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Aim: To uncover differences in small non-coding RNAs (sncRNAs) in individuals with type 2 diabetes (T2D) categorized into five clusters based on individual characteristics, which may aid in the identification of those prone to rapid progression., Materials and Methods: In the Hoorn Diabetes Care System (DCS) cohort, participants were clustered by age, body mass index (BMI), and glycated haemoglobin, C-peptide and high-density lipoprotein (HDL) cholesterol levels, yielding severe insulin-deficient diabetes, severe insulin-resistant diabetes (SIRD), mild obesity-related diabetes (MOD), mild diabetes, and mild diabetes with high HDL cholesterol clusters (n = 412). Utilizing plasma sncRNA-sequencing, we identified distinct cluster-specific sncRNAs. Validation was performed in a smaller DCS Hoorn dataset (n = 138). To elucidate their potential functions, we examined tissue expression, identified potential targets or (co-)regulated proteins, conducted gene set enrichment analyses on the targets through Reactome, and examined tissue expression of the (co-)regulated proteins., Results: The insulin-resistant cluster exhibited aberrant expression of 10 sncRNAs, while the high BMI cluster featured eight differentially expressed sncRNAs. Multiple (co-)regulated proteins were identified for sncRNAs associated with both clusters. Proteins associated with both clusters showed enrichment for metabolism. Proteins that specifically and only associated with the SIRD cluster showed enrichment for immune-related signalling. Furthermore, MOD cluster-specific associated proteins showed enrichment for the complement system., Conclusions: Our research showed differential sncRNA levels among type 2 diabetes clusters. This may reflect and could deepen our understanding of molecular mechanisms, in development, progression, and risk factors for each cluster., (© 2024 The Author(s). Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Puumala orthohantavirus (PUUV) infection in humans can result in hemorrhagic fever with renal syndrome. Endothelial cells (ECs) are primarily infected with increased vascular permeability as a central aspect of pathogenesis. Historically, most studies included ECs cultured under static two-dimensional (2D) conditions, thereby not recapitulating the physiological environment due to their lack of flow and inherent pro-inflammatory state. Here, we present a high-throughput model for culturing primary human umbilical vein ECs in 3D vessels-on-chip in which we compared host responses of these ECs to those of static 2D-cultured ECs on a transcriptional level. The phenotype of ECs in vessels-on-chip more closely resembled the in vivo situation due to higher similarity in expression of genes encoding described markers for disease severity and coagulopathy, including IDO1 , LGALS3BP , IL6 and PLAT , and more diverse endothelial-leukocyte interactions in the context of PUUV infection. In these vessels-on-chip, PUUV infection did not directly increase vascular permeability, but increased monocyte adhesion. This platform can be used for studying pathogenesis and assessment of possible therapeutics for other endotheliotropic viruses even in high biocontainment facilities.

Torque Teno Virus (TTV) is a non-pathogenic anellovirus, highly prevalent in healthy populations. Variations in its viral load have been associated with states of diminished immunity, as occurs after organ transplantation. It is hypothesized that TTV-load might be used as a diagnostic tool to guide prescription and dosing of immunosuppressive drugs. Not much is known about the effects of combined immunosuppressive drugs on TTV replication in renal transplantation. Belatacept was introduced to counter side-effects of calcineurin inhibitors (CNI). It was never widely adopted, mainly because its association with increased risk of rejection. To investigate the differential effects of a regimen based on calcineurin inhibitors versus belatacept on TTV-loads, we measured TTV-levels in 105 patients from two randomized controlled trials in kidney transplant recipients (KTRs). We observed that time after transplantation was inversely related to TTV-levels of patients that remained on a CNI-containing regime, whereas this decline over time was diminished after conversion to belatacept. In addition, a correlation with tacrolimus-trough levels and age were found. Our study is the first report on the impact of conversion from CNI to belatacept on TTV-levels in KTR. In conclusion, the time-related decline in TTV-levels is mitigated after conversion from CNI to belatacept., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Endothelial dysfunction, prevalent in cardiovascular diseases (CVDs) and linked to conditions like diabetes, hypertension, obesity, renal failure, or hypercholesterolemia, is characterized by diminished nitric oxide (NO) bioavailability-a key signaling molecule for vascular homeostasis. Current two-dimensional (2D) in vitro studies on NO synthesis by endothelial cells (ECs) lack the crucial laminar shear stress, a vital factor in modulating the NO-generating enzyme, endothelial nitric oxide synthase (eNOS), under physiological conditions. Here we developed a tracer-based metabolomics approach to measure NO-specific metabolites with mass spectrometry (MS) and show the impact of fluid flow on metabolic parameters associated with NO synthesis using 2D and 3D platforms. Specifically, we tracked the conversion of stable-isotope labeled NO substrate L-Arginine to L-Citrulline and L-Ornithine to determine eNOS activity. We demonstrated clear responses in human coronary artery endothelial cells (HCAECs) cultured with 13 C 6 , 15 N 4 -L-Arginine, and treated with eNOS stimulator, eNOS inhibitor, and arginase inhibitor. Analysis of downstream metabolites, 13 C 6 , 15 N 3 L-Citrulline and 13 C 5 , 15 N 2 L-Ornithine, revealed distinct outcomes. Additionally, we evaluated the NO metabolic status in static 2D culture and 3D microvessel models with bidirectional and unidirectional fluid flow. Our 3D model exhibited significant effects, particularly in microvessels exposed to the eNOS stimulator, as indicated by the 13 C 6 , 15 N 3 L-Citrulline/ 13 C 5 , 15 N 2 L-Ornithine ratio, compared to the 2D culture. The obtained results indicate that the 2D static culture mimics an endothelial dysfunction status, while the 3D model with a unidirectional fluid flow provides a more representative physiological environment that provides a better model to study endothelial dysfunction., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Cancer increases the risk of venous thromboembolism, and glioblastoma is one of the cancer types with the highest risk of venous thromboembolism (10%-30%). Tumor-intrinsic features are believed to affect vascular permeability and hypercoagulability, but novel models are required to study the pathophysiological dynamics underlying cancer-associated thrombosis at the molecular level., Objectives: We have developed a novel cancer-on-a-chip model to examine the effects of glioblastoma cells on the deregulation of blood coagulation., Methods: This was accomplished by coculturing vessel-forming human umbilical vein endothelial cells with glioblastoma spheroids overexpressing tissue factor (TF), the initiator of coagulation (U251 lentivirus, LV-TF) or an LV-control (U251 LV-Ctrl) in an OrganoPlate Graft platform., Results: Using a modified thrombin generation assay inside the cancer-on-a-chip, we found that U251 LV-Ctrl and U251 LV-TF spheroids promoted an increased procoagulant state in plasma, as was shown by a 3.1- and 7.0-fold increase in endogenous thrombin potential, respectively. Furthermore, the anticoagulant drug rivaroxaban and TF coagulation-blocking antibody 5G9 inhibited the activation of blood coagulation in U251 LV-TF spheroid-containing graft plates, as was shown by a reduced endogenous thrombin potential (4.0- and 4.4-fold, respectively)., Conclusion: With this study, we present a novel 3-dimensional cancer-on-a-chip model that has the potential to be used in the discovery of new anticoagulant drugs and identification of optimal anticoagulant strategies for glioblastoma and other cancer types., (© 2024 The Author(s).)

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed. High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions. In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Aim: To investigate the association of plasma metabolites with incident and prevalent chronic kidney disease (CKD) in people with type 2 diabetes and establish whether this association is causal., Materials and Methods: The Hoorn Diabetes Care System cohort is a large prospective cohort consisting of individuals with type 2 diabetes from the northwest part of the Netherlands. In this cohort we assessed the association of baseline plasma levels of 172 metabolites with incident (N total  = 462/N case  = 81) and prevalent (N total  = 1247/N case  = 120) CKD using logistic regression. Additionally, replication in the UK Biobank, body mass index (BMI) mediation and causality of the association with Mendelian randomization was performed., Results: Elevated levels of total and individual branched-chain amino acids (BCAAs)-valine, leucine and isoleucine-were associated with an increased risk of incident CKD, but with reduced odds of prevalent CKD, where BMI was identified as an effect modifier. The observed inverse effects were replicated in the UK Biobank. Mendelian randomization analysis did not provide evidence for a causal relationship between BCAAs and prevalent CKD., Conclusions: Our study shows the intricate relationship between plasma BCAA levels and CKD in individuals with type 2 diabetes. While an association exists, its manifestation varies based on disease status and BMI, with no definitive evidence supporting a causal link between BCAAs and prevalent CKD., (© 2024 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Aim: Sodium glucose co-transporter-2 (SGLT2) inhibitors stimulate renal excretion of sodium and glucose and exert renal protective effects in patients with (non-)diabetic chronic kidney disease (CKD) and may as well protect against acute kidney injury (AKI). The mechanism behind this kidney protective effect remains unclear. Juxtaglomerular cells of renin lineage (CoRL) have been demonstrated to function as progenitors for multiple adult glomerular cell types in kidney disease. This study assesses the impact of SGLT2 inhibition on the repopulation of glomerular cells by CoRL and examines their phenotypic commitment., Methods: Experiments were performed in Ren1cre-tdTomato lineage-trace mice. Either 5/6 nephrectomy (5/6NX) modeling CKD or bilateral ischaemia reperfusion injury (bIRI) mimicking AKI was applied, while the SGLT2 inhibitor empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days., Results: Both 5/6NX and bIRI-induced kidney injury increased the number of glomerular CoRL-derived cells. SGLT2 inhibition improved kidney function after 5/6NX, indicated by decreased blood creatinine and urea levels, but not after bIRI. In line with this, empagliflozin in 5/6NX animals resulted in less glomerulosclerosis, while it did not affect histopathological features in bIRI. Treatment with empagliflozin resulted in an increase in the number of CoRL-derived glomerular cells in both 5/6NX and bIRI conditions. Interestingly, SGLT2 inhibition led to more CoRL-derived podocytes in 5/6NX animals, whereas empagliflozin-treated bIRI mice presented with increased levels of parietal epithelial and mesangial cells derived from CoRL., Conclusion: We conclude that SGLT2 inhibition by empagliflozin promotes CoRL-mediated glomerular repopulation with selective CoRL-derived cell types depending on the type of experimental kidney injury. These findings suggest a previously unidentified mechanism that could contribute to the renoprotective effect of SGLT2 inhibitors., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Introduction: The impact of cardiovascular disease (CVD) risk factors, encompassing various biological determinants and unhealthy lifestyles, on the functional dynamics of circulating monocytes-a pivotal cell type in CVD pathophysiology remains elusive. In this study, we aimed to elucidate the influence of CVD risk factors on monocyte transcriptional responses to an infectious stimulus., Methods: We conducted a comparative analysis of monocyte gene expression profiles from the CTMM - CIRCULATING CELLS Cohort of coronary artery disease (CAD) patients, at baseline and after lipopolysaccharide (LPS) stimulation. Gene co-expression analysis was used to identify gene modules and their correlations with CVD risk factors, while pivotal transcription factors controlling the hub genes in these modules were identified by regulatory network analyses. The identified gene module was subjected to a drug repurposing screen, utilizing the LINCS L1000 database., Results: Monocyte responsiveness to LPS showed a highly significant, negative correlation with blood pressure levels (ρ< -0.4; P<10 -80 ). We identified a ZNF12/ZBTB43-driven gene module closely linked to diastolic blood pressure, suggesting that monocyte responses to infectious stimuli, such as LPS, are attenuated in CAD patients with elevated diastolic blood pressure. This attenuation appears associated with a dampening of the LPS-induced suppression of oxidative phosphorylation. Finally, we identified the serine-threonine inhibitor MW-STK33-97 as a drug candidate capable of reversing this aberrant LPS response., Conclusions: Monocyte responses to infectious stimuli may be hampered in CAD patients with high diastolic blood pressure and this attenuated inflammatory response may be reversed by the serine-threonine inhibitor MW-STK33-97. Whether the identified gene module is a mere indicator of, or causal factor in diastolic blood pressure and the associated dampened LPS responses remains to be determined., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lu, Donners, de Baaij, Jin, Otten, Manca, van Zonneveld, Jukema, Kraaijeveld, Kuiper, Pasterkamp, Mees, Sluimer, Cavill, Karel, Goossens and Biessen.)

Belatacept, a modified form of CTLA-Ig that blocks CD28-mediated co-stimulation of T cells, is an immune-suppressant that can be used as an alternative to calcineurin inhibitors (CNIs). In kidney transplant recipients, belatacept has been associated with improved renal function and reduced cardiovascular toxicity. Monocytes as well as T-lymphocytes play causal roles in the pathophysiology of atherosclerotic disease. We hypothesized that the beneficial impact of the use of belatacept over CNIs on cardiovascular risk could be partly explained by the impact of belatacept therapy on these circulating leukocytes. Hence, we phenotyped circulating leukocytes in transplanted patients with a stable renal function that were randomized between either continuation of CNI or conversion to belatacept in two international studies in which we participated. In 41 patients, we found that belatacept-treated patients consistently showed lower numbers of B-lymphocytes, T-lymphocytes as well as CD14-negative monocytes (CD14NM), especially in non-diabetic patients. Our observation that this decrease was associated to plasma concentrations of TNFα is consistent with a model where CD14NM-production of TNFα is diminished by belatacept-treatment, due to effects on the antigen-presenting cell compartment., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the current worldwide pandemic and the associated coronavirus disease 2019 with potentially lethal outcome. Although effective vaccines strongly contributed to reduce disease severity, establishing a toolbox to control current and newly emerging coronaviruses of epidemic concern requires the development of novel therapeutic compounds, to treat severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA genome using antisense oligonucleotides (ASOs). We demonstrate that selected locked nucleic acid gapmers have the potency to reduce the in vitro intracellular viral load by up to 96%. Our promising results strongly support the case for further development of our preselected ASOs as therapeutic or prophylactic antiviral agents.

Background and Aims: This study aims to identify sex-specific transcriptional differences and signaling pathways in circulating monocytes contributing to cardiovascular disease., Methods and Results: We generated sex-biased gene expression signatures by comparing male versus female monocytes of coronary artery disease (CAD) patients (n = 450) from the Center for Translational Molecular Medicine-Circulating Cells Cohort. Gene set enrichment analysis demonstrated that monocytes from female CAD patients carry stronger chemotaxis and migratory signature than those from males. We then inferred cytokine signaling activities based on CytoSig database of 51 cytokine and growth factor regulation profiles. Monocytes from females feature a higher activation level of EGF, IFN1, VEGF, GM-CSF, and CD40L pathways, whereas IL-4, INS, and HMGB1 signaling was seen to be more activated in males. These sex differences were not observed in healthy subjects, as shown for an independent monocyte cohort of healthy subjects (GSE56034, n = 485). More pronounced GM-CSF signaling in monocytes of female CAD patients was confirmed by the significant enrichment of GM-CSF-activated monocyte signature in females. As we show these effects were not due to increased plasma levels of the corresponding ligands, sex-intrinsic differences in monocyte signaling regulation are suggested. Consistently, regulatory network analysis revealed jun-B as a shared transcription factor activated in all female-specific pathways except IFN1 but suppressed in male-activated IL-4., Conclusions: We observed overt CAD-specific sex differences in monocyte transcriptional profiles and cytokine- or growth factor-induced responses, which provide insights into underlying mechanisms of sex differences in CVD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality ex vivo . In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel ex vivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients., Methods: Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features., Results: Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) class could be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in which mitochondrial changes were most discriminative., Conclusion: Morphological profiling presents a viable tool to assess the endothelium ex vivo . We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient., (© 2024 The Authors.)

Post-transcriptional regulation by non-coding RNAs (ncRNAs) can modulate the expression of genes involved in kidney physiology and disease. A large variety of ncRNA species exist, including microRNAs, long non-coding RNAs, piwi-interacting RNAs, small nucleolar RNAs, circular RNAs and yRNAs. Despite early assumptions that some of these species may exist as by-products of cell or tissue injury, a growing body of literature suggests that these ncRNAs are functional and participate in a variety of processes. Although they function intracellularly, ncRNAs are also present in the circulation, where they are carried by extracellular vesicles, ribonucleoprotein complexes or lipoprotein complexes such as HDL. These systemic, circulating ncRNAs are derived from specific cell types and can be directly transferred to a variety of cells, including endothelial cells of the vasculature and virtually any cell type in the kidney, thereby affecting the function of the host cell and/or its response to injury. Moreover, chronic kidney disease itself, as well as injury states associated with transplantation and allograft dysfunction, is associated with a shift in the distribution of circulating ncRNAs. These findings may provide opportunities for the identification of biomarkers with which to monitor disease progression and/or the development of therapeutic interventions., (© 2023. Springer Nature Limited.)

The gut microbiota has emerged as an important modulator of cardiovascular and renal homeostasis. The composition of gut microbiota in patients suffering from chronic kidney disease (CKD) is altered, where a lower number of bacteria producing short chain fatty acids (SCFAs) is observed. It is known that SCFAs, such as butyrate and acetate, have protective effects against cardiovascular diseases and CKD but their mechanisms of action remain largely unexplored. In the present study, we investigated the effect of butyrate and acetate on glomerular endothelial cells. Human glomerular microvascular endothelial cells (hgMVECs) were cultured and exposed to butyrate and acetate and their effects on cellular proliferation, mitochondrial mass and metabolism, as well as monolayer integrity were studied. While acetate did not show any effects on hgMVECs, our results revealed that butyrate reduces the proliferation of hgMVECs, strengthens the endothelial barrier through increased expression of VE-cadherin and Claudin-5 and promotes mitochondrial biogenesis. Moreover, butyrate reduces the increase in oxygen consumption induced by lipopolysaccharides (LPS) , revealing a protective effect of butyrate against the detrimental effects of LPS. Taken together, our data show that butyrate is a key player in endothelial integrity and metabolic homeostasis.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Aims/hypothesis: The aim of this study was to identify differentially expressed long non-coding RNAs (lncRNAs) and mRNAs in whole blood of people with type 2 diabetes across five different clusters: severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD), mild obesity-related diabetes (MOD), mild diabetes (MD) and mild diabetes with high HDL-cholesterol (MDH). This was to increase our understanding of different molecular mechanisms underlying the five putative clusters of type 2 diabetes., Methods: Participants in the Hoorn Diabetes Care System (DCS) cohort were clustered based on age, BMI, HbA 1c , C-peptide and HDL-cholesterol. Whole blood RNA-seq was used to identify differentially expressed lncRNAs and mRNAs in a cluster compared with all others. Differentially expressed genes were validated in the Innovative Medicines Initiative DIabetes REsearCh on patient straTification (IMI DIRECT) study. Expression quantitative trait loci (eQTLs) for differentially expressed RNAs were obtained from a publicly available dataset. To estimate the causal effects of RNAs on traits, a two-sample Mendelian randomisation analysis was performed using public genome-wide association study (GWAS) data., Results: Eleven lncRNAs and 175 mRNAs were differentially expressed in the MOD cluster, the lncRNA AL354696.2 was upregulated in the SIDD cluster and GPR15 mRNA was downregulated in the MDH cluster. mRNAs and lncRNAs that were differentially expressed in the MOD cluster were correlated among each other. Six lncRNAs and 120 mRNAs validated in the IMI DIRECT study. Using two-sample Mendelian randomisation, we found 52 mRNAs to have a causal effect on anthropometric traits (n=23) and lipid metabolism traits (n=10). GPR146 showed a causal effect on plasma HDL-cholesterol levels (p = 2×10 -15 ), without evidence for reverse causality., Conclusions/interpretation: Multiple lncRNAs and mRNAs were found to be differentially expressed among clusters and particularly in the MOD cluster. mRNAs in the MOD cluster showed a possible causal effect on anthropometric traits, lipid metabolism traits and blood cell fractions. Together, our results show that individuals in the MOD cluster show aberrant RNA expression of genes that have a suggested causal role on multiple diabetes-relevant traits., (© 2023. The Author(s).)

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions., (Copyright © 2023. Published by Elsevier B.V.)

Objective: Atrial fibrillation (AF) often progresses from paroxysmal AF (PAF) to more permanent forms. To improve personalised medicine, we aim to develop a new AF progression risk prediction model in patients with PAF., Methods: In this interim-analysis of the Reappraisal of AF: Interaction Between HyperCoagulability, Electrical Remodelling, and Vascular Destabilisation in the Progression of AF study, patients with PAF undergoing extensive phenotyping at baseline and continuous rhythm monitoring during follow-up of ≥1 year were analysed. AF progression was defined as (1) progression to persistent or permanent AF or (2) progression of PAF with >3% burden increase. Multivariable analysis was done to identify predictors of AF progression., Results: Mean age was 65 (58-71) years, 179 (43%) were female. Follow-up was 2.2 (1.6-2.8) years, 51 of 417 patients (5.5%/year) showed AF progression. Multivariable analysis identified, PR interval, impaired left atrial function, mitral valve regurgitation and waist circumference to be associated with AF progression. Adding blood biomarkers improved the model (C-statistic from 0.709 to 0.830) and showed male sex, lower levels of factor XIIa:C1-esterase inhibitor and tissue factor pathway inhibitor, and higher levels of N-terminal pro-brain natriuretic peptide, proprotein convertase subtilisin/kexin type 9 and peptidoglycan recognition protein 1 were associated with AF progression., Conclusion: In patients with PAF, AF progression occurred in 5.5%/year. Predictors for progression included markers for atrial remodelling, sex, mitral valve regurgitation, waist circumference and biomarkers associated with coagulation, inflammation, cardiomyocyte stretch and atherosclerosis. These prediction models may help to determine risk of AF progression and treatment targets, but validation is needed., Trial Registration Number: NCT02726698., Competing Interests: Competing interests: US reports grants from Roche Diagnostics, EP Solutions, Dutch Heart Foundation, European Union, personal fees from Roche Diagnostics, EP Solutions, other from YourRhythmics BV, outside the submitted work. In addition, US has a patent Noninvasive classification of AF licensed to YourRhythmics. JDeG reports grants and personal fees from Atricure, Bayer, Daiichi Sankyo, Johnson&Johnson, grants from Boston Scientific, personal fees from Novartis and Servier, outside the submitted work. JL reports consultancy agreement Medtronic. HTC reports grants from Bayer and Pfizer and consultancy agreements for Pfizer, Alveron, STAGO, Leo Pharma, Daiichi Sankyo, Gilead/Galapagos, Portola/Aexia and Coagulation Profile. All other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Background: Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs)., Objective: To investigate the potential effect of different DOACs on vascular integrity., Methods: To model the impact of DOACs on vascular integrity, we utilized platelet-free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro-coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro- and anti-barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics., Results: We provide evidence that the anti-FXa DOAC rivaroxaban and the anti-thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom-made peptide that blocks thrombin's exosite-I., Conclusions: Proteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease-activated receptor-1 mimicking peptide blocking thrombin's exosite-I., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: The prevalence of end-stage renal disease (ESRD) is increasing worldwide, with the majority of new ESRD cases diagnosed in patients >60 years of age. These older patients are at increased risk for impaired cognitive functioning, potentially through cerebral small vessel disease (SVD). Novel markers of vascular integrity may be of clinical value for identifying patients at high risk for cognitive impairment., Methods: We aimed to associate the levels of angiopoietin-2 (Ang-2), asymmetric dimethylarginine and a selection of eight circulating angiogenic microRNAs (miRNAs) with SVD and cognitive impairment in older patients reaching ESRD that did not yet initiate renal replacement therapy (n = 129; mean age 75.3 years, mean eGFR 16.4 mL/min). We assessed brain magnetic resonance imaging changes of SVD (white matter hyperintensity volume, microbleeds and the presence of lacunes) and measures of cognition in domains of memory, psychomotor speed and executive function in a neuropsychological test battery., Results: Older patients reaching ESRD showed an unfavourable angiogenic profile, as indicated by aberrant levels of Ang-2 and five angiogenic miRNAs (miR-27a, miR-126, miR-132, miR-223 and miR-326), compared with healthy persons and patients with diabetic nephropathy. Moreover, Ang-2 was associated with SVD and with the domains of psychomotor speed and executive function, while miR-223 and miR-29a were associated with memory function., Conclusions: Taken together, these novel angiogenic markers might serve to identify older patients with ESRD at risk of cognitive decline, as well as provide insights into the underlying (vascular) pathophysiology., (© The Author(s) 2020. Published by Oxford University Press on behalf of the ERA.)

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing and mostly occurring in patients aged 65 years or older. The main cause of death in these patients is cardiovascular disease (CVD). Novel markers of vascular integrity may thus be of clinical value for identifying patients at high risk for CVD. Here we associated the levels of selected circulating angiogenic miRNAs, angiopoietin-2 (Ang-2) and asymmetric dimethylarginine (ADMA) with cardiovascular structure and function (as determined by cardiovascular MRI) in 67 older patients reaching ESKD that were included from 'The Cognitive decline in Older Patients with End stage renal disease' (COPE) prospective, multicentered cohort study. We first determined the association between the vascular injury markers and specific heart conditions and observed that ESKD patients with coronary heart disease have significantly higher levels of circulating ADMA and miR-27a. Moreover, circulating levels of miR-27a were higher in patients with atrial fibrillation. In addition, the circulating levels of the vascular injury markers were associated with measures of cardiovascular structure and function obtained from cardiovascular MRI: pulse wave velocity (PWV), ejection fraction (EF) and cardiac index (CI). We found Ang-2 and miR-27a to be strongly correlated to the PWV, while Ang-2 also associated with ejection fraction. Finally, we observed that in contrast to miR-27a, Ang-2 was not associated with a vascular cause of the primary kidney disease, suggesting Ang-2 may be an ESKD-specific marker of vascular injury. Taken together, among older patients with ESKD, aberrant levels of vascular injury markers (miR-27a, Ang-2 and ADMA) associated with impaired cardiovascular function. These markers may serve to identify individuals at higher risk of CVD, as well as give insight into the underlying (vascular) pathophysiology.

Circular RNAs (circRNAs) are a class of endogenously expressed regulatory RNAs with a single-stranded circular structure. They are generated by back splicing and their expression can be tightly regulated by RNA binding proteins. Cytoplasmic circRNAs can function as molecular sponges that inhibit microRNA-target interactions and protein function or as templates for the efficient generation of peptides via rolling circle amplification. They can also act as molecular scaffolds that enhance the reaction kinetics of enzyme-substrate interactions. In the nucleus, circRNAs might facilitate chromatin modifications and promote gene expression. CircRNAs are resistant to degradation and can be packaged in extracellular vesicles and transported in the circulation. Initial studies suggest that circRNAs have roles in kidney disease and associated cardiovascular complications. They have been implicated in hypertensive nephropathy, diabetic kidney disease, glomerular disease, acute kidney injury and kidney allograft rejection, as well as in microvascular and macrovascular complications of chronic kidney disease, including atherosclerotic vascular disease. In addition, several circRNAs have been reported to have oncogenic or tumour suppressor roles or to regulate drug resistance in kidney cancer. The available data suggest that circRNAs could be promising diagnostic and/or prognostic biomarkers and potential therapeutic targets for kidney disease, cardiovascular disease and kidney cancer., (© 2021. Springer Nature Limited.)

Accumulating evidence pinpoints sex differences in stroke incidence, etiology and outcome. Therefore, more understanding of the sex-specific mechanisms that lead to ischemic stroke and aggravation of secondary damage after stroke is needed. Our current mechanistic understanding of cerebral ischemia states that endothelial quiescence in neurovascular units (NVUs) is a major physiological parameter affecting the cellular response to neuron, astrocyte and vascular smooth muscle cell (VSMC) injury. Although a hallmark of the response to injury in these cells is transcriptional activation, noncoding RNAs such as microRNAs exhibit cell-type and context dependent regulation of gene expression at the post-transcriptional level. This review assesses whether sex-specific microRNA expression (either derived from X-chromosome loci following incomplete X-chromosome inactivation or regulated by estrogen in their biogenesis) in these cells controls NVU quiescence, and as such, could differentiate stroke pathophysiology in women compared to men. Their adverse expression was found to decrease tight junction affinity in endothelial cells and activate VSMC proliferation, while their regulation of paracrine astrocyte signaling was shown to neutralize sex-specific apoptotic pathways in neurons. As such, these microRNAs have cell type-specific functions in astrocytes and vascular cells which act on one another, thereby affecting the cell viability of neurons. Furthermore, these microRNAs display actual and potential clinical implications as diagnostic and prognostic biomarkers in ischemic stroke and in predicting therapeutic response to antiplatelet therapy. In conclusion, this review improves the current mechanistic understanding of the molecular mechanisms leading to ischemic stroke in women and highlights the clinical promise of sex-specific microRNAs as novel diagnostic biomarkers for (silent) ischemic stroke.

This study provides a method to assess the impact of circulating plasma factors on microvascular integrity by using a recently developed microvessel-on-a-chip platform featuring the human endothelium that is partly surrounded by the extracellular matrix. The system is high-throughput, which allows parallel analysis of organ-level microvessel pathophysiology, including vascular leakage. Ethylenediaminetetraacetic acid plasma samples are mixed with inhibitors for recalcification of the plasma samples to avoid activation of the coagulation- or complement system. Moreover, the assay is validated by spiking vascular endothelial growth factor, histamine, or tumor necrosis factor alpha to recalcified plasma and confirms their modulation of microvessel barrier function at physiologically relevant concentrations. Finally, this study shows that perfusing the microvessels with recalcified plasma samples of coronavirus disease-2019 patients, with a confirmed proinflammatory profile, results in markedly increased leakage of the microvessels. The assay provides opportunities for diagnostic screening of inflammatory or endothelial disrupting plasma factors associated with endothelial dysfunction., (© 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Objective: Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice., Methods: Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes., Results: Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose)., Conclusion: This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.

Objective: Besides hyperlipidemia, inflammation is an important determinant in the initiation and the progression of atherosclerosis. As Neuroimmune Guidance Cues (NGCs) are emerging as regulators of atherosclerosis, we set out to investigate the expression and function of inflammation-regulated NGCs. Methods and results: NGC expression in human monocytes and endothelial cells was assessed using a publicly available RNA dataset. Next, the mRNA levels of expressed NGCs were analyzed in primary human monocytes and endothelial cells after stimulation with IL1β or TNFα. Upon stimulation a total of 14 and 19 NGCs in monocytes and endothelial cells, respectively, were differentially expressed. Since plexin A4 (PLXNA4) was strongly downregulated in endothelial cells under inflammatory conditions, the role of PLXNA4 in endothelial function was investigated. Knockdown of PLXNA4 in endothelial cells markedly impaired the integrity of the monolayer leading to more elongated cells with an inflammatory phenotype. In addition, these cells showed an increase in actin stress fibers and decreased cell-cell junctions. Functional assays revealed decreased barrier function and capillary network formation of the endothelial cells, while vascular leakage and trans-endothelial migration of monocytes was increased. Conclusion: The current study demonstrates that pro-inflammatory conditions result in differential expression of NGCs in endothelial cells and monocytes, both culprit cell types in atherosclerosis. Specifically, endothelial PLXNA4 is reduced upon inflammation, while PLXNA4 maintains endothelial barrier function thereby preventing vascular leakage of fluids as well as cells. Taken together, PLXNA4 may well have a causal role in atherogenesis that deserves further investigation., Competing Interests: GH has served as consultant and speaker for biotech and pharmaceutical companies that develop molecules that influence lipoprotein metabolism, including Regeneron, Pfizer, MSD, Sanofi, and Amgen. Until April 2019, GH has served as PI for clinical trials conducted with A.O. Amgen, Sanofi, Eli Lilly, Novartis, Kowa, Genzyme, Cerenis, Pfizer, Dezima, Astra Zeneca. The Department of Vascular Medicine receives the honoraria and investigator fees for sponsor studies/lectures for companies with approved lipid lowering therapy in the Netherlands. Since April 2019, GH is partly employed by Novo Nordisk and the AMC. GH has no active patents nor share or ownership of listed companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vreeken, Bruikman, Stam, Cox, Nagy, Zhang, Postma, van Zonneveld, Hovingh and van Gils.)

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated β-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3'UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3'UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages.

Simultaneous pancreas-kidney transplantation (SPKT) replaces kidney function and restores endogenous insulin secretion in patients with diabetic nephropathy (DN). Here, we aimed to identify circulating long noncoding RNAs (lncRNAs) that are associated with DN and vascular injury in the context of SPKT. Based on a pilot study and a literature-based selection of vascular injury-related lncRNAs, we assessed 9 candidate lncRNAs in plasma samples of patients with diabetes mellitus with a kidney function >35 mL/min/1.73 m 2 (DM; n = 12), DN (n = 14), SPKT (n = 35), healthy controls (n = 15), and renal transplant recipients (KTx; n = 13). DN patients were also studied longitudinally before and 1, 6, and 12 months after SPKT. Of 9 selected lncRNAs, we found MALAT1, LIPCAR, and LNC-EPHA6 to be higher in DN compared with healthy controls. SPKT caused MALAT1, LIPCAR, and LNC-EPHA6 to normalize to levels of healthy controls, which was confirmed in the longitudinal study. In addition, we observed a strong association between MALAT1, LNC-EPHA6, and LIPCAR and vascular injury marker soluble thrombomodulin and a subset of angiogenic microRNAs (miR-27a, miR-130b, miR-152, and miR-340). We conclude that specific circulating lncRNAs associate with DN-related vascular injury and normalize after SPKT, suggesting that lncRNAs may provide a promising novel monitoring strategy for vascular integrity in the context of SPKT., (© 2020 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.

The molecular basis of atherosclerosis is not fully understood and mice studies have shown that Ephrins and EPH receptors play a role in the atherosclerotic process. We set out to assess the role for monocytic EPHB2 and its Ephrin ligands in human atherosclerosis and show a role for EPHB2 in monocyte functions independently of its EphrinB ligands. Immunohistochemical staining of human aortic sections at different stages of atherosclerosis showed that EPHB2 and its ligand EphrinB are expressed in atherosclerotic plaques and that expression proportionally increases with plaque severity. Functionally, stimulation with EPHB2 did not affect endothelial barrier function, nor did stimulation with EphrinB1 or EphrinB2 affect monocyte-endothelial interactions. In contrast, reduced expression of EPHB2 in monocytes resulted in decreased monocyte adhesion to endothelial cells and a decrease in monocyte transmigration, mediated by an altered morphology and a decreased ability to phosphorylate FAK. Our results suggest that EPHB2 expression in monocytes results in monocyte accumulation by virtue of an increase of transendothelial migration, which can subsequently contribute to atherosclerotic plaque progression., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFpEF) are microcirculation defects following diabetes mellitus (DM). Unrecognized HFpEF is more prevalent in women with diabetes compared to men with diabetes and therefore sex-specific diagnostic strategies are needed. Previously, we demonstrated altered plasma miRs in DM patients with microvascular injury [defined by elevated plasma Angiopoietin-2 (Ang-2) levels]. This study hypothesized the presence of sex-differences in plasma miRs and Ang-2 in diabetic (female) patients with LVDD or HFpEF. After a pilot study, we assessed 16 plasma miRs in patients with LVDD (n = 122), controls (n = 244) and female diabetic patients (n = 10). Subsequently, among these miRs we selected and measured plasma miR-34a, -224 and -452 in diabetic HFpEF patients (n = 53) and controls (n = 52). In LVDD patients, miR-34a associated with Ang-2 levels (R 2 0.04, R = 0.21, p = 0.001, 95% CI 0.103-0.312), with plasma levels being diminished in patients with DM, while women with an eGFR < 60 ml/min and LVDD had lower levels of miR-34a, -224 and -452 compared to women without an eGFR < 60 ml/min without LVDD. In diabetic HFpEF women (n = 28), plasma Ang-2 levels and the X-chromosome located miR-224/452 cluster increased compared to men. We conclude that plasma miR-34a, -224 and -452 display an association with the microvascular injury marker Ang-2 and are particularly targeted to women with LVDD or HFpEF.

Eph receptors and their ephrin ligands are important guidance molecules during neurological and vascular development. In recent years, it has become clear that the Eph protein family remains functional in adult physiology. A subset of Ephs and ephrins is highly expressed by endothelial cells. As endothelial cells form the first barrier between the blood and surrounding tissues, maintenance of a healthy endothelium is crucial for tissue homeostasis. This review gives an overview of the current insights of the role of ephrin ligands and receptors in endothelial function and leukocyte recruitment in the (patho)physiology of adult vascular biology.

Acute rejection (AR) of a kidney graft in renal transplant recipients is associated with microvascular injury in graft dysfunction and, ultimately, graft failure. Circulating long noncoding RNAs (lncRNAs) may be suitable markers for vascular injury in the context of AR. Here, we first investigated the effect of AR after kidney transplantation on local vascular integrity and demonstrated that the capillary density markedly decreased in AR kidney biopsies compared to pre-transplant biopsies. Subsequently, we assessed the circulating levels of four lncRNAs (LNC-RPS24, LNC-EPHA6, MALAT1, and LIPCAR), that were previously demonstrated to associate with vascular injury in a cohort of kidney recipients with a stable kidney transplant function ( n = 32) and recipients with AR ( n = 15). The latter were followed longitudinally six and 12 months after rejection. We found higher levels of circulating LNC-EPHA6 during rejection, compared with renal recipients with a stable kidney function ( p = 0.017), that normalized one year after AR. In addition, LNC-RPS24, LNC-EPHA6, and LIPCAR levels correlated significantly with the vascular injury marker soluble thrombomodulin. We conclude that AR and microvascular injury are associated with higher levels of circulating LNC-EPHA6, which emphasizes the potential role of lncRNAs as biomarker in the context of AR.

TNFα signaling in the vascular endothelium elicits multiple inflammatory responses that drive vascular destabilization and leakage. Bioactive lipids are main drivers of these processes. In vitro mechanistic studies of bioactive lipids have been largely based on two-dimensional endothelial cell cultures that, due to lack of laminar flow and the growth of the cells on non-compliant stiff substrates, often display a pro-inflammatory phenotype. This complicates the assessment of inflammatory processes. Three-dimensional microvessels-on-a-chip models provide a unique opportunity to generate endothelial microvessels in a more physiological environment. Using an optimized targeted liquid chromatography-tandem mass spectrometry measurements of a panel of pro- and anti-inflammatory bioactive lipids, we measure the profile changes upon administration of TNFα. We demonstrate that bioactive lipid profiles can be readily detected from three-dimensional microvessels-on-a-chip and display a more dynamic, less inflammatory response to TNFα, that resembles more the human situation, compared to classical two-dimensional endothelial cell cultures., Competing Interests: AJ, JS, WY, WS, AM, Av No competing interests declared, TH co-founder of MIMETAS and has some shares in MIMETAS., (© 2020, Junaid et al.)

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo . Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

The central dogma in molecular biology states that genetic information is transmitted from DNA to RNA to proteins, but not the other way round. Thanks to a recent technological revolution - the 'RNAissance' - it has, however, become clear that RNA is not solely a messenger for passing on the genetic information necessary for protein synthesis, but that RNA also plays an important role in sickness and health. In the past 5 years alone more than 100 therapies with (complementary) RNA molecules have been investigated in Phase 1 trials, and a quarter of these have also been investigated in Phase 2 or 3 trials. The dramatic increase in the number of pharmaceutical companies that are developing RNA therapeutics illustrates the enormous potential of these medicines. Once the toxicity and the costs of RNA therapeutics can be limited, these medicines - personalized or not - could soon be prescribed for patients with a wide range of chronic conditions.

The body's salt and fluid balance is regulated by the renin-angiotensin-aldosterone system. Generation of prostaglandin-E2 (PGE2) in a cyclo-oxygenase-2 (COX-2)-dependent manner in the macula densa, the salt-sensing cells of the kidney, plays a dominant role in renin regulation. Here we show that miR-132 directly targets Cox-2 and affects subsequent PGE2 and renin levels. MiR-132 is induced and reduced by low- and high salt treatment, respectively, in a p38- and ERK1/2-independent and CREB- and salt inducible kinase-dependent manner. Silencing of miR-132 in mice increases macula densa COX-2 expression and elevates PGE2 and renin levels, which are abrogated by the selective COX-2-inhibitor Celecoxib. Furthermore, a low or high salt diet induces and reduces macula densa miR-132 expression, while low salt diet combined with silencing miR-132 further increases renin levels. Taken together, we demonstrate a posttranscriptional regulatory role for salt-dependent miR-132 in fine-tuning the steady-state levels of renin.

Background and Aims: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis., Methods: To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used., Results: Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and β3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-κB pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration., Conclusions: Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia., Competing Interests: none declared

In normal physiology, endothelial cells (ECs) form a vital barrier between the blood and underlying tissue controlling leukocyte diapedesis and vascular inflammation. Emerging data suggest that neuronal guidance cues, typically expressed during development, have roles outside the nervous system in vascular biology and immune responses. In particular, Class III semaphorins have been reported to affect EC migration and angiogenesis. While ECs express high levels of semaphorin 3F (SEMA3F), little is known about its function in mature ECs. Here we show that SEMA3F expression is reduced by inflammatory stimuli and increased by laminar flow. Endothelial cells exposed to laminar flow secrete SEMA3F, which subsequently binds to heparan sulfates on the surface of ECs. However, under pro-inflammatory conditions, reduced levels of SEMA3F make ECs more prone to monocyte diapedesis and display impaired barrier function as measured with an electric cell-substrate impedance sensing system and a microfluidic system. In addition, we demonstrate that SEMA3F can directly inhibit the migration of activated monocytes. Taken together, our data suggest an important homeostatic function for EC-expressed SEMA3F, serving as a mediator of endothelial quiescence.

In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice ( qk v ) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre ( QKI FL/FL;LysM-Cre mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qk v and QKI FL/FL;LysM-Cre mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKI FL/FL;LysM-Cre mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.

Objective: Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether Netrin-1 is associated with different stages of atherosclerosis. Approach and Results: Plasma Netrin-1 levels were measured in different patient cohorts: (1) 22 patients with high cardiovascular risk who underwent arterial wall inflammation assessment using positron-emission tomography / computed tomography, (2) 168 patients with a positive family history of premature atherosclerosis in whom coronary artery calcium scores were obtained, and (3) 104 patients with chest pain who underwent coronary computed tomography angiography imaging to evaluate plaque vulnerability and burden. Netrin-1 plasma levels were negatively correlated with arterial wall inflammation (β, -0.01 [95% CI, 0.02 to -0.01] R 2 , 0.61; P <0.0001), and concentrations of Netrin-1 were significantly lower when atherosclerosis was present compared with individuals without atherosclerosis (28.01 versus 10.51 ng/mL, P <0.001). There was no difference in Netrin-1 plasma concentrations between patients with stable versus unstable plaques (11.17 versus 11.74 ng/mL, P =0.511). However, Netrin-1 plasma levels were negatively correlated to total plaque volume (β, -0.09 [95% CI, -0.11 to -0.08] R 2 , 0.57, P <0.0001), calcified plaque volumes (β, -0.10 [95% CI, -0.12 to -0.08] R 2 , 0.53; P <0.0001), and noncalcified plaque volumes (β, -0.08 [95% CI, -0.10 to -0.06] R 2 , 0.41; P <0.0001). Treatment of inflammatory stimulated endothelial cells with plasma with high Netrin-1 level resulted in reduced endothelial inflammation and consequently, less monocyte adhesion., Conclusions: Netrin-1 plasma levels are lower in patients with subclinical atherosclerosis and in patients with arterial wall inflammation. Netrin-1 is not associated with plaque vulnerability; however, it is negatively correlated to plaque burden, suggesting that Netrin-1 is involved in some, but not all, stages of atherosclerosis.

Ebola virus, for which we lack effective countermeasures, causes hemorrhagic fever in humans, with significant case fatality rates. Lack of experimental human models for Ebola hemorrhagic fever is a major obstacle that hinders the development of treatment strategies. Here, we model the Ebola hemorrhagic syndrome in a microvessel-on-a-chip system and demonstrate its applicability to drug studies. Luminal infusion of Ebola virus-like particles leads to albumin leakage from the engineered vessels. The process is mediated by the Rho/ROCK pathway and is associated with cytoskeleton remodeling. Infusion of Ebola glycoprotein (GP 1,2 ) generates a similar phenotype, indicating the key role of GP 1,2 in this process. Finally, we measured the potency of a recently developed experimental drug FX06 and a novel drug candidate, melatonin, in phenotypic rescue. Our study confirms the effects of FX06 and identifies melatonin as an effective, safe, inexpensive therapeutic option that is worth investigating in animal models and human trials., Competing Interests: Declaration of Interests Authors declare no conflict of interest related to the content of this manuscript. T.H. is shareholder in Mimetas BV, which was involved in the fabrication of the chips used in this study., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The durability of prosthetic arteriovenous (AV) grafts for hemodialysis access is low, predominantly due to stenotic lesions in the venous outflow tract and infectious complications. Tissue engineered blood vessels (TEBVs) might offer a tailor-made autologous alternative for prosthetic grafts. We have designed a method in which TEBVs are grown in vivo, by utilizing the foreign body response to subcutaneously implanted polymeric rods in goats, resulting in the formation of an autologous fibrocellular tissue capsule (TC). One month after implantation, the polymeric rod is extracted, whereupon TCs (length 6 cm, diameter 6.8 mm) were grafted as arteriovenous conduit between the carotid artery and jugular vein of the same goats. At time of grafting, the TCs were shown to have sufficient mechanical strength in terms of bursting pressure (2382 ± 129 mmHg), and suture retention strength (SRS: 1.97 ± 0.49 N). The AV grafts were harvested at 1 or 2 months after grafting. In an ex vivo whole blood perfusion system, the lumen of the vascular grafts was shown to be less thrombogenic compared to the initial TCs and ePTFE grafts. At 8 weeks after grafting, the entire graft was covered with an endothelial layer and abundant elastin expression was present throughout the graft. Patency at 1 and 2 months was comparable with ePTFE AV-grafts. In conclusion, we demonstrate the remodeling capacity of cellularized in vivo engineered TEBVs, and their potential as autologous alternative for prosthetic vascular grafts., (Copyright © 2019. Published by Elsevier Ltd.)

Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m 2 ), and patients with DN (eGFR <30 mL/min/1.73 m 2 ). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132)., (© 2019 by the American Diabetes Association.)

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

Background: A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function., Methods: To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy., Results: Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2., Conclusions: Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy., (Copyright © 2019 by the American Society of Nephrology.)