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1.
AI Civil Eng ; 1(1): 7, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-38013884

RESUMEN

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.

2.
Vascul Pharmacol ; 141: 106922, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34592427

RESUMEN

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.


Asunto(s)
Sepsis , Choque Séptico , Animales , Aorta/metabolismo , Senescencia Celular , Masculino , Ratas , Ratas Wistar , Sepsis/complicaciones
3.
Transplant Proc ; 53(5): 1736-1743, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33934912

RESUMEN

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.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Senescencia Celular , Islotes Pancreáticos/metabolismo , Animales , Apoptosis/genética , Supervivencia Celular , Micropartículas Derivadas de Células/fisiología , Células Cultivadas , Senescencia Celular/genética , Vasos Coronarios/citología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Porcinos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
4.
Am J Respir Cell Mol Biol ; 65(2): 167-175, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33798037

RESUMEN

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.


Asunto(s)
Coagulación Intravascular Diseminada/metabolismo , Interferón-alfa/metabolismo , Factor de Transcripción STAT1/metabolismo , Choque Séptico/metabolismo , Transducción de Señal , Anciano , Animales , Coagulación Intravascular Diseminada/genética , Femenino , Humanos , Interferón-alfa/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Factor de Transcripción STAT1/genética , Choque Séptico/genética , Choque Séptico/terapia
5.
J Cell Mol Med ; 24(13): 7266-7281, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32520423

RESUMEN

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.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Senescencia Celular , Células Endoteliales/patología , Endotelio Vascular/fisiopatología , Inflamación/patología , Neutrófilos/metabolismo , Daño por Reperfusión/fisiopatología , Angiotensinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Coagulación Sanguínea/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Micropartículas Derivadas de Células/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Neutrófilos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas Wistar , Bazo/efectos de los fármacos , Bazo/patología , Porcinos , Acetato de Tetradecanoilforbol/farmacología , Tromboplastina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo
6.
Transpl Immunol ; 59: 101273, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32097721

RESUMEN

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.


Asunto(s)
Bronquiolitis Obliterante/metabolismo , Micropartículas Derivadas de Células/metabolismo , Trasplante de Pulmón , Pulmón/patología , Monocitos/metabolismo , Complicaciones Posoperatorias/metabolismo , Mucosa Respiratoria/metabolismo , Adulto , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/patología , Cadherinas/metabolismo , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/patología , Complicaciones Posoperatorias/patología , Mucosa Respiratoria/patología
7.
Am J Transplant ; 20(1): 40-51, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31319009

RESUMEN

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.


Asunto(s)
Micropartículas Derivadas de Células/patología , Diabetes Mellitus Tipo 1/terapia , Rechazo de Injerto/diagnóstico , Células Secretoras de Insulina/patología , Trasplante de Islotes Pancreáticos/efectos adversos , Leucocitos/patología , Complicaciones Posoperatorias/diagnóstico , Adulto , Anciano , Femenino , Estudios de Seguimiento , Rechazo de Injerto/etiología , Supervivencia de Injerto , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Proyectos Piloto , Complicaciones Posoperatorias/etiología , Pronóstico , Factores de Riesgo
8.
Shock ; 51(1): 97-104, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29461465

RESUMEN

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 8.27 ±â€Š3.39 and 10.48 ±â€Š3.99 pmol/s/10 cells at days 1 and 7, respectively, significantly higher than in control PBMCs (5.37 ±â€Š1.46 pmol/s/10 cells, P < 0.05). 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 6 h, septic plasmas showed a decreased O2 consumption of CEM (4.73 ±â€Š1.46 vs. 6.58 ±â€Š1.53, P < 0.05) as well as in control group PBMCs (1.76 ±â€Š0.36 vs. 2.70 ±â€Š0.42, P < 0.05), 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.


Asunto(s)
Linfocitos/metabolismo , Mitocondrias/metabolismo , Plasma/metabolismo , Choque Séptico/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular , Supervivencia sin Enfermedad , Femenino , Humanos , Linfocitos/patología , Masculino , Persona de Mediana Edad , Mitocondrias/patología , Consumo de Oxígeno , Choque Séptico/mortalidad , Tasa de Supervivencia
9.
Langmuir ; 34(5): 1981-1991, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29334739

RESUMEN

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.

10.
Hypertens Res ; 40(12): 966-975, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28878301

RESUMEN

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.


Asunto(s)
Antihipertensivos/química , Antihipertensivos/farmacología , Endotelio Vascular/efectos de los fármacos , Ácidos Grasos Omega-3/química , Ácidos Grasos Omega-3/farmacología , Hipertensión/prevención & control , Estrés Oxidativo/efectos de los fármacos , Angiotensina II , Animales , Evaluación Preclínica de Medicamentos , Hipertensión/inducido químicamente , Masculino , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Distribución Aleatoria , Ratas Wistar
11.
J Cell Mol Med ; 21(11): 2759-2772, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28524456

RESUMEN

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.


Asunto(s)
Anexina A1/genética , Micropartículas Derivadas de Células/química , Células Secretoras de Insulina/efectos de los fármacos , Proteína C/farmacología , Proteínas Serina-Treonina Quinasas/genética , Receptores de Endotelina/genética , Receptores de Lipoxina/genética , Animales , Anexina A1/metabolismo , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Vasos Coronarios/química , Vasos Coronarios/citología , Vasos Coronarios/metabolismo , Células Endoteliales/química , Células Endoteliales/citología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cultivo Primario de Células , Sustancias Protectoras/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptores de Endotelina/metabolismo , Receptores de Lipoxina/metabolismo , Transducción de Señal , Porcinos , Técnicas de Cultivo de Tejidos
12.
Ann Transplant ; 22: 177-186, 2017 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-28360408

RESUMEN

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.


Asunto(s)
Envejecimiento/fisiología , Endotelio Vascular/fisiología , Estrés Oxidativo/fisiología , Páncreas/metabolismo , Animales , Caspasa 3/metabolismo , Glucagón/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo
13.
Mol Cell Biochem ; 418(1-2): 91-102, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27344165

RESUMEN

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.


Asunto(s)
Apoptosis/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Páncreas Exocrino/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Tacrolimus/farmacología , Animales , Línea Celular , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas
14.
J Cell Mol Med ; 20(2): 231-42, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26607759

RESUMEN

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.


Asunto(s)
Membrana Celular/fisiología , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Inflamación/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Insulina/metabolismo , Tromboplastina/metabolismo , Animales , Caspasa 3/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Péptido 1 Similar al Glucagón/metabolismo , Hiperglucemia/metabolismo , Hiperglucemia/patología , Inflamación/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Liraglutida/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Ratas , Proteínas SNARE/metabolismo
15.
Adv Chronic Kidney Dis ; 22(6): 453-8, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26524950

RESUMEN

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.


Asunto(s)
Aneurisma Falso/cirugía , Aneurisma/cirugía , Angioplastia/métodos , Derivación Arteriovenosa Quirúrgica/métodos , Constricción Patológica/terapia , Fallo Renal Crónico/terapia , Complicaciones Posoperatorias/terapia , Diálisis Renal , Stents , Trombosis/cirugía , Humanos
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