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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.
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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 13C6, 15N4-L-Arginine, and treated with eNOS stimulator, eNOS inhibitor, and arginase inhibitor. Analysis of downstream metabolites, 13C6, 15N3 L-Citrulline and 13C5, 15N2 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 13C6, 15N3 L-Citrulline/13C5, 15N2 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.
Subject(s)
Endothelial Cells , Metabolomics , Microvessels , Nitric Oxide Synthase Type III , Nitric Oxide , Humans , Nitric Oxide/metabolism , Metabolomics/methods , Microvessels/metabolism , Nitric Oxide Synthase Type III/metabolism , Endothelial Cells/metabolism , Arginine/metabolism , Lab-On-A-Chip Devices , Cells, Cultured , Citrulline/metabolismABSTRACT
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.
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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.
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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 (Ntotal = 462/Ncase = 81) and prevalent (Ntotal = 1247/Ncase = 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.
Subject(s)
Diabetes Mellitus, Type 2 , Renal Insufficiency, Chronic , Humans , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/epidemiology , Risk Factors , Prospective Studies , Amino Acids, Branched-Chain/adverse effects , Amino Acids, Branched-Chain/metabolism , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/epidemiology , Renal Insufficiency, Chronic/chemically inducedABSTRACT
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.
Subject(s)
Coronary Artery Disease , Hypertension , Humans , Coronary Artery Disease/metabolism , Monocytes/metabolism , Gene Regulatory Networks , Lipopolysaccharides/pharmacology , Hypertension/genetics , Arteries/metabolism , Serine/metabolism , Threonine/genetics , Protein Serine-Threonine Kinases/metabolism , Kruppel-Like Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Acute Kidney Injury , Benzhydryl Compounds , Glucosides , Red Fluorescent Protein , Renal Insufficiency, Chronic , Sodium-Glucose Transporter 2 Inhibitors , Humans , Mice , Animals , Renin/metabolism , Sodium-Glucose Transporter 2 , Renal Insufficiency, Chronic/metabolism , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Glucose , Sodium/metabolismABSTRACT
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.
Subject(s)
COVID-19 , Oligonucleotides, Antisense , Humans , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/therapeutic use , SARS-CoV-2/genetics , RNA, Viral/genetics , COVID-19/genetics , COVID-19/therapyABSTRACT
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.
Subject(s)
Cardiovascular Diseases , Endothelial Cells , Humans , Butyric Acid/pharmacology , Lipopolysaccharides , Cell ProliferationABSTRACT
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.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Renal Insufficiency, Chronic , Humans , Endothelial Cells , RNA, Untranslated/genetics , MicroRNAs/genetics , Renal Insufficiency, Chronic/genetics , Gene Expression Regulation , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
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.
Subject(s)
Cardiovascular Diseases , Coronary Artery Disease , Humans , Male , Female , Monocytes/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Sex Characteristics , Interleukin-4 , Cytokines/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Autoimmune Diseases , COVID-19 , Humans , Autoantibodies , Autoimmunity , Receptors, G-Protein-Coupled/metabolismABSTRACT
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, HbA1c, 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.
Subject(s)
Diabetes Mellitus, Type 2 , Insulins , RNA, Long Noncoding , Humans , Diabetes Mellitus, Type 2/genetics , Lipid Metabolism/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Genome-Wide Association Study , Cholesterol, HDL , Gene Expression , Obesity/complications , Obesity/genetics , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Receptors, G-Protein-Coupled/metabolismABSTRACT
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.
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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.
Subject(s)
Atrial Fibrillation , Dabigatran , Administration, Oral , Anticoagulants/therapeutic use , Atrial Fibrillation/drug therapy , Dabigatran/adverse effects , Factor Xa/therapeutic use , Factor Xa Inhibitors/adverse effects , Humans , Receptor, PAR-1 , Rivaroxaban/adverse effects , Thrombin/therapeutic useABSTRACT
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.
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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.
Subject(s)
Cerebral Small Vessel Diseases , Cognitive Dysfunction , Kidney Failure, Chronic , MicroRNAs , Aged , Angiopoietin-2/genetics , Cerebral Small Vessel Diseases/complications , Cerebral Small Vessel Diseases/epidemiology , Cerebral Small Vessel Diseases/genetics , Cognition , Cognitive Dysfunction/genetics , Humans , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/genetics , Magnetic Resonance Imaging/methods , MicroRNAs/genetics , Neuropsychological TestsABSTRACT
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.
Subject(s)
Ischemic Stroke/complications , MicroRNAs/genetics , Nervous System Diseases/pathology , Neurovascular Coupling , Vascular Diseases/pathology , Animals , Humans , Nervous System Diseases/etiology , Nervous System Diseases/metabolism , Vascular Diseases/etiology , Vascular Diseases/metabolismABSTRACT
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.
Subject(s)
COVID-19 , Microfluidics , Endothelium, Vascular , Humans , Plasma , SARS-CoV-2 , Vascular Endothelial Growth Factor AABSTRACT
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.