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Purpose Hydralazine, doxazosin, and verapamil are currently recommended by the Endocrine Society as acceptable bridging treatment in those in whom full cessation of antihypertensive medication is infeasible during screening for primary aldosteronism (PA). This is under the assumption that they cause minimal to no effect on the aldosterone-to-renin ratio, the most widely used screening test for PA. However, limited evidence is available regarding the effects of these particular drugs on said ratio. Methods In the present study, we retrospectively assessed the changes in aldosterone, renin, and aldosterone-to-renin values in essential hypertensive participants before and after treatment with either hydralazine (n = 26) or doxazosin (n = 20) or verapamil (n = 15). All samples were taken under highly standardized conditions. Results Hydralazine resulted in a borderline significant rise in active plasma renin concentration (19 vs 25 mIU/L, p = 0.067) and a significant fall in the aldosterone-to-renin ratio (38 vs 24, p = 0.017). Doxazosin caused declines in both plasma aldosterone concentration (470 vs 330 pmol/L, p = 0.028) and the aldosterone-to-renin ratio (30 vs 20, p = 0.020). With respect to verapamil, we found no statistically significant effect on any of these outcome variables. Conclusion We conclude that the assumption that these drugs can be used with little consequence to the aldosterone-to-renin cannot be substantiated. While it is possible that they are indeed the best option when full antihypertensive drug cessation is infeasible, the potential effects of these drugs must still be taken into account when interpreting the aldosterone-to-renin ratio.

Real world data (RWD) refers to healthcare information that is routinely collected in electronic healthcare records (EHR), hospital and pharmacy records, patient and disease registries, and health insurance databases. The collection and analysis of this vast amount of data is an important complement to that obtained from conventional randomised controlled trials (RCT). Real world data has been used for healthcare quality improvements, to conduct clinical trials, to support drug and device development, and to inform medical guidelines. The utility of RWD may be facilitated by common data models, which standardise format and content, and allow data from different health systems to be analysed together.The European Society of Cardiology (ESC) supports the use of RWD in collaboration with national cardiac societies, regulatory authorities, and industry to encourage continuous quality of care improvements at the hospital and country level, to conduct registry-based randomised clinical trials (R-RCT) and to facilitate safety surveillance of novel drugs and devices.The European Medicines Agency (EMA) is developing systems and processes to enable the use of RWD that can help in trial planning, defining clinical contexts, and enhancing outcome assessments. RWD can also contribute to the measurement of the impact of regulatory actions, such as contraindications or restriction of indications by looking at medicines use patterns over time across European Member States. A number of other initiatives from the European Commission and the EMA are underway to strengthen the EU's health security framework, and foster the collection and utilisation of RWD.

Background: Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways. Methods: To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes. Results: In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in I CaL , I NaL , and Ca 2+ spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca 2+ handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion. Conclusions: CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.

BACKGROUND: There is near-global consensus that all newborns be given parenteral vitamin K1 (VK1 ) at birth as prophylaxis against VK deficiency bleeding (VKDB). Breastmilk has a low VK content and cases of late VKDB are reported in exclusively breastmilk-fed preterm infants despite VK prophylaxis at birth.OBJECTIVES: To assess the prevalence of functional VK insufficiency in preterm infants based on elevated under-γ-carboxylated (Glu) species of Gla-proteins, factor II (PIVKA-II) and osteocalcin (GluOC), synthesized by liver and bone respectively.PATIENTS/METHODS: Prospective, multi-center, observational study in preterm infants born RESULTS: Post discharge, breastmilk-fed babies had significantly lower serum VK1 (0.15 vs. 1.81 μg/L), higher PIVKA-II (0.10 vs. 0.02 AU/mL) and higher %GluOC (63.6% vs. 8.1%) than those receiving a formula/mixed-feed diet. Pre-discharge (based on elevated PIVKA-II), only 1 (2%) of 45 breastmilk-fed infants was VK insufficient. Post-discharge, 8 (67%) of 12 exclusively breastmilk-fed babies were VK insufficient versus only 1 (4%) of 25 formula/mixed-fed babies.CONCLUSIONS: Preterm infants who remain exclusively or predominantly human breastmilk-fed post neonatal unit discharge are at high risk of developing subclinical VK deficiency in early infancy. Routine post-discharge VK1 supplementation of breastfed infants to provide intakes comparable to those from formula milks should prevent this deficiency.

Atrial fibrillation (AF) is more prevalent in athletes, and currently, the mechanisms are not fully understood. Atrial fibrillation inducibility and stability was investigated in trained and untrained Standardbred racehorses. The horses underwent echocardiography for evaluation of atrial size. High-density mapping during AF was performed, and the presence of structural remodeling, as well as the expression of inflammatory and pro-inflammatory markers in the atria, was studied. Atrial fibrillation sustained significantly longer after tachypacing in the trained horses, whereas no difference in AF inducibility was found. The untrained horses displayed a significant difference in the AF complexity when comparing right and left atria, whereas such difference was not observed in the trained animals. No evidence of increased structural remodeling or inflammation could be identified. Left atrial dimensions were not significantly increased. The increased AF sustainability in trained horses was not related to fibrosis or inflammation as seen in other animal exercise models. Graphical abstract

Introduction Patients with ventricular tachyarrhythmias (VT) are at high risk of sudden cardiac death. When appropriate, catheter ablation is modestly effective, with relatively high VT recurrence and complication rates. Personalized models that incorporate imaging and computational approaches have advanced VT management. However, 3D patient-specific functional electrical information is typically not considered. We hypothesize that incorporating non-invasive 3D electrical and structural characterization in a patient-specific model improves VT-substrate recognition and ablation targeting.Materials and methods In a 53-year-old male with ischemic cardiomyopathy and recurrent monomorphic VT, we built a structural-functional model based on high-resolution 3D late-gadolinium enhancement (LGE) cardiac magnetic resonance imaging (3D-LGE CMR), multi-detector computed tomography (CT), and electrocardiographic imaging (ECGI). Invasive data from high-density contact and pace mapping obtained during endocardial VT-substrate modification were also incorporated. The integrated 3D electro-anatomic model was analyzed off-line.Results Merging the invasive voltage maps and 3D-LGE CMR endocardial geometry led to a mean Euclidean node-to-node distance of 5 & PLUSMN; 2 mm. Inferolateral and apical areas of low bipolar voltage (< 1.5 mV) were associated with high 3D-LGE CMR signal intensity (> 0.4) and with higher transmurality of fibrosis. Areas of functional conduction delay or block (evoked delayed potentials, EDPs) were in close proximity to 3D-LGE CMR-derived heterogeneous tissue corridors. ECGI pinpointed the epicardial VT exit at & SIM;10 mm from the endocardial site of origin, both juxtaposed to the distal ends of two heterogeneous tissue corridors in the inferobasal left ventricle. Radiofrequency ablation at the entrances of these corridors, eliminating all EDPs, and at the VT site of origin rendered the patient non-inducible and arrhythmia-free until the present day (20 months follow-up). Off-line analysis in our model uncovered dynamic electrical instability of the LV inferolateral heterogeneous scar region which set the stage for an evolving VT circuit.Discussion and conclusion We developed a personalized 3D model that integrates high-resolution structural and electrical information and allows the investigation of their dynamic interaction during arrhythmia formation. This model enhances our mechanistic understanding of scar-related VT and provides an advanced, non-invasive roadmap for catheter ablation. This study was supported by the Special Research Fund (BOF) of Hasselt University (BOF17DOCMA15) and the Maastricht University Medical Center (MUMC+) to JS, the Hein Wellens Foundation, Health Foundation Limburg (Maastricht, The Netherlands), and a Veni grant from the Netherlands Organization for Scientific Research (TTW16772) to MC, the Netherlands CardioVascular Research Initiative (CVON2017-13 VIGILANCE and CVON2018B030 PREDICT2), Den Haag, The Netherlands to PV, and a Veni grant from the Netherlands Organization for Scientific Research (NWO/ZonMw 0915016181013) to RTB.

IntroductionThe transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a “pattern regulatory function,” by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo.Methods and resultsEndothelial Adam10 deficiency (Adam10ecko) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes.DiscussionCollectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases.

Introduction: High pacing frequency or irregular activity due to arrhythmia produces complex optical mapping signals and challenges for processing. The objective is to establish an automated activation time-based analytical framework applicable to optical mapping images of complex electrical behavior.Methods: Optical mapping signals with varying complexity from sheep (N = 7) ventricular preparations were examined. Windows of activation centered on each action potential upstroke were derived using Hilbert transform phase. Upstroke morphology was evaluated for potential multiple activation components and peaks of upstroke signal derivatives defined activation time. Spatially and temporally clustered activation time points were grouped in to wave fronts for individual processing. Each activation time point was evaluated for corresponding repolarization times. Each wave front was subsequently classified based on repetitive or non-repetitive events. Wave fronts were evaluated for activation time minima defining sites of wave front origin. A visualization tool was further developed to probe dynamically the ensemble activation sequence.Results: Our framework facilitated activation time mapping during complex dynamic events including transitions to rotor-like reentry and ventricular fibrillation. We showed that using fixed AT windows to extract AT maps can impair interpretation of the activation sequence. However, the phase windowing of action potential upstrokes enabled accurate recapitulation of repetitive behavior, providing spatially coherent activation patterns. We further demonstrate that grouping the spatio-temporal distribution of AT points in to coherent wave fronts, facilitated interpretation of isolated conduction events, such as conduction slowing, and to derive dynamic changes in repolarization properties. Focal origins precisely detected sites of stimulation origin and breakthrough for individual wave fronts. Furthermore, a visualization tool to dynamically probe activation time windows during reentry revealed a critical single static line of conduction slowing associated with the rotation core.Conclusion: This comprehensive analytical framework enables detailed quantitative assessment and visualization of complex electrical behavior.

Advanced science 10(5), 2203053 (2023). doi:10.1002/advs.202203053, Published by Wiley-VCH, Weinheim

Ex vivo characterisation of arterial biomechanics enables detailed discrimination of the various cellular and extracellular contributions to arterial stiffness. However, ex vivo biomechanical studies are commonly performed under quasi-static conditions, whereas dynamic biomechanical behaviour (as relevant in vivo) may differ substantially. Hence, we aim to (1) develop an integrated set-up for quasi-static and dynamic biaxial biomechanical testing, (2) quantify set-up reproducibility, and (3) illustrate the differences in measured arterial stiffness between quasi-static and dynamic conditions. Twenty-two mouse carotid arteries were mounted between glass micropipettes and kept fully vasodilated. While recording pressure, axial force (F), and inner diameter, arteries were exposed to (1) quasi-static pressure inflation from 0 to 200 mmHg; (2) 300 bpm dynamic pressure inflation (peaking at 80/120/160 mmHg); and (3) axial stretch (λz) variation at constant pressures of 10/60/100/140/200 mmHg. Measurements were performed in duplicate. Single-point pulse wave velocities (PWV; Bramwell-Hill) and axial stiffness coefficients (cax = dF/dλz) were calculated at the in vivo value of λz. Within-subject coefficients of variation were ~ 20%. Dynamic PWVs were consistently higher than quasi-static PWVs (p cax increased with increasing pressure. We demonstrated the feasibility of ex vivo biomechanical characterisation of biaxially-loaded murine carotid arteries under pulsatile conditions, and quantified reproducibility allowing for well-powered future study design.

The prevalence of cardiovascular disease increases exponentially with age, highlighting the contribution of aging mechanisms to cardiac diseases. Although model organisms which share human disease pathologies can elucidate mechanisms driving disease, they do not provide us with innate examples how cardiac aging might be slowed or attenuated. The identification of animal models that preserve cardiac function throughout most of life offers an alternative approach to study mechanisms which might slow cardiac aging. One such species may be the naked mole-rat (NMR), a mouse-sized (40 g) rodent with extraordinary longevity (> 37 years), and constant mortality hazard over its four decades of life. We used a cross-sectional study design to measure a range of physiological parameters in NMRs between 2 and 34 years of age and compared these findings with those of mice aged between 3 months and 2.5 years. We observed a rapid decline in body fat content and bone mineral density in old mice, but no changes in NMRs. Similarly, rhythm disorders (premature atrial and ventricular complexes) occurred in aged mice but not in NMRs. Magnetic resonance and ultrasound imaging showed age-dependent increases in cardiac hypertrophy and diastolic dysfunction in mice which were absent in NMRs. Finally, cardiac stress tests showed an age-dependent decline in normalized cardiac output in mice, which was absent in NMRs. Unlike mice, that manifest several aspects of human cardiac aging, NMRs maintain cardiac function and reserve capacity throughout their long lives and may offer insights on how to delay or prevent cardiac aging.

Frontiers in physiology 13, 779826 (2022). doi:10.3389/fphys.2022.779826 special issue: "How Can a Better Understanding of Mechanisms Enable Phenotyping of Human Atrial Fibrillation and Improve Clinical Treatments?", Published by Frontiers Research Foundation, Lausanne

Background Respiratory failure and thromboembolism are frequent in severe acute respiratory syndrome coronavirus 2–infected patients. Vitamin K activates both hepatic coagulation factors and extrahepatic endothelial anticoagulant protein S, required for thrombosis prevention. In times of vitamin K insufficiency, hepatic procoagulant factors are preferentially activated over extrahepatic proteins. Vitamin K also activates matrix Gla protein (MGP), which protects against pulmonary and vascular elastic fiber damage. We hypothesized that vitamin K may be implicated in coronavirus disease 2019 (COVID-19), linking pulmonary and thromboembolic disease. Methods A total of 135 hospitalized COVID-19 patients were compared with 184 historic controls. Inactive vitamin K–dependent MGP (desphospho-uncarboxylated [dp-uc] MGP) and prothrombin (PIVKA-II) were measured inversely related to extrahepatic and hepatic vitamin K status, respectively. Desmosine was measured to quantify the rate of elastic fiber degradation. Arterial calcification severity was assessed using computed tomography. Results dp-ucMGP was elevated in COVID-19 patients compared with controls (P Conclusions dp-ucMGP was severely increased in COVID-19 patients, indicating extrahepatic vitamin K insufficiency, which was related to poor outcome; hepatic procoagulant factor II remained unaffected. These data suggest pneumonia-induced extrahepatic vitamin K depletion leading to accelerated elastic fiber damage and thrombosis in severe COVID-19 due to impaired activation of MGP and endothelial protein S, respectively.

The Esaote MyLab70 ultrasound system has been extensively used to evaluate arterial properties. Since it is reaching end-of-service-life, ongoing studies are forced to seek an alternative, with some opting for the Esaote MyLabOne. Biases might exist between the two systems, which, if uncorrected, could potentially lead to the misinterpretation of results. This study aims to evaluate a potential bias between the two devices. Moreover, by comparing two identical MyLabOne systems, this study also aims to investigate whether biases estimated between the MyLabOne and MyLab70 employed in this study could be generalized to any other pair of similar scanners. Using a phantom set-up, we performed n = 60 measurements to compare MyLab70 to MyLabOne and n = 40 measurements to compare the two MyLabOne systems. Comparisons were performed to measure diameter, wall thickness, and distension. Both comparisons led to significant biases for the diameter (relative bias: −0.27% and −0.30% for the inter- and intra-scanner model, respectively, p < 0.05) and wall thickness (relative bias: 0.38% and −1.23% for inter- and intra-scanner model, respectively p < 0.05), but not for distension (relative bias: 0.48% and −0.12% for inter- and intra-scanner model, respectively, p > 0.05). The biases estimated here cannot be generalized to any other pair of similar scanners. Therefore, longitudinal studies with large sample sizes switching between scanners should perform a preliminary comparison to evaluate potential biases between their devices. Furthermore, caution is warranted when using biases reported in similar comparative studies. Further work should evaluate the presence and relevance of similar biases in human data.

We aimed to investigate the association between Red Blood Cell Distribution Width (RDW) and Neutrophil-to-Lymphocyte Ratio (NLR), simple, rapidly assessed markers from the complete blood count with vascular calcification (VC)/stiffness and cardiovascular disease (CVD) in chronic kidney disease (CKD). Dephosphorylated, uncarboxylated matrix Gla-protein (dp-ucMGP), and central/peripheral hemodynamics’ parameters were measured in 158 CKD patients, including Hemodialysis and Peritoneal Dialysis. Spearman’s rho analysis showed that RDW correlated with C-reactive protein (CRP) (r = 0.29, p < 0.001), dp-ucMGP (r = 0.43, p = < 0.0001), central diastolic blood pressure (DBP) (r = −0.19, p = 0.02), and albuminuria (r = −0.17, p = 0.03). NLR correlated with the duration of CVD (r = 0.32, p < 0.001), CRP (r = 0.27, p = 0.01), dp-ucMGP (r = 0.43, p < 0.0001), central DBP (r = −0.32, p < 0.0001) and eGFR (r = −0.25, p = 0.04). In multiple regression models, circulating dp-ucMGP was an independent predictor of RDW (β = 0.001, p = 0.001) and NLR (β = 0.002, p = 0.002). In CKD patients, RDW and NLR are associated with traditional and novel markers of VC and CVD.

Background: Reliable assays to measure direct oral anticoagulant (DOAC) levels and their activity in critical situations are needed. Drug levels alone are not representative of the effect of DOACs on an individual's coagulation. We developed a technique that provides direct assessment of the global effect of rivaroxaban on the individual's coagulation in addition to plasma concentrations.Methods: DOAC concentrations were determined in fifty patients using rivaroxaban, with the new assay, Xross-CAT. The effect of rivaroxaban on coagulation (activity) was measured with thrombin generation (TG) in platelet poor plasma using 5 pM tissue factor on the same device. The levels were validated with the Biophen DiXal assay. The prothrombin time (PT) and dilute Russell viper venom time (dRVVT) were performed to estimate the effect on coagulation.Results: The variability of Xross-CAT was below 12%. Xross-CAT correlates well with Biophen DiXaI (r(s) = 0.885). The bias, determined by Bland-Altman analysis, was 4.9% and the Passing-Bablok equation was y = 1.1x - 2.1. The correlation of plasma levels with TG was moderate (ETP r(s) = -0.548; Peak r(s) = -0.559), as for the PT (r(s) = 0.739) and the dRVVT (r(s) = 0.692).Conclusions: Xross-CAT shows a good correlation with Biophen DiXaI that was previously confirmed to accurately assess rivaroxaban levels. Bleeding and thrombotic complications are not necessarily associated with drug levels and could be influenced by concomitant risk factors. The main benefit of Xross-CAT is that it can be performed simultaneously with thrombin generation, providing an overview of the global anticoagulation status of a patient in relation to circulating DOAC levels.

Outline of this thesisThis thesis focuses on measurements of circulating MGP species and their potential diagnostic and clinical value. Chapter 1 gives an introduction of MGP and vitamin K. In Chapter 2, the mono-antibody MGP ELISA measuring total uncarboxylated MGP (t-ucMGP) levels is presented and characterised, with application of t-ucMGP measurements in patient populations with cardiovascular disease (angioplasty and aortic valve stenosis) and vascular calcification (hemodialysis and calciphylaxis). Further investigation of t-ucMGP as a biomarker in dialysis patients is described in Chapters 3 and 4. Chapter 3 describes the correlation between t-ucMGP levels and the extent of coronary artery calcification in hemodialysis patients. In Chapter 4, the association between t-ucMGP levels and (cardiovascular) mortality in a large Dutch cohort of hemodialysis and peritoneal dialysis patients is investigated. In Chapter 5, we compared the four available MGP assays, including two novel dual-antibody MGP ELISA. All assays were applied in patients with cardiovascular disease, rheumatic diseases, and end-stage renal disease. To determine which assay would be suited to assess vascular vitamin K status, the effect of changes in vitamin K status on circulating MGP species was also investigated. For this, the assays were applied in two groups of healthy subjects with opposite vitamin K status: subjects receiving vitamin K supplementation and those receiving vitamin K antagonists. In Chapter 6, we investigated the functions of MGP in humans by phenotypic characterization of a newly-identified Dutch patient with Keutel syndrome. In order to further elucidate the difference in phenotype between MGP deficient mice and men, we characterized the circulating MGP species of this patient and his first-degree relatives. Additionally, MGP species were determined in tissues from the first described Keutel syndrome patient in the literature. Chapter 7 describes the investigation of the vitamin K intake and the hepatic and extra-hepatic vitamin K status in a Dutch cohort of hemodialysis patients.

This dissertation has shown by means of a goat model that the behaviour of atrial fibrillation is determined to a large extent by underlying muscle fibres. It was also found that paroxysmal atrial fibrillation in humans is far less complex than long-standing atrial fibrillation, although paroxysmal atrial fibrillation varies considerably in complexity. It was shown that only one particular type of fibrosis is relevant to the behaviour of atrial fibrillation rather than all types of fibrosis as previously assumed. Furthermore, it was shown why a particular type of ablation therapy for atrial fibrillation is less effective. Finally, a new software programme was developed allowing atrial fibrillation to be analysed very quickly.

Background While single‐omics analyses on human atherosclerotic plaque have been very useful to map stage‐ or disease‐related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale‐intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model. Methods In this study, we compared protein and gene makeup of low‐ versus high‐risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. Results We identified a protein‐gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA−PDGFRα+ fibroblast‐like cell content (p = 2.4E‐05) and Arg1+ macrophage content (p = 2.2E‐04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia‐1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. Conclusions In conclusion, our integrative omics study has identified an intraplaque hemorrhage‐associated cardiovascular signature that provides excellent stratification of low‐ from high‐risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF‐regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization., This multiomics analysis identified a cardiovascular signature in carotid atherosclerotic lesions, which provides excellent stratification of low‐/high‐risk carotid plaques.This study highlights the advantages of multiomics analysis in terms of model robustness, biological significance, and clinical translatability.The prediction model pointed to an SRF‐regulated disease network providing valuable new insights that expedite the design of targeted intervention in plaque rupture.

Summary Background Endovascular renal denervation reduces blood pressure in patients with mild-to-moderate hypertension, but its efficacy in patients with true resistant hypertension has not been shown. We aimed to assess the efficacy and safety of endovascular ultrasound renal denervation in patients with hypertension resistant to three or more antihypertensive medications. Methods In a randomised, international, multicentre, single-blind, sham-controlled trial done at 28 tertiary centres in the USA and 25 in Europe, we included patients aged 18–75 years with office blood pressure of at least 140/90 mm Hg despite three or more antihypertensive medications including a diuretic. Eligible patients were switched to a once daily, fixed-dose, single-pill combination of a calcium channel blocker, an angiotensin receptor blocker, and a thiazide diuretic. After 4 weeks of standardised therapy, patients with daytime ambulatory blood pressure of at least 135/85 mm Hg were randomly assigned (1:1) by computer (stratified by centres) to ultrasound renal denervation or a sham procedure. Patients and outcome assessors were masked to randomisation. Addition of antihypertensive medications was allowed if specified blood pressure thresholds were exceeded. The primary endpoint was the change in daytime ambulatory systolic blood pressure at 2 months in the intention-to-treat population. Safety was also assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov , NCT02649426 . Findings Between March 11, 2016, and March 13, 2020, 989 participants were enrolled and 136 were randomly assigned to renal denervation (n=69) or a sham procedure (n=67). Full adherence to the combination medications at 2 months among patients with urine samples was similar in both groups (42 [82%] of 51 in the renal denervation group vs 47 [82%] of 57 in the sham procedure group; p=0·99). Renal denervation reduced daytime ambulatory systolic blood pressure more than the sham procedure (−8·0 mm Hg [IQR –16·4 to 0·0] vs –3·0 mm Hg [–10·3 to 1·8]; median between-group difference –4·5 mm Hg [95% CI –8·5 to –0·3]; adjusted p=0·022); the median between-group difference was –5·8 mm Hg (95% CI –9·7 to –1·6; adjusted p=0·0051) among patients with complete ambulatory blood pressure data. There were no differences in safety outcomes between the two groups. Interpretation Compared with a sham procedure, ultrasound renal denervation reduced blood pressure at 2 months in patients with hypertension resistant to a standardised triple combination pill. If the blood pressure lowering effect and safety of renal denervation are maintained in the long term, renal denervation might be an alternative to the addition of further antihypertensive medications in patients with resistant hypertension. Funding ReCor Medical.

1380 Objectives: The purpose of this study was to investigate how gender, BMI, patient cross-sectional area and heart size affect the influence of time-of-flight (TOF) and point spread function (PSF) modeling in image reconstruction on regional absolute myocardial blood flow (MBF) measured with 82Rb PET in an obese population. Methods: Dynamic rest/stress 82Rb cardiac PET studies were performed for 30 obese subjects on a TOF PET scanner. Subject characteristics were (mean ± SD for continuous variables): gender 9 male, 21 female; weight 138.9 ± 32.8 kg; BMI 47.8 ± 9.5 kg/m2; heart size 92.4 ± 55.3 ml; cross-sectional area in a transaxial slice containing the heart 1133 ± 233 cm2. Images were reconstructed in four ways: (1) OSEM without TOF or PSF modeling (OSEM), (2) OSEM with TOF (TOF), (3) OSEM with PSF modeling (PSF) and (4) OSEM with TOF and PSF modeling (PSFTOF). All were with 3 iterations, 8 subsets and with a post-reconstruction 8mm 3D Gaussian filter. A one tissue compartmental model was used to compute MBF. Average rest and stress flow and myocardial flow reserve were computed in five regions: anterior, septal, inferior, lateral and apical. Results from TOF, PSF and PSFTOF were compared with those from OSEM, with subjects grouped by gender, and by tertiles (10 subjects each) for BMI (I: 32.5-40.5, II: 41.1-52.3, III: 53.3-75.6 (kg/m2)), cross-sectional area (I: 532 - 1086, II: 1089 - 1157, III: 1187 - 1662 (cm2)) and heart size (I: 19 -56, II: 66 - 93, III: 97 - 238 (ml)). The MBF or myocardial flow reserve (MFR) in a given region was compared with the corresponding OSEM value and the change was expressed as a percent. Results: The general trends for all subjects with TOF were similar MBF increases in the apical (+22.0%) and septal walls (+19.4%), and a slightly lower increase in the anterior wall (+11.3 %). Increases were small in the inferior (+4.6%) and lateral (+4.9%) walls. There was little effect of PSF modeling in any region. MBF trends with PSFTOF were similar to those for TOF alone. Rest and stress increases with TOF tended to be matched, so that MFR was little changed. Trends were examined for the groupings by patient characteristics. (1) Gender. MBF showed a greater increase with TOF for females (mean +20.9%) compared to males (mean +14.9%) in the apical, septal and anterior walls. Results with PSFTOF were similar. (2) BMI. The MBF increase with TOF in the apical, anterior and septal walls was greatest for tertile II (mean +26.9%) compared with tertile I (mean +13.6%) and tertile III (mean +16.7%). (3) Cross-sectional area. The MBF increase with TOF in the apical, anterior and septal walls was similar for tertiles II (mean +22.1%) and III (mean +22.6%), and was greater than that for tertile I (mean +12.6%). (4) Heart size. The MBF increase with TOF in the apical, anterior and septal walls was greater for smaller heart sizes: tertile I (mean +21.2%), tertile II (+20.7%) and tertile III (+15.3%). These results are for our patient population and particular hardware, software and processing implementation. Conclusions: Patient gender, BMI, cross-sectional area and heart size affect the degree to which MBF values change with TOF. The largest changes are observed for females, patients with larger BMI, larger cross-sectional area and smaller hearts. The greatest changes are found in the apical, septal and lateral walls. The studied patient characteristics have little effect on flow reserve. It is important to consider these patient-dependent effects when evaluating absolute MBF in a clinical environment, and to recognize that equipment or protocol changes may affect MBF results.

Introduction Although physical exercise is protective against cardiovascular disease, it can also provoke sudden cardiac death (exercise paradox). Epidemiological studies suggest that systemic hypoxia at high altitude is a risk factor for venous thromboembolism. Forthcoming, this study investigated the effect of repeated exercise at high altitude on blood coagulation, platelet function, and fibrinolysis. Methods Six trained male volunteers were recruited. Participants ascended from sea level to 3,375 m altitude. They performed four exercise tests at 65 to 80% of their heart-rate reserve during 2 hours: one time at sea level and three times on consecutive days at 3,375 m altitude. Thrombin generation (TG) was measured in whole blood (WB) and platelet-rich and platelet-poor plasma. Coagulation factor levels were measured. Platelet activation was measured as αIIbβ3 activation and P-selectin expression. Fibrinolysis was studied using a clot-lysis assay. Results Normoxic exercise increased plasma peak TG through increased factor VIII (FVIII), and increased von Willebrand factor (VWF) and active VWF levels. Platelet granule release potential was slightly decreased. After repetitive hypoxic exercise, the increase in (active) VWF tapered, and there was no more distinct exercise-related increase in peak. Platelet aggregation potential and platelet-dependent TG decreased at high altitude. There were no effects on fibrinolysis upon exercise and/or hypoxia. Conclusion Strenuous exercise induces a procoagulant state that is mediated by the endothelium, by increasing VWF and secondarily raising FVIII levels. After repetitive exercise, the amplitude of the endothelial response to exercise diminishes. A hypoxic environment appears to further attenuate the procoagulant changes by decreasing platelet activation and platelet-dependent TG.

Background and aims: Obese individuals have a higher risk of developing atherosclerosis, possibly driven by adipose tissue (AT) inflammation. We recently showed that AT macrophages (ATMs), which accumulate in the expanding obese AT, produce mediators causing immune cell recruitment from the bone marrow. In the current study, we evaluated whether ATMs are directly involved in atherosclerotic plaque development.Methods: Lean ldlr(-/-) acceptor mice received visceral AT (vAT) from lean, obese, or ATM-depleted obese ldlr(-/-) mice. Acceptor mice were fed high cholesterol diet (HCD) for 4 weeks before and 8 weeks after AT transplantation to induce atherosclerosis. Atherosclerotic plaque development was studied 8 weeks after transplantation.Results: Transplanting donor vAT from obese mice increased circulating triglycerides and B-cells, but decreased Ly6c(-) monocytes. Plasma cholesterol, Ly6c(+) monocytes, T-cells, NK-cells and eosinophils were unaffected. Depleting ATMs from obese AT using clodronate liposomes prior to vAT transplantation prevented the increase in triglycerides and B-cells and decrease in Ly6c(-) monocytes, but did increase eosinophils. Circulating Cxcl1 was reduced by obese AT transplantation and Ifn-gamma tended to be increased while Tnf and Il-1 beta were unaffected. ATM-depleted obese AT transplantation also reduced Cxcl1, but increased circulating Tnf levels. However, obese AT transplantation with or without depletion of ATMs did not influence atherosclerotic plaque size, phenotype, or stability.Conclusions: ATMs from obese vAT do not affect atherosclerotic plaque development or phenotype.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

Introduction: Congenital atrioventricular block (CAVB) is a rare disorder with a significant morbidity and mortality. Consensus regarding the prescription and efficacy of prenatal corticosteroids is lacking. This nationwide study was initiated to evaluate the effects of prenatal treatment with corticosteroids on the outcome of CAVB in The Netherlands.Methods: All fetuses identified with isolated congenital AVB-II degrees or AVB-III degrees in any of the eight academic fetal heart centers of The Netherlands between 2003 and 2013 were included and reviewed.Results: Fifty-six fetuses were included. Fourteen (25%) fetuses were treated with dexamethasone. We found no differences between the steroid-treated and untreated cases regarding in utero progression of the AVB (63% vs 67% respectively), survival to birth (86% vs 84%), pacemaker implantations (74% vs 58%) or long-term dilated cardiomyopathy (13% vs 17%). Steroid treated fetuses demonstrated more in utero growth restriction (38% vs 11%).Conclusion: No benefit from prenatal corticosteroid treatment was demonstrated for fetuses with isolated CAVB in this study. However, we found negative side effects. Our data provide no evidence to support the routine administration of corticosteroids for the treatment of fetal CAVB. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Heparin was discovered around 1922 by Howell (Baltimore) and was further developed by the teams of Best (Toronto) and Jorpes (Stockholm). Kakkar (London) propagated its routine use for the prevention of postoperative thrombosis from 1971 onwards. The discovery of low molecular weight heparins (1976, Johnson, London) and their development in the subsequent years led to the present arsenal of clinically useful drugs.In 1976, three teams independently found that a specific structure in heparin binds tightly to antithrombin. This enabled the teams of Lindahl (Stockholm) and Casu (Milan) to determine the pentasaccharide structure responsible for this binding and Petitou, from the Choay team (Paris), to synthesize it (1983).It was found (Olson and others) that heparin facilitates the interaction between antithrombin and a clotting enzyme by allosteric changes in the antithrombin (important for factor Xa) and by facilitating the approach of the enzyme to antithrombin via its "sliding" along the heparin molecule (important for thrombin). Antithrombin action therefore requires a minimum length of seven sugar units next to the pentasaccharide whereas anti-factor Xa action does not. The effect of heparin is almost entirely due to anti-thrombin action (Beguin), so anti-factor Xa activity does not reflect the concentration of anticoagulant heparin. The anticoagulant effect is poorly reflected by the activated partial thromboplastin time. Because present clinical use is based on the latter tests, it is not generally known that the individual response to heparin shows an extremely wide variation. Individualization of heparin dosage is likely to improve clinical results.

Contains fulltext : 168089.pdf (Publisher’s version ) (Open Access) Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems.

Introduction: An inadequate wound healing following myocardial infarction (MI) is one of the main etiologies of heart failure (HF) development. Interventions aiming at improving this process may contribute to preserving cardiac function after MI. Our group, as well as others, have demonstrated the crucial role of Wnt/frizzled signaling in post-MI remodeling. In this overview, we provide the results of different studies aimed at confirming an initial study from our group, in which we observed beneficial effects of administration of a peptide fragment of Wnt5a, UM206, on infarct healing in a mouse MI model.Methods: Mice were subjected to permanent left coronary artery ligation, and treated with saline (control) or UM206, administered via osmotic minipumps. Cardiac function was assessed by echocardiography and hemodynamic measurements, while infarct size and myofibroblast content were characterized by (immuno)histochemistry.Results: In total, we performed seven follow-up studies, but we were unable to reproduce the beneficial effects of UM206 on infarct healing in most of them. Variations in dose and timing of UM206 administration, its manufacturer and the genetic background of the mice could not restore the phenotype. An in-depth analysis of the datasets revealed that the absence of effect of UM206 coincided with a lack of adverse cardiac remodeling and HF development in all experimental groups, irrespective of the treatment.Discussion: Irreproducibility of experimental observations is a major issue in biomedical sciences. It can arise from a relatively low number of experimental observations in the original study, a faulty hypothesis or a variation in the experimental model that cannot be controlled. In this case, the lack of adverse cardiac remodeling and lung weight increases in the follow-up studies point out to altered experimental conditions as the most likely explanation.

Atherosclerosis is a progressive inflammatory vascular disorder, complicated by plaque rupture and subsequently atherothrombosis. In vitro studies indicate that key clotting proteases, such as factor Xa (FXa), can promote atherosclerosis, presumably mediated through protease activated receptors (PARs). Although experimental studies showed reduced onset of atherosclerosis upon FXa inhibition, the effect on pre-existing plaques has never been studied. Therefore, we investigated effects of FXa inhibition by rivaroxaban on both newly-formed and pre-existing atherosclerotic plaques in apolipoprotein-e deficient (ApoE−/−) mice. Female ApoE−/− mice (age: 8–9 weeks, n = 10/group) received western type diet (WTD) or WTD supplemented with rivaroxaban (1.2 mg/g) for 14 weeks. In a second arm, mice received a WTD for 14 weeks, followed by continuation with either WTD or WTD supplemented with rivaroxaban (1.2 mg/g) for 6 weeks (total 20 weeks). Atherosclerotic burden in aortic arch was assessed by haematoxilin & eosin immunohistochemistry (IHC); plaque vulnerability was examined by IHC against macrophages, collagen, vascular smooth muscle cells (VSMC) and matrix metalloproteinases (MMPs). In addition, PAR1 and -2 expressions and their main activators thrombin and FXa in the plaque were determined in the plaque. Administration of rivaroxaban at human therapeutic concentrations reduced the onset of atherosclerosis (−46%, p 0.05), and promoted a regression of pre-existing plaques in the carotids (−24%, p 0.001). In addition, the vulnerability of pre-existing plaques was reduced by FXa inhibition as reflected by reduced macrophages (−39.03%, p 0.05), enhanced collagen deposition (+38.47%, p 0.05) and diminished necrotic core (−31.39%, p 0.05). These findings were accompanied with elevated vascular smooth muscle cells and reduced MMPs. Furthermore, expression of PARs and their activators, thrombin and FXa was diminished after rivaroxaban treatment. Pharmacological inhibition of FXa promotes regression of advanced atherosclerotic plaques and enhances plaque stability. These data suggest that inhibition of FXa may be beneficial in prevention and regression of atherosclerosis, possibly mediated through reduced activation of PARs.

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3′ to the 5′ end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5′ end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling., Raso et al. show that miR-148a is elevated in concentric hypertrophy and decreased in dilated cardiomyopathy. Adeno-associated viral delivery of miR-148a protects the mouse heart from cardiac dilation. Mechanistically, miR-148a regulates the sensitivity of the heart to extracellular cytokines.

Background: Sunitinib is an oral tyrosine kinase inhibitor used for cancer treatment. Patients treated with sunitinib are at higher bleeding risk. As tyrosine kinases are essential for platelet signalling, the effects of sunitinib on platelet function in vitro and in cancer patients on treatment were investigated.Patients and methods: Blood samples were collected from eight healthy volunteers and eight patients diagnosed with metastatic renal cell cancer (RCC) before and 2 weeks on treatment with sunitinib. Platelets from 15 additional healthy individuals were preincubated with sunitinib or vehicle to perform in vitro experiments. Immunofluorescence imaging, western blotting, light transmission aggregometry, whole blood perfusion over collagen, flow cytometry and ELISA were performed.Results: Confocal microscopy indicated that platelets sequester sunitinib in vitro and in patients. In platelets from healthy controls, tyrosine phosphorylation was inhibited by sunitinib. Also, sunitinib dose dependently reduced collagen- and ADP-induced aggregation, collagen-dependent thrombus formation and collagen-induced secretion of platelet-derived growth factor and β-thromboglobulin. In blood from RCC patients before treatment, thrombus formation and procoagulant activity under flow were 47% and 80% higher than in healthy controls. After 14 d of sunitinib treatment, platelet count was moderately, but significantly decreased (from 243 to 144 × 109/l). At the same time, collagen-induced platelet aggregation as well as thrombus formation and phosphatidylserine exposure under flow were significantly reduced (by 45%, 16% and 61%, respectively).Conclusions: Sunitinib uptake by platelets inhibits collagen receptor-induced aggregation and thrombus formation via reduction of protein tyrosine phosphorylation and α-granule secretion. This dysfunction may contribute to the higher bleeding tendency observed in sunitinib-treated patients.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Objectives To determine whether audio and visual distraction can affect the pain perception of a patient undergoing endovenous thermal ablation under tumescent anesthesia. Methods Patients underwent an endovenous thermal ablation procedure and were randomized into two groups: non-distraction (AVD−) and with distraction (AVD+). Visual analogue scale pain score and hospital anxiety depression scale scores were used to measure outcome. The recruited patients were asked to submit an anticipated visual analogue scale pain score prior to treatment as well as the actual experienced post-operative visual analogue scale pain score. Results There was no significant difference between the AVD− and AVD+ groups post-operative pain score 2.85 (SD 1.7) versus 2.60 (SD 2.3), p = 0.68. Pain score in women with distraction (AVD+) decreased from 3.81 to 2.42. The pain score in men with distraction (AVD+) increases from 1.88 to 2.82. In the AVD− group, a significant difference was found between men and women ( p = 0.014). Disregarding gender, a significant difference was found between the anticipated pain score and the actual pain score in both the AVD− group ( p = 0.009) and AVD+ group ( p = 0.021). There was a correlation between depression and the pain score, which was not seen between pain and anxiety score. Conclusion The results suggested that audiovisual distraction has no influence on the visual analogue scale pain score during endovenous thermal ablation under tumescent anesthesia. Despite this, patients appreciate and evaluate audio and visual distraction as pleasant to extremely pleasant when applied.

Purpose We discuss a case of a brachiocephalic vein (BCV) perforation after Tesio® central venous catheter insertion. Method and Results An 80-year-old patient underwent an ultrasound-guided hemodialysis (HD) catheter placement via his left internal jugular vein (IJV). One day postoperatively, the patient became hemodynamically unstable immediately after HD initiation. As a vascular event was feared, an emergency CT scan was performed demonstrating a BCV perforation. The patient underwent a sternotomy, the lines were removed and the venous laceration was closed. The patient recovered well. Conclusions In spite of ultrasound guidance, fluoroscopy for guidewire and sheath advancement, venous blood aspiration and a normal appearing postoperative x-ray, traumatic central venous catheter placement is still possible. Tenting of the BCV wall during catheter advancement possibly caused the venous perforation. A ‘how-to’ for correct catheter placement via the IJV is provided and potential pitfalls during each procedural step are discussed.

Citrus pectin is widely used as a gelling/thickening agent and stabilizer in the food and pharmaceutical industries. The usefulness of citrus pectin, however, has been limited due to its large size, an obstacle that can be partially overcome with pH-modified citrus pectin (MCP). In the present study, we fractionated MCP into four fractions (MCP-1, −2, −3, and −4) and then sub-fractions of type-I rhamnogalacturonan-rich pectins (RG-I: MCP-1a, MCP-2a, MCP-3Sa and MCP-4a) and homogalacturonan-rich pectins (HG: MCP-2b, MCP-3Sb, MCP-3P, MCP-4b). Upon analysis of their chemical structures, we found that the RG-I sub-fractions contain (1 → 4)-linked β- D -galactan and (1 → 5)-linked α- L -arabinans or type-I arabinogalactan side-chains. Because parent MCP is known to inhibit hemagglutination mediated by galectin-3 (a lectin associated with cancer progression and metastasis), we used this assay to define which MCP fraction best promotes anti-galectin-3 activity. Our results demonstrate that MCP-2 was the most inhibitory with a MIC of 0.06 μg/ml (ten-fold more potent than parent MCP), and that RG-I-rich pectins with (1 → 4)-linked β- D -galactan side-chains were more active than the other RG-I-rich and HG-rich pectins. These findings provide new insight into structure-activity relationships of MCP and provide impetus towards the development of MCP-based food stabilizers and galectin-3 targeted therapeutics for use in the clinical setting.

Objective Air plethysmography (APG) is a functional, noninvasive test that can assess volumetric changes in the lower limb and might therefore be used as a diagnostic tool in chronic deep venous disease. However, use of APG in chronic deep venous obstructive disease remains debatable. This study assessed the clinical value of APG in identifying chronic deep venous obstruction. Methods All patients referred to our tertiary, outpatient clinic between January 2011 and August 2013 with chronic venous complaints and suspected outflow obstruction underwent an outflow fraction (OF), ejection fraction (EF), and residual volume fraction (RVF) test using APG. Duplex ultrasound and magnetic resonance venography were used to establish whether and where obstruction was present. Diagnostic values of these tests were assessed for obstructions at different levels of the deep venous system. Results A total of 312 limbs in 248 patients were tested. Mean age was 45.5 ± 14.0 years, and 62.5% were female. In post-thrombotic disease, specificity and positive predictive value for OF were as high as 98.4% and 95.0%, respectively; however, sensitivity was 34.8% and negative predictive value was 29.6%, with no clinically relevant positive or negative likelihood ratios. No clinically relevant differences were observed in stratifying for level of obstruction. EF and RVF were as inconclusive. Neither could these parameters be used in diagnosing nonthrombotic iliac vein compression. Conclusions We found a poor correlation between OF, EF, or RVF, determined by APG, and the presence of chronic deep venous obstruction. Therefore, use of its relative parameters is unwarranted in daily clinical practice.

SummaryThrombin generation (TG) is decreased in children. TG is determined by two underlying processes: the conversion of prothrombin to thrombin and the inactivation of thrombin. Therefore, lower TG capacity in children can either be caused by a reduction of prothrombin conversion, an increase of thrombin inactivation, or both. In 36 children and 8 adults, TG and the factors that determine thrombin inactivation (antithrombin, α2 Macroglobulin (α2M) and fibrinogen) were measured. Prothrombin conversion, thrombin inhibitor complex formation, and the overall thrombin decay capacity were determined. In silico modelling was performed to determine the contribution prothrombin conversion and thrombin inactivation to deviant paediatric TG. Both the amount of prothrombin converted and the maximal prothrombin conversion rate are significantly reduced in children as compared to adults. This is partly due to the prothrombin levels being lower and partly to a lower prothrombin conversion rate. The overall thrombin decay capacity is not significantly different in children, but α2Macroglobulin plays a more important role than it does in adults. In silico experiments demonstrate that reduced prothrombin conversion and to a lesser extent elevated α2M levels provide an explanation for low TG in children. Young age has a dual effect on prothrombin conversion. Lower plasma prothrombin levels result in decreased prothrombin conversion but the rate of prothrombin conversion is also decreased, i. e. the development of prothrombinase is lower than in adults.