RESUMO
Septic shock and disseminated intravascular coagulation (DIC) are known to be characterized by an endothelial cell dysfunction. The molecular mechanisms underlying this relationship are, however, poorly understood. In this work, we aimed to investigate human circulating IFN-α in patients with septic shock-induced DIC and tested the potential role of endothelial Stat1 (signal transducer and activator of transcription 1) as a therapeutic target in a mouse model of sepsis. For this, circulating type I, type II, and type III IFNs and procoagulant microvesicles were quantified in a prospective cohort of patients with septic shock. Next, we used a septic shock model induced by cecal ligation and puncture in wild-type mice, in Ifnar1 (type I IFN receptor subunit 1)-knockout mice, and in Stat1 conditional knockout mice. In human samples, we observed higher concentrations of circulating IFN-α and IFN-α1 in patients with DIC compared with patients without DIC, whereas concentrations of IFN-ß, IFN-γ, IFN-λ1, IFN-λ2, and IFN-λ3 were not different. IFN-α concentration was positively correlated with CD105 microvesicle concentrations, reflecting endothelial injury. In Ifnar1-/- mice, cecal ligation and puncture did not induce septic shock and was characterized by lesser endothelial cell injury, with lower aortic inflammatory cytokine expression, endothelial inflammatory-related gene expression, and fibrinolysis. In mice in which Stat1 was specifically ablated in endothelial cells, a marked protection against sepsis was also observed, suggesting the relevance of an endothelium-targeted strategy. Our work highlights the key roles of type I IFNs as pathogenic players in septic shock-induced DIC and the potential pertinence of endothelial STAT1 as a therapeutic target.
Assuntos
Coagulação Intravascular Disseminada/metabolismo , Interferon-alfa/metabolismo , Fator de Transcrição STAT1/metabolismo , Choque Séptico/metabolismo , Transdução de Sinais , Idoso , Animais , Coagulação Intravascular Disseminada/genética , Feminino , Humanos , Interferon-alfa/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fator de Transcrição STAT1/genética , Choque Séptico/genética , Choque Séptico/terapiaRESUMO
Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated ß-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Senescência Celular , Células Endoteliais/patologia , Endotélio Vascular/fisiopatologia , Inflamação/patologia , Neutrófilos/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Angiotensinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos Wistar , Baço/efeitos dos fármacos , Baço/patologia , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Tromboplastina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet-transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the ß-score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin-secreting ß-cells typified by polysialic acid of neural cell adhesion molecule (PSA-NCAM), and data were compared with values of the ß-score. Similar ranges of PSA-NCAM+ -MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2-fold elevation in PSA-NCAM+ -MVs preceded each ß-score drop along with a concomitant rise in insulin needs, suggesting ß-cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)+ -MVs, endothelial CD105+ -MVs, neutrophil CD66b+ -MVs, monocyte CD 14+ -MVs, and T4 lymphocyte CD4+ -MVs occurred before each ß-score drop, CD8+ -MVs increased only in F, and B lymphocyte CD19+ -MVs remained undetectable. In conclusion, PSA-NCAM+ -MVs are noninvasive early markers of transplant dysfunction, while ASGPR+ -MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.
Assuntos
Micropartículas Derivadas de Células/patologia , Diabetes Mellitus Tipo 1/terapia , Rejeição de Enxerto/diagnóstico , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Leucócitos/patologia , Complicações Pós-Operatórias/diagnóstico , Adulto , Idoso , Feminino , Seguimentos , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Complicações Pós-Operatórias/etiologia , Prognóstico , Fatores de RiscoRESUMO
In this paper, superparamagnetic iron oxide nanoparticles (SPIONs, around 6 nm) encapsulated in poly(methyl methacrylate) nanoparticles (PMMA NPs) with controlled sizes ranging from 100 to 200 nm have been successfully produced. The hybrid polymeric NPs were prepared following two different methods: (1) nanoprecipitation and (2) nanoemulsification-evaporation. These two methods were implemented in two different microprocesses based on the use of an impact jet micromixer and an elongational-flow microemulsifier. SPIONs-loaded PMMA NPs synthesized by the two methods presented completely different physicochemical properties. The polymeric NPs prepared with the micromixer-assisted nanoprecipitation method showed a heterogeneous dispersion of SPIONs inside the polymer matrix, an encapsulation efficiency close to 100 wt %, and an irregular shape. In contrast, the polymeric NPs prepared with the microfluidic-assisted nanoemulsification-evaporation method showed a homogeneous dispersion, an almost complete encapsulation, and a spherical shape. The properties of the polymeric NPs have been characterized by dynamic light scattering, thermogravimetric analysis, and transmission electron microscope. In vitro cytotoxicity assays were also performed on the nanohybrids and pure PMMA NPs.
RESUMO
Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f ß-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or ß-cells were applied to H2 O2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in ß-cells. MP from aPC-treated EC (eMaPC ) exhibited high EPCR and annexin A1 content, protected ß-cells, restored insulin secretion and were captured by 80% of ß cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H2 O2 -treated rat islets with increased viability (62% versus 48% H2 O2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.
Assuntos
Anexina A1/genética , Micropartículas Derivadas de Células/química , Células Secretoras de Insulina/efeitos dos fármacos , Proteína C/farmacologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Endotelina/genética , Receptores de Lipoxinas/genética , Animais , Anexina A1/metabolismo , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Vasos Coronários/química , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Células Endoteliais/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cultura Primária de Células , Substâncias Protetoras/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Receptores de Endotelina/metabolismo , Receptores de Lipoxinas/metabolismo , Transdução de Sinais , Suínos , Técnicas de Cultura de TecidosRESUMO
Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and ß-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f ß-cell function, TF activity mediated by MPs and their modulation by 1 µM liraglutide were examined in a cell cross-talk model. Methyl-ß-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative ß-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events.
Assuntos
Membrana Celular/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Inflamação/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Tromboplastina/metabolismo , Animais , Caspase 3/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Inflamação/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Liraglutida/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ratos , Proteínas SNARE/metabolismoRESUMO
To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine ß-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1-100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-ß-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-ß-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype.
Assuntos
Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Pâncreas Exócrino/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Tacrolimo/farmacologia , Animais , Linhagem Celular , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RatosRESUMO
Current research on Digital Twin (DT) is largely focused on the performance of built assets in their operational phases as well as on urban environment. However, Digital Twin has not been given enough attention to construction phases, for which this paper proposes a Digital Twin framework for the construction phase, develops a DT prototype and tests it for the use case of measuring the productivity and monitoring of earthwork operation. The DT framework and its prototype are underpinned by the principles of versatility, scalability, usability and automation to enable the DT to fulfil the requirements of large-sized earthwork projects and the dynamic nature of their operation. Cloud computing and dashboard visualisation were deployed to enable automated and repeatable data pipelines and data analytics at scale and to provide insights in near-real time. The testing of the DT prototype in a motorway project in the Northeast of England successfully demonstrated its ability to produce key insights by using the following approaches: (i) To predict equipment utilisation ratios and productivities; (ii) To detect the percentage of time spent on different tasks (i.e., loading, hauling, dumping, returning or idling), the distance travelled by equipment over time and the speed distribution; and (iii) To visualise certain earthwork operations.
RESUMO
BACKGROUND: Ischemia-driven islet isolation procedure is one of the limiting causes of pancreatic islet transplantation. Ischemia-reperfusion process is associated with endothelium dysfunction and the release of pro-senescent microvesicles. We investigated whether pro-senescent endothelial microvesicles prompt islet senescence and dysfunction in vitro. MATERIAL AND METHODS: Pancreatic islets were isolated from male young rats. Replicative endothelial senescence was induced by serial passaging of primary porcine coronary artery endothelial cells, and microvesicles were isolated either from young passage 1 (P1) or senescent passage 3 (P3) endothelial cells. Islet viability was assessed by fluorescence microscopy, apoptosis by flow cytometry, and Western blot. Function was assessed by insulin secretion and islet senescence markers p53, p21, and p16 by Western blot. Microvesicles were stained by the PKH26 lipid fluorescent probe and their islet integration assessed by microscopy and flow cytometry. RESULTS: Regardless of the passage, half microvesicles were integrated in target islets after 24 hours incubation. Insulin secretion significantly decreased after treatment by senescent microvesicles (P3: 1.7 ± 0.2 vs untreated islet: 2.7 ± 0.2, P < .05) without altering the islet viability (89.47% ± 1.69 vs 93.15% ± 0.97) and with no significant apoptosis. Senescent microvesicles significantly doubled the expression of p53, p21, and p16 (P < .05), whereas young microvesicles had no significant effect. CONCLUSION: Pro-senescent endothelial microvesicles specifically accelerate the senescence of islets and alter their function. These data suggest that islet isolation contributes to endothelial driven islet senescence.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Senescência Celular , Ilhotas Pancreáticas/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular , Micropartículas Derivadas de Células/fisiologia , Células Cultivadas , Senescência Celular/genética , Vasos Coronários/citologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Suínos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Major adverse cardiovascular events among sepsis survivors is an emerging health issue. Because endothelial senescence leads to vascular dysfunction and atherothrombosis, sepsis could be associated to vascular stress-induced premature senescence and thus with long-term cardiovascular events. MATERIALS & METHODS: Adult Wistar male rats were submitted to cecal ligation and puncture, or a SHAM operation. Markers of inflammation, oxidative stress and endothelial senescence were assessed at 3, 7 and 90 days (D), and vascular reactivity was assessed in conductance and resistance vessels at D90. Expression of proteins involved in senescence and inflammation was assessed by Western blot analysis and confocal microscopy, oxidative stress by dihydroethidium probing. RESULTS: Pro-inflammatory endothelial ICAM-1 and VCAM-1 were up-regulated by three-fold in CLP vs. SHAM at D7 and remained elevated at D90. Oxidative stress followed a similar pattern but was detected in the whole vascular wall. Sepsis accelerated premature senescence in aorta vascular tissue as shown by the significant up-regulation of p53 and down-stream p21 and p16 senescent markers at D7, values peaking at D90 whereas the absence of significant variation in activated caspase-3 confirmed p53 as a prime inducer of senescence. In addition, p53 was mainly expressed in the endothelium. Sepsis-induced long-term vascular dysfunction was confirmed in aorta and main mesenteric artery, with a major alteration of the endothelial-dependent nitric oxide pathway. CONCLUSIONS: Septic shock-induced long-term vascular dysfunction is associated with endothelial and vascular senescence. Our model could prove useful for investigating senotherapies aiming at reducing long-term cardiovascular consequences of septic shock.
Assuntos
Sepse , Choque Séptico , Animais , Aorta/metabolismo , Senescência Celular , Masculino , Ratos , Ratos Wistar , Sepse/complicaçõesRESUMO
Long term survival post lung transplantation (LTx) is limited by the occurrence of bronchiolitis obliterans syndrome (BOS). One mechanism involved is the epithelial-mesenchymal transition (EMT). Membrane microparticles (MPs) are known to be involved in some respiratory diseases and in other organs allograft rejection episodes. We hypothesized that leukocyte-derived MPs likely contribute to EMT. To emphasize this physiological concept, our objectives were to: (1) confirm the presence of EMT on explanted lungs from patients who underwent a second LTx for BOS; 2) characterize circulating MPs in transplanted patients, with or without BOS; (3) evaluate in vitro the effect of monocyte-derived MPs in EMT of human bronchial epithelial cells. Our IHC analysis on explanted graft lungs revealed significant pathological signs of EMT with an inhomogeneous destruction of the bronchial epithelium, with decreased expression of the epithelial protein E-cadherin and increased expression of the mesenchymal protein Vimentin. The immunophenotyping of MPs demonstrated that the concentration of MPs carrying E-cadherin was lower in patients affected by BOS (p = .007). In vitro, monocyte-derived MPs produced with LPS were associated with decreased E-cadherin expression (p < .05) along with significant morphological and functional cell modifications. MPs may play a role in EMT onset in bronchial epithelium following LTx.
Assuntos
Bronquiolite Obliterante/metabolismo , Micropartículas Derivadas de Células/metabolismo , Transplante de Pulmão , Pulmão/patologia , Monócitos/metabolismo , Complicações Pós-Operatórias/metabolismo , Mucosa Respiratória/metabolismo , Adulto , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/patologia , Caderinas/metabolismo , Regulação para Baixo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Complicações Pós-Operatórias/patologia , Mucosa Respiratória/patologiaRESUMO
INTRODUCTION: In septic shock patients, postseptic immunosuppression state after the systemic inflammatory response syndrome is responsible for nosocomial infections, with subsequent increased mortality. The aim of the present study was to assess the underlying cellular mechanisms of the postseptic immunosuppression state, by investigating mitochondrial functions of peripheral blood mononuclear cells (PBMCs) from septic shock patients over 7 days. MATERIALS AND METHODS: Eighteen patients admitted to a French intensive care unit for septic shock were included. At days 1 and 7, PBMCs were isolated by Ficoll density gradient centrifugation. Mitochondrial respiration of intact septic PBMCs was assessed versus control group PBMCs, by measuring O2 consumption in plasma, using high-resolution respirometry. Mitochondrial respiration was then compared between septic plasmas and control plasmas for control PBMCs, septic PBMCs, and lymphoid cell-line (CEM). To investigate the role of plasma, we measured several plasma cytokines, among them High-Mobility Group Box 1 (HMGB1), by enzyme-linked immunosorbent assays. RESULTS: Basal O2 consumption of septic shock PBMCs was of 8.27â±â3.39 and 10.48â±â3.99âpmol/s/10 cells at days 1 and 7, respectively, significantly higher than in control PBMCs (5.37â±â1.46âpmol/s/10 cells, Pâ<â0.05). Septic patient PBMCs showed a lower response to oligomycin, suggesting a reduced ATP-synthase activity, as well as an increased response to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) suggesting an increased mitochondrial respiratory capacity. At 6âh, septic plasmas showed a decreased O2 consumption of CEM (4.73â±â1.46 vs. 6.58â±â1.53, Pâ<â0.05) as well as in control group PBMCs (1.76â±â0.36 vs. 2.70â±â0.42, Pâ<â0.05), and triggered a decreased ATP-synthase activity but an increased response to FCCP. These differences are not explained by different cell survival. High HMGB1 levels were significantly associated with reduced PBMCs mitochondrial respiration. CONCLUSIONS: Septic plasma impairs mitochondrial respiration in immune cells, with a possible role of the proinflammatory protein HMGB1, leading to a subsequent compensation, probably by enzymatic activation. This compensation result is an improvement of global mitochondrial respiratory capacity, but without restoring ATP-synthase activity.
Assuntos
Linfócitos/metabolismo , Mitocôndrias/metabolismo , Plasma/metabolismo , Choque Séptico/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Intervalo Livre de Doença , Feminino , Humanos , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Consumo de Oxigênio , Choque Séptico/mortalidade , Taxa de SobrevidaRESUMO
BACKGROUND In organ transplantation, particularly pancreas transplantation, donor age is a determinant factor for graft survival. Physiological aging is crucial in the progressive deterioration of organs in adulthood. We compared the senescence and function features of pancreas and vascular tissues in young rats and middle-aged rats. MATERIAL AND METHODS Islet morphology and the area of cells secreting insulin or glucagon was investigated using immunohistology in young rats (12 weeks) and middle-aged rats (52 weeks) (n=8). Senescence markers, oxidative stress (ROS), and tissue factor (TF) were measured in the rat pancreases. Circulating microparticles (MPs) were measured as surrogates of vascular cell injury. Vascular function was studied in mesenteric arterial rings. RESULTS Larger islets were twice as frequent in young rats versus middle-aged rats. In middle-aged rats there was a significant decrease of the ß-cells/islet area ratio. Western blot analysis showed an increased expression of p53, p21, and p16 senescence markers (2-, 7- and 3-fold respectively) with no modification in caspase-3 activation. A 30% decrease of endothelial nitric oxide synthase (eNOS) was observed together with a 4-fold increase in TF expression. ROS formation increased significantly (2-fold) in middle-aged rats and their main source, determined by pharmacological inhibition, was NADPH oxidase and uncoupled nitric-oxide (NO) synthase. No sign of vascular injury (microparticles) or dysfunction was evidenced. CONCLUSIONS Modification in islet morphology and function were detected in middle-aged rats before any measurement of macro-vascular dysfunction. The data indicate a pancreatic senescence in the process of aging associated with uncontrolled accumulation of oxidative species that suggests a determining role of donor age in transplantation.
Assuntos
Envelhecimento/fisiologia , Endotélio Vascular/fisiologia , Estresse Oxidativo/fisiologia , Pâncreas/metabolismo , Animais , Caspase 3/metabolismo , Glucagon/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Eicosapentaenoic acid:docosahexaenoic acid (EPA:DHA) 6:1, an omega-3 polyunsaturated fatty acid formulation, has been shown to induce a sustained formation of endothelial nitric oxide (NO) synthase-derived NO, a major vasoprotective factor. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Male Wister rats received orally corn oil or EPA:DHA 6:1 (500 mg kg-1 per day) before chronic infusion of Ang II (0.4 mg kg-1 per day). Systolic blood pressure was determined by tail cuff sphingomanometry, vascular reactivity using a myograph, oxidative stress using dihydroethidium and protein expression by immunofluorescence and western blot analysis. Ang II-induced hypertension was associated with reduced acetylcholine-induced relaxations of secondary branch mesenteric artery rings affecting the endothelium-dependent hyperpolarization (EDH)- and the NO-mediated relaxations, both of which were improved by the NADPH oxidase inhibitor VAS-2870. The Ang II treatment induced also endothelium-dependent contractile responses (EDCFs), which were abolished by the cyclooxygenase (COX) inhibitor indomethacin. An increased level of vascular oxidative stress and expression of NADPH oxidase subunits (p47phox and p22phox), COX-1 and COX-2, endothelial NO synthase and Ang II type 1 receptors were observed in the Ang II group, whereas SKCa and connexin 37 were downregulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction by improving both the NO- and EDH-mediated relaxations, and by reducing EDCFs and the expression of target proteins. The present findings indicate that chronic intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidase- and COX-derived oxidative stress.
Assuntos
Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-3/farmacologia , Hipertensão/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Angiotensina II , Animais , Avaliação Pré-Clínica de Medicamentos , Hipertensão/induzido quimicamente , Masculino , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Distribuição Aleatória , Ratos WistarRESUMO
Vascular stenosis is most often the culprit behind hemodialysis vascular access dysfunction, and although percutaneous transluminal angioplasty remains the gold standard treatment for vascular stenosis, over the past decade the use of stents as a treatment option has been on the rise. Aside from the 2 Food and Drug Administration-approved stent grafts for the treatment of venous graft anastomosis stenosis, use of all other stents in vascular access dysfunction is off-label. Kidney Disease Outcomes Quality Initiative recommends limiting stent use to specific conditions, such as elastic lesions and recurrent stenosis; otherwise, additional adapted indications are in procedure-related complications, such as grade 2 and 3 hematomas. Published reports have shown the potential use of stents in a variety of conditions leading to vascular access dysfunction, such as venous graft anastomosis stenosis, cephalic arch stenosis, central venous stenosis, dialysis access aneurysmal elimination, cardiac implantable electronic device-induced stenosis, and thrombosed arteriovenous grafts. Although further research is needed for many of these conditions, evidence for recommendations has been clear in some; for instance, we know now that stents should be avoided along cannulation sites and should not be used in eliminating dialysis access aneurysms. In this review article, we evaluate the available evidence for the use of stents in each of the aforementioned conditions leading to hemodialysis vascular access dysfunctions.