 antibodies that specifically recognize domain 1–5 or the non-functional membrane-proximal domain 6. The ratio between the domains was significantly lower in ACS than in SA and CTRL (P = 0.002 ACS vs. SA; P = 0.002 ACS vs. CTRL). After stimulation with anti-CD3/CD28, the 1–5/6 domain ratio was significantly lower in ACS than in SA (P = 0.005). ELISA of supernatants obtained from T-cell receptor-stimulated CD4+ T-cells showed higher production of MMP-9 in ACS than in SA (P

Heart valve diseases (HVDs) arise from a number of different processes that affect both the structure and function of the valve apparatus. Despite diverse aetiologies, treatments for HVDs are limited to percutaneous or surgical interventions. The search for medical therapies to prevent or slow the progression of HVDs has been hampered by our poor understanding of the progression from subclinical to symptomatic phases, and our limited knowledge of the molecular signals that control the susceptibility of valve interstitial cells to pathological remodeling. Clinical evidence has suggested a link between certain neurotransmitters and valvular diseases of the heart. The fenfluramine-phentermine appetite suppressants popular in the 1980s were linked to mitral valve dysfunction, and ergot-derived dopamine agonists for Parkinson's disease have been associated with an increased risk of mitral and aortic valve regurgitation. The effect does not appear to be limited to medications, as valvular pathologies have also been observed in patients with carcinoid tumours of serotonin-producing enterochromaffin cells. The role of neurotransmitter molecules in valve pathology has not been adequately characterized and may represent a target for future medical therapies. Here we present current evidence from both clinical and basic science suggesting a link between neurotransmitters and HVDs, opening the door to future research in this area.

Aims Rapid over-activation of β-adrenergic receptor (β-AR) upon stress leads to cardiac inflammation, a prevailing factor that underlies heart injury. However, mechanisms by which acute β-AR stimulation induce cardiac inflammation still remain unknown. Here, we set out to identify the crucial role of inflammasome/interleukin (IL)-18 in initiating and maintaining cardiac inflammatory cascades upon β-AR insult. Methods and results Male C57BL/6 mice were injected with a single dose of β-AR agonist, isoproterenol (ISO, 5 mg/kg body weight) or saline subcutaneously. Cytokine array profiling demonstrated that chemokines dominated the initial cytokines upregulation specifically within the heart upon β-AR insult, which promoted early macrophage infiltration. Further investigation revealed that the rapid inflammasome-dependent activation of IL-18, but not IL-1β, was the critical up-stream regulator for elevated chemokine expression in the myocardium upon ISO induced β1-AR-ROS signalling. Indeed, a positive correlation was observed between the serum levels of norepinephrine and IL-18 in patients with chest pain. Genetic deletion of IL-18 or the up-stream inflammasome component NLRP3 significantly attenuated ISO-induced chemokine expression and macrophage infiltration. In addition, IL-18 neutralizing antibodies selectively abated ISO-induced chemokines, proinflammatory cytokines and adhesion molecules but not growth factors. Moreover, blocking IL-18 early after ISO treatment effectively attenuated cardiac inflammation and fibrosis. Conclusion Inflammasome-dependent activation of IL-18 within the myocardium upon acute β-AR over-activation triggers cytokine cascades, macrophage infiltration and pathological cardiac remodelling. Blocking IL-18 at the early stage of β-AR insult can successfully prevent inflammatory responses and cardiac injuries.

Aims C-type natriuretic peptide (CNP) is an essential endothelium-derived signalling species that governs vascular homoeostasis; CNP is also expressed in the heart but an intrinsic role for the peptide in cardiac function is not established. Herein, we employ unique transgenic strains with cell-specific deletion of CNP to define a central (patho)physiological capacity of CNP in maintaining heart morphology and contractility. Methods and results Cardiac structure and function were explored in wild type (WT), cardiomyocyte (cmCNP−/−), endothelium (ecCNP−/−), and fibroblast (fbCNP−/−)—specific CNP knockout mice, and global natriuretic peptide receptor (NPR)-B−/−, and NPR-C−/− animals at baseline and in experimental models of myocardial infarction and heart failure (HF). Endothelium-specific deletion of CNP resulted in impaired coronary responsiveness to endothelium-dependent- and flow-mediated-dilatation; changes mirrored in NPR-C−/− mice. Ex vivo, global ischaemia resulted in larger infarcts and diminished functional recovery in cmCNP−/− and NPR-C−/−, but not ecCNP−/−, vs. WT. The cardiac phenotype of cmCNP−/−, fbCNP−/−, and NPR-C−/− (but not ecCNP−/− or NPR-B−/−) mice was more severe in pressure overload- and sympathetic hyperactivation-induced HF compared with WT; these adverse effects were rescued by pharmacological CNP administration in WT, but not NPR-C−/−, mice. At a molecular level, CNP/NPR-C signalling is impaired in human HF but attenuates activation of well-validated pro-hypertrophic and pro-fibrotic pathways. Conclusion C-type natriuretic peptide of cardiomyocyte, endothelial and fibroblast origins co-ordinates and preserves cardiac structure, function, and coronary vasoreactivity via activation of NPR-C. Targeting NPR-C may prove an innovative approach to treating HF and ischaemic cardiovascular disorders.

Background Diseases of the aortic valve are a common reason for heart surgery. Aortic stenosis (AS) is associated with pressure and aortic regurgitation (AR) with a volume overload of the left ventricle (LV). Over time both pathologies lead to systolic and diastolic heart failure, while progressive downregulation of β-adrenoceptors occurs. While LV re-remodeling occurs in the majority of patients after aortic valve surgery, LV dysfunction persists in one fourth of such patients and leads to a terminal heart failure. We aimed to investigate whether differential remodeling in the protein kinase A (PKA) dependent inotropic response in myocytes and myocardial tissue obtained from patients undergoing aortic valve surgery is associated with the LV re-remodeling after surgery. Methods Preoperatively, pro BNP levels were measured and left ventricular strain analysis via echocardiography was performed. Interventricular septal biopsy was obtained intraoperatively in 10 patients who underwent aortic valve surgery. In-vitro contractility was analyzed in myocardial tissue paced with 4 Hz at 37 °C. Freshly isolated cells were transduced with an adenovirus expressing a cytosolic Förster resonance energy transfer (FRET) based cAMP biosensor (Epac1-camps). After 48 hours of culture, Föster-resonance energy transfer (FRET) was used for the first time to measure cAMP in 60 isolated human ventricular myocytes. Isoprenaline (10 nM – 10 μM) was used for β-adrenoceptor activation and forskolin (10 μmol) to activate adenylyl cyclase directly. Results We found a significantly downregulated β-adrenergic sensitivity in cardiomyocytes of patients with aortic valve disease, although contractile response to forskolin was maintained. Furthermore, we found a clear association between reduced sensitivity to isoprenaline (i.e., high EC50 values) and low maximum effect size to isoprenaline in myocardial tissue of patients with aortic valve disease, pointing out relevant β-adrenoceptor dysfunction. There were no significant differences in basal myocardial force between tissue samples of patients with AR and AS. Conclusion Collectively, our data show a profound remodelling in the cAMP/PKA pathway in patients with aortic valve disease. These disturbances may have an impact on the postoperative ventricular function and possibly on the long-term LV re-remodelling after aortic valve surgery.

DESCRIPTION The lack of enrollment of AYA patients on clinical trials is well documented and multivariant. Here we present the basic science, examination of its relevance to the AYA population specifically, and the parallel deployment of two international clinical trials via a pediatric neuro-oncology and adult brain tumor consortium. DISCUSSION In February of 2017, the laboratory of Ranjit Bindra, MD, PhD, published a manuscript describing the finding that tumors with IDH1/2 mutations induce a BRCAness state leading to PARP inhibitor (PARPi) sensitivity and synergistic interactions with temozolomide chemotherapy [2]. Despite IDH1/2 mutations being rare in the pediatric high-grade glioma population, three independent groups confirmed that the incidence is significantly increased to ~30% in the adolescent and young adult (AYA) population. Upon discovery of a high blood-brain-barrier penetrant, high potency PARPi by BeiGene Pharmaceuticals, an international trial was launched through the Pacific Pediatric Neuro-Oncology Consortium (PNOC) [3] to test this drug in an AYA specific trial recruiting patients ages 13 to 25, with a concurrent trial being run for patients older than 25 years of age through the Adult Brain Tumor Consortium (ABTC) [4]. While most trials that enroll AYA patients are forced to assess them as a unique cohort in post-analysis, if at all, the PNOC trial mentioned above was designed from the ground up with the AYA population in mind. It has allowed us to base initial dosing, recruitment strategies, psychosocial assessments, and outcomes, specifically on the AYA population. Ultimately, we expect their distinctive biology to yield unique results when compared to the ABTC trial. We propose that this is a model that could potentially be replicated in other disease processes and early phase drugs with the buy-in of the pharmaceutical industry and early phase consortiums. Acknowledgements: BeiGene Pharmaceuticals, PNOC, ABTC, CureSearch, Gateway Foundation

Introduction The use of current available treatment for male erectile dysfunction (ED) has some limitations that are related to efficacy and adverse effects. Nanotechnology has been used as a new tool in medicine to improve these limitations and new medications potentially to alleviate and cure ED. Aim To review the currently literature on new nano medications for ED based on scientific and clinical studies, efficacy, safety, mechanisms of action, and to identify gaps for future research. Methods A comprehensive literature review was conducted via Google Scholar, Science Direct, and PubMed on English publications using different keywords such as “erectile dysfunction”, “emerging treatments”, “nanotechnology”, and “herbal medicine”. The retrieved papers were organized into groups according to the sections covered in this review paper. Main Outcomes Measures We reviewed novel ED treatments such as nanotechnological phosphodiesterase inhibitors, papaverine hydrochloride, sialorphin, adipose tissue-derived stem cells, sonic hedgehog, and herbal medicine. Results Numerous preclinical studies have addressed novel phosphodiesterase 5 inhibitors nanoparticle, and their recent delivery systems. Nitric oxide, sialorphin, sonic hedgehog, and herbal medicine loaded nanoparticles and nano adipose tissue-derived stem cells as a potential new treatment for ED. In addition, papaverine-containing nanoparticles have been reported. A limited number of randomized clinical studies have determined the mechanism of these treatments. Conclusion A literature review on the application of nanotechnology in ED therapy was successfully conducted. New nano medications are promising to treat ED. However, further studies are warranted to further assess their efficacy and safety.

Background Brain metastases (BrM) with a highly invasive (HI) histological growth pattern are associated with poor prognosis compared to minimally invasive (MI) masses. Compared to MI lesions, HI BrM form greater contacts with cells in the peritumoral brain, particularly reactive astrocytes (RAs). RAs expressing phosphorylated STAT3 (pSTAT3+RAs) have been shown to promote BrM colonization. Here, we investigate the role of pSTAT3+RAs in promoting invasive growth of HI BrM. Methods We performed immunohistochemistry to identify pSTAT3+RAs in HI and MI human and patient-derived xenograft BrM. We assessed how pharmacological STAT3 inhibition or RA-specific STAT3 genetic ablation affected HI and MI BrM growth in vivo. scRNA-seq data generated from HI BrM astrocytes were integrated with published RA secretome data to identify STAT3 targets expressed by RAs that may drive invasion. Cancer cell invasion was modeled in vitro using a brain slice-tumor co-culture assay. Results HI BrM display increased pSTAT3-positivity within RAs when compared to MI lesions. Pharmacological STAT3 inhibition with Legasil (Silibinin) or genetic ablation decreased in vivo growth of HI, but not MI, BrM. Brain slice cultures treated with STAT3-activating cytokines induced cancer cell invasion, a response that was ablated following STAT3 inhibition. Chi3L1 was identified as a STAT3 target expressed by RAs. Cancer cells treated with recombinant Chi3L1 showed greater invasion into brain slice cultures compared to untreated cells. Conclusions pSTAT3+RAs are over-represented in HI BrM, rendering HI BrM preferentially sensitive to STAT3 inhibition. pSTAT3+RAs functionally contribute to BrM invasion within the brain, in part through Chi3L1-mediated activity. This work identifies STAT3 and Chi3L1 as clinically relevant therapeutic targets in management of HI BrM.