RESUMO
BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.
Assuntos
Canais Iônicos , Óxido Nítrico , Veia Porta , Canais de Cátion TRPV , Animais , Masculino , Camundongos , Endotélio/metabolismo , Óxido Nítrico Sintase/metabolismo , Pressão Osmótica , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Vasodilatação , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
OBJECTIVE: The prevalence of obesity is growing globally. Adiposity increases the risk for metabolic syndrome, type 2 diabetes and cardiovascular disease. Adipose tissue distribution influences systemic metabolism and impacts metabolic disease risk. The link between sexual dimorphisms of adiposity and metabolism is poorly defined. We hypothesise that depot-specific adipose tissue mitochondrial function contributes to the sexual dimorphism of metabolic flexibility in obesity. METHODS: Male and female mice fed high fat diet (HFD) or standard diet (STD) from 8-18 weeks of age underwent whole animal calorimetry and high-resolution mitochondrial respirometry analysis on adipose tissue depots. To determine translatability we used RT-qPCR to examine key brown adipocyte-associated gene expression: peroxisome proliferator-activated receptor co-activator 1α, Uncoupling protein 1 and cell death inducing DFFA like effector a in brown adipose tissue (BAT) and subcutaneous adipose tissue (sWAT) of 18-week-old mice and sWAT from human volunteers. RESULTS: Male mice exhibited greater weight gain compared to female mice when challenged with HFD. Relative to increased body mass, the adipose to body weight ratio for BAT and sWAT depots was increased in HFD-fed males compared to female HFD-fed mice. Oxygen consumption, energy expenditure, respiratory exchange ratio and food consumption did not differ between males and females fed HFD. BAT mitochondria from obese females showed increased Complex I & II respiration and maximal respiration compared to lean females whereas obese males did not exhibit adaptive mitochondrial BAT respiration. Sexual dimorphism in BAT-associated gene expression in sWAT was also associated with Body Mass Index in humans. CONCLUSIONS: We show that sexual dimorphism of weight gain is reflected in mitochondrial respiration analysis. Female mice have increased metabolic flexibility to adapt to changes in energy intake by regulating energy expenditure through increased complex II and maximal mitochondrial respiration within BAT when HFD challenged and increased proton leak in sWAT mitochondria.
Assuntos
Tecido Adiposo , Mitocôndrias/metabolismo , Obesidade/metabolismo , Caracteres Sexuais , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , CamundongosAssuntos
Anemia Hemolítica Congênita/sangue , Eritrócitos/fisiologia , Hidropisia Fetal/sangue , Canais Iônicos/fisiologia , Anemia Hemolítica Congênita/complicações , Anemia Hemolítica Congênita/genética , Animais , Cálcio/sangue , Hemorreologia , Hidropisia Fetal/genética , Transporte de Íons , Cinética , Camundongos , Mutação de Sentido Incorreto , Tamanho do Órgão , Fragilidade Osmótica , Mutação Puntual , Baço/patologia , Esplenomegalia/etiologia , ÁguaRESUMO
PIEZO1 is a subunit of mechanically-activated, nonselective cation channels. Gain-of-function PIEZO1 mutations are associated with dehydrated hereditary stomatocytosis (DHS), a type of anaemia, due to abnormal red blood cell function. Here, we hypothesised additional effects on the heart. Consistent with this hypothesis, mice engineered to contain the M2241R mutation in PIEZO1 to mimic a DHS mutation had increased cardiac mass and interventricular septum thickness at 8-12 weeks of age, without altered cardiac contractility. Myocyte size was greater and there was increased expression of genes associated with cardiac hypertrophy (Anp, Acta1 and ß-MHC). There was also cardiac fibrosis, increased expression of Col3a1 (a gene associated with fibrosis) and increased responses of isolated cardiac fibroblasts to PIEZO1 agonism. The data suggest detrimental effects of excess PIEZO1 activity on the heart, mediated in part by amplified PIEZO1 function in cardiac fibroblasts.
Assuntos
Cardiomegalia , Mutação com Ganho de Função , Canais Iônicos , Animais , Cardiomegalia/genética , Fibrose , Canais Iônicos/genética , CamundongosRESUMO
Piezo1 forms mechanically activated nonselective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial cell-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial cell apoptosis, with no effect on TSP1, a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without an effect on Tsp1. In endothelial cells, Piezo1 was required for normal expression of endothelial NO synthase. The data suggest an endothelial cell-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.
Assuntos
Células Endoteliais , Canais Iônicos , Condicionamento Físico Animal , Animais , Capilares/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Músculos , Pericitos/metabolismo , Condicionamento Físico Animal/fisiologiaRESUMO
Mechanical force is a determinant of Notch signalling but the mechanism of force detection and its coupling to Notch are unclear. We propose a role for Piezo1 channels, which are mechanically-activated non-selective cation channels. In cultured microvascular endothelial cells, Piezo1 channel activation by either shear stress or a chemical agonist Yoda1 activated a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a Ca2+-regulated transmembrane sheddase that mediates S2 Notch1 cleavage. Consistent with this observation, we found Piezo1-dependent increase in the abundance of Notch1 intracellular domain (NICD) that depended on ADAM10 and the downstream S3 cleavage enzyme, γ-secretase. Conditional endothelial-specific disruption of Piezo1 in adult mice suppressed the expression of multiple Notch1 target genes in hepatic vasculature, suggesting constitutive functional importance in vivo. The data suggest that Piezo1 is a mechanism conferring force sensitivity on ADAM10 and Notch1 with downstream consequences for sustained activation of Notch1 target genes and potentially other processes.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Canais Iônicos/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Estresse Mecânico , Fatores de Transcrição HES-1/genéticaRESUMO
Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.
Assuntos
Canais Iônicos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Humanos , CamundongosRESUMO
OBJECTIVE: Activated factor XIII (FXIII) crosslinks fibrin to enhance the mechanical strength of a blood clot and increase its resistance to fibrinolysis. The prevalence of a common variant in the FXIII-A gene (V34L) has been reported to be lower in patients with myocardial infarction and ischemic stroke than in controls, suggesting a protective role for this polymorphism in vascular diseases. The current study investigated 6 single-nucleotide polymorphisms (SNPs) within the FXIII A-subunit gene to locate functional polymorphism(s) responsible for variation in FXIII activation. METHODS AND RESULTS: A total of 201 dizygotic twin pairs were genotyped for 1 promoter and all common nonsynonymous coding polymorphisms in the FXIII A-subunit gene: -246G>A, V34L, Y204F, P564L, V650I, and E651Q. Tests of linkage, association, and combined linkage and association were performed using QTDT software. Significant linkage to the V34L polymorphism (P=5 x 10(-12)) as well as association (P=3 x 10(-49)) was observed. Adjusting for association while performing linkage made the linkage signal disappear for the V34L polymorphism (from chi2=47.55, P=5x10(-12) to chi2=1.30, P=0.25). Only haplotypes containing the 34L allele showed association with FXIII activation. CONCLUSIONS: Testing multiple SNPs in the FXIII A-subunit gene indicates that V34L is the main functional polymorphism influencing FXIII activation.
Assuntos
Mapeamento Cromossômico , Fator XIII/genética , Ligação Genética , Polimorfismo de Nucleotídeo Único , Gêmeos Dizigóticos/genética , Adulto , Idoso , Alelos , Feminino , Haplótipos , Humanos , Leucina , Pessoa de Meia-Idade , Polimorfismo Genético , ValinaRESUMO
Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.
Assuntos
Canais Iônicos/fisiologia , Condicionamento Físico Animal/fisiologia , Animais , Pressão Sanguínea , Sinalização do Cálcio , Células Cultivadas , Células Endoteliais/metabolismo , Células HEK293 , Homeostase/genética , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Miócitos de Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Vasoconstrição/fisiologiaRESUMO
UNLABELLED: Activation of the receptor for advanced glycation end-products (RAGE) leads to a cascade of pro-inflammatory and pro-coagulant responses which are important in the pathogenesis of the vascular complications of diabetes mellitus. It is known that pro-inflammatory mechanisms underpin the development of type 2 diabetes. Our hypothesis is that RAGE may be involved in the evolution of insulin resistance in addition to mediating glucotoxic complications of diabetes mellitus. METHODS: To investigate the relationship between RAGE allelic variation and insulin resistance, the Gly82Ser variant and three promoter variants (-429, -374, 63 bp deletion) were studied in 480 subjects of known relationship from 89 families characterised for insulin resistance (using homeostasis model assessment [HOMA]) and for atherothrombotic risk. Carriage of the -429 C allele was weakly associated with increased insulin resistance (p = 0.02) when pedigree analysis was performed using SOLAR software. RESULTS: Insulin resistance was estimated to have a heritability of 25.8% before the addition of covariates. Analysis of the relationship between RAGE and insulin resistance indicated that the -429 polymorphism reduced the unexplained heritability of insulin resistance after adjusting for covariates (age, sex, body mass index) from 17.5% of the total variance to 15.6% of the total variance. CONCLUSIONS: These preliminary results indicate that the RAGE gene may affect the development of insulin resistance or be in linkage disequilibrium with a locus involved in this process.
Assuntos
Resistência à Insulina/genética , Polimorfismo Genético , Receptores Imunológicos/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Feminino , Genótipo , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Linhagem , Receptor para Produtos Finais de Glicação Avançada , Reino UnidoRESUMO
Advanced glycation endproducts (AGEs) form at an accelerated rate in diabetes and contribute to the development of macrovascular disease. Their precursors are detoxified by the glyoxalase system. Perturbations of the glyoxalase-1 gene may alter AGEs' interactions and affect pro-thrombotic factors. We screened the glyoxalase-1 gene for mutations and measured pro-thrombotic markers in 537 subjects from 89 healthy probands. Common single nucleotide polymorphisms (SNPs) were identified at positions -7 (C to T) and 20203 (C to A) from the translation start site. These SNPs were in Hardy-Weinberg equilibrium (CC=105, CA=266, AA=148; p>0.05; CC=126, CT=279, TT=114; p>0.05, respectively) and in linkage disequilibrium (D=27%, p<0.01), with mutant allele frequencies of 48% and 52% respectively. A significant association was found between SNPs at position 20203 and PAI-1 antigen concentrations and -7 and factor XIII A2B2 complex (p=0.001 and p=0.042). After Bonferroni correction a significant association remained between the SNP at 20203 and PAI-1 concentrations (p=0.005), and after adjustment for pedigree the association accounted for 1.3% of its heritability (p=0.04). No significant associations were found for SNP -7 T to C and factor VII activity, tPa concentrations, fibrinogen concentrations or factor XIII concentrations and SNP 20203 C to A and factor VII concentrations, PAI-1 concentrations, tPa concentrations or fibrinogen concentrations. Common variants in the glyoxalase-1 gene are associated with some pro-thrombotic factors, account for part of their heritability in healthy pedigrees and may alter susceptibility to macrovascular complications.
Assuntos
Fatores de Coagulação Sanguínea/análise , Lactoilglutationa Liase/genética , Adulto , Análise de Variância , Antígenos/sangue , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/imunologia , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/genética , Fator XIII/análise , Feminino , Frequência do Gene , Predisposição Genética para Doença , Testes Genéticos , Genótipo , Humanos , Masculino , Mutação , Linhagem , Inibidor 1 de Ativador de Plasminogênio/imunologia , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to N(G)-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P < 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P < 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P < 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/fisiologia , Receptor IGF Tipo 1/fisiologia , Animais , Aorta/fisiologia , Glicemia/metabolismo , Pressão Sanguínea , Feminino , Homeostase , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Receptor IGF Tipo 1/deficiência , Receptor IGF Tipo 1/genética , Vasoconstrição/efeitos dos fármacosRESUMO
OBJECTIVE: To identify novel polymorphisms in the genes encoding the transcription factors CCAAT/enhancer binding protein alpha, beta and delta ( CEBPA, CEBPB, CEBPD) and investigate associations between polymorphisms and obesity-related phenotypes. METHODS: Denaturing high-performance liquid chromatography (HPLC) was used to screen for novel gene variants and polymorphisms were genotyped in stored DNA from participants of the Leeds Family Study (537 subjects from 89 families). Genotype and haplotype analyses were carried out in STATA and PBAT, respectively. RESULTS: Twenty-five polymorphisms were identified; 11 in CEBPA, 12 in CEBPB and 2 in CEBPD. Several allelic variants were associated at a nominal 5% level with waist-to-hip ratio (-919G>A in CEBPA, -412G>T and 646C>T in CEBPB), leptin (1558G>A in CEBPA, -1051A>G and 1383T>- in CEBPB) and adiponectin (1382G>T and 1903G>T in CEBPB). Effects of CEBPA and CEBPB allelic variants were independent, but variants within each gene were in linkage disequilibrium. Several associations were observed between other obesity-related traits and allelic variants in CEBPA and CEBPB, but not CEBPD. CONCLUSION: These findings suggest that common allelic variants in CEBPA and CEBPB could influence abdominal obesity and related metabolic abnormalities associated with type 2 diabetes and cardiovascular disease in healthy White Northern European families, although results require independent confirmation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Obesidade/genética , Polimorfismo de Nucleotídeo Único , Adiponectina/sangue , Cromatografia Líquida de Alta Pressão , Inglaterra/epidemiologia , Predisposição Genética para Doença , Haplótipos , Humanos , Leptina/sangue , Modelos Lineares , Desequilíbrio de Ligação , Obesidade/sangue , Obesidade/etnologia , Obesidade/fisiopatologia , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Medição de Risco , Fatores de Risco , Relação Cintura-Quadril , População Branca/genéticaRESUMO
In yeast and higher plants, separate genes encode the cytosolic and mitochondrial forms of glyoxalase II. In contrast, although glyoxalase II activity has been detected both in the cytosol and mitochondria of mammals, only a single gene encoding glyoxalase II has been identified. Previously it was thought that this gene (the hydroxyacylglutathione hydrolase gene), comprised 8 exons that are transcribed into mRNA and that the resulting mRNA species encoded a single cytosolic form of glyoxalase II. Here we show that this gene gives rise to two distinct mRNA species transcribed from 9 and 10 exons, respectively. The 9-exon-derived transcript encodes two protein species: mitochondrially targeted glyoxylase II, which is initiated from an AUG codon in a previously uncharacterized part of the mRNA sequence, and cytosolic glyoxalase II, which is initiated by internal ribosome entry at a downstream AUG codon. The transcript deriving from 10 exons has an in-frame termination codon between the two initiating AUG codons and hence only encodes the cytosolic form of the protein. Confocal fluorescence microscopy indicates that the mitochondrially targeted form of glyoxalase II is directed to the mitochondrial matrix. Analysis of glyoxalase II mRNA sequences from a number of species indicates that dual initiation from alternative AUG codons is conserved throughout vertebrates.