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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.
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Células Endoteliais , Metabolômica , Microvasos , Óxido Nítrico Sintase Tipo III , Óxido Nítrico , Humanos , Óxido Nítrico/metabolismo , Metabolômica/métodos , Microvasos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Células Endoteliais/metabolismo , Arginina/metabolismo , Dispositivos Lab-On-A-Chip , Células Cultivadas , Citrulina/metabolismoRESUMO
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.
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Diabetes Mellitus Tipo 2 , Insuficiência Renal Crônica , Humanos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Fatores de Risco , Estudos Prospectivos , Aminoácidos de Cadeia Ramificada/efeitos adversos , Aminoácidos de Cadeia Ramificada/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/induzido quimicamenteRESUMO
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.
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Diabetes Mellitus Tipo 2 , Insulinas , RNA Longo não Codificante , Humanos , Diabetes Mellitus Tipo 2/genética , Metabolismo dos Lipídeos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Estudo de Associação Genômica Ampla , HDL-Colesterol , Expressão Gênica , Obesidade/complicações , Obesidade/genética , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
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.
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Doenças Cardiovasculares , Células Endoteliais , Humanos , Ácido Butírico/farmacologia , Lipopolissacarídeos , Proliferação de CélulasRESUMO
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.
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Doenças de Pequenos Vasos Cerebrais , Disfunção Cognitiva , Falência Renal Crônica , MicroRNAs , Idoso , Angiopoietina-2/genética , Doenças de Pequenos Vasos Cerebrais/complicações , Doenças de Pequenos Vasos Cerebrais/epidemiologia , Doenças de Pequenos Vasos Cerebrais/genética , Cognição , Disfunção Cognitiva/genética , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/genética , Imageamento por Ressonância Magnética/métodos , MicroRNAs/genética , Testes NeuropsicológicosRESUMO
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 (ß, -0.01 [95% CI, 0.02 to -0.01] R2, 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 (ß, -0.09 [95% CI, -0.11 to -0.08] R2, 0.57, P<0.0001), calcified plaque volumes (ß, -0.10 [95% CI, -0.12 to -0.08] R2, 0.53; P<0.0001), and noncalcified plaque volumes (ß, -0.08 [95% CI, -0.10 to -0.06] R2, 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.
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Aterosclerose/sangue , Doença da Artéria Coronariana/sangue , Vasos Coronários/diagnóstico por imagem , Netrina-1/sangue , Aterosclerose/diagnóstico , Biomarcadores/sangue , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , PrognósticoRESUMO
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.
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AVC Isquêmico/complicações , MicroRNAs/genética , Doenças do Sistema Nervoso/patologia , Acoplamento Neurovascular , Doenças Vasculares/patologia , Animais , Humanos , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismoRESUMO
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 m2 (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.
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Diabetes Mellitus , Nefropatias Diabéticas , Transplante de Rim , MicroRNAs , Transplante de Pâncreas , RNA Longo não Codificante , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/cirurgia , Humanos , Transplante de Rim/efeitos adversos , Estudos Longitudinais , Masculino , Pâncreas , Projetos Piloto , RNA Longo não Codificante/genéticaRESUMO
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.
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Ácido Hialurônico/biossíntese , Glomérulos Renais/anatomia & histologia , Glomérulos Renais/fisiologia , Animais , Endotélio/metabolismo , Humanos , Glomérulos Renais/metabolismo , Camundongos , UrotélioRESUMO
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.
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Células Endoteliais/metabolismo , Efrinas/metabolismo , Receptores da Família Eph/metabolismo , Adulto , Células Endoteliais/citologia , Hemodinâmica , Homeostase , Humanos , Transdução de SinaisRESUMO
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.
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Inibidores da Angiogênese/farmacologia , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Endotélio/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Endotélio/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
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Biomarcadores/sangue , Rejeição de Enxerto/genética , Transplante de Rim/efeitos adversos , RNA Longo não Codificante/genética , Adulto , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/patologia , Humanos , Rim/metabolismo , Rim/patologia , Rim/cirurgia , Masculino , Pessoa de Meia-Idade , Transplante Homólogo/efeitos adversosRESUMO
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.
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Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Migração Transendotelial e Transepitelial , Endotélio Vascular/patologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Monócitos/patologiaRESUMO
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.
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Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Efrinas/sangue , Aprendizado de Máquina , Monócitos/metabolismo , Netrina-1/sangue , Semaforinas/sangue , Adulto , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Estudos de Casos e Controles , Estudos de Coortes , Efrinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Netrina-1/genética , Semaforinas/genética , TranscriptomaRESUMO
Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (â¼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; â¼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.
Assuntos
Benzazepinas/farmacologia , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Neprilisina/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Obstrução Ureteral/tratamento farmacológico , Animais , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/enzimologia , Rim/patologia , Nefropatias/enzimologia , Nefropatias/genética , Nefropatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Células NIH 3T3 , Neprilisina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Obstrução Ureteral/enzimologia , Obstrução Ureteral/genética , Obstrução Ureteral/patologiaRESUMO
The pathophysiology of arteriovenous fistula (AVF) maturation failure is not completely understood but impaired outward remodeling (OR) and intimal hyperplasia are thought to be contributors. This adverse vascular response after AVF surgery results from interplay between vascular smooth muscle cells (VSMCs), the extracellular matrix (ECM), and inflammatory cells. Relaxin (RLN) is a hormone that acts on the vasculature via interaction with RLN/insulin-like peptide family receptor 1 (RXFP1), resulting in vasodilatation, ECM remodeling, and decreased inflammation. In the present study, we evaluated the consequences of RXFP1 knockout ( Rxfp1-/-) on AVF maturation in a murine model of AVF failure. Rxfp1-/- mice showed a 22% decrease in vessel size at the venous outflow tract 14 d after AVF surgery. Furthermore, a 43% increase in elastin content was observed in the lesions of Rxfp1-/- mice and coincided with a 41% reduction in elastase activity. In addition, Rxfp1-/- mice displayed a 6-fold increase in CD45+ leukocytes, along with a 2-fold increase in monocyte chemoattractant protein 1 (MCP1) levels, when compared with wild-type mice. In vitro, VSMCs from Rxfp1-/- mice exhibited a synthetic phenotype, as illustrated by augmentation of collagen, fibronectin, TGF-ß, and platelet-derived growth factor mRNA. In addition, VSMCs derived from Rxfp1-/- mice showed a 5-fold increase in cell migration. Finally, RXFP1 and RLN expression levels were increased in human AVFs when compared with unoperated cephalic veins. In conclusion, RXFP1 deficiency hampers elastin degradation and results in induced vascular inflammation after AVF surgery. These processes impair OR in murine AVF, suggesting that the RLN axis could be a potential therapeutic target for promoting AVF maturation.-Bezhaeva, T., de Vries, M. R., Geelhoed, W. J., van der Veer, E. P., Versteeg, S., van Alem, C. M. A., Voorzaat, B. M., Eijkelkamp, N., van der Bogt, K. E., Agoulnik, A. I., van Zonneveld, A.-J., Quax, P. H. A., Rotmans, J. I. Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.
RESUMO
OBJECTIVE: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. APPROACH AND RESULTS: Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of ß1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. CONCLUSIONS: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a ß1 integrin-dependent manner.
Assuntos
Antígenos CD/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Doenças das Artérias Carótidas/metabolismo , Células Endoteliais/metabolismo , Integrina beta1/metabolismo , Mecanotransdução Celular , Semaforinas/metabolismo , Animais , Antígenos CD/genética , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Células Endoteliais/patologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Migração e Rolagem de Leucócitos , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Placa Aterosclerótica , Fluxo Sanguíneo Regional , Semaforinas/deficiência , Semaforinas/genética , Células THP-1 , Regulação para CimaRESUMO
Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.
Assuntos
Arginina Vasopressina/metabolismo , Homeostase/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/genética , Vasopressinas/metabolismo , Animais , Aquaporina 2/metabolismo , Expressão Gênica/genética , Hipotálamo/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Concentração Osmolar , Receptores de Vasopressinas/metabolismo , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
This commentary highlights the article by Wilflingseder et al that investigates the relationship between in vivo inhibition of miR-182 by antisense oligonucleotides and improved post-injury kidney function.