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/metabolismoRESUMEN
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 RiesgoRESUMEN
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 TejidosRESUMEN
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/metabolismoRESUMEN
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 , RatasRESUMEN
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/metabolismoRESUMEN
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/patologíaRESUMEN
Patient premedication with carbidopa seems to improve the accuracy of 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-FDOPA) PET for insulinoma diagnosis. However, the risk of PET false-negative results in the presence of carbidopa is a concern. Consequently, we aimed to evaluate the effect of carbidopa on 18F-FDOPA uptake in insulinoma ß-cells and an insulinoma xenograft model in mice. METHODS: 18F-FDOPA in vitro accumulation was assessed in the murine ß-cell line RIN-m5F. In vivo small-animal PET experiments were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F cells. Experiments were conducted with and without carbidopa pretreatment. RESULTS: Incubation of RIN-m5F cells with 80 µM carbidopa did not significantly affect the cellular accumulation of 18F-FDOPA. Tumor xenografts were clearly detectable by small-animal PET in all cases. Insulinoma xenografts in carbidopa-treated mice showed significantly higher 18F-FDOPA uptake than those in nontreated mice. Regardless of carbidopa premedication, the xenografts were characterized by an early increase in 18F-FDOPA uptake and then a progressive reduction over time. CONCLUSION: Carbidopa did not influence in vitro 18F-FDOPA accumulation in RIN-m5F cells but improved insulinoma imaging in vivo. Our findings increase current knowledge about the 18F-FDOPA uptake profile of RIN-m5F cells and a related xenograft model. To our knowledge, the present work represents the first preclinical research specifically focused on insulinomas, with potential translational implications.
Asunto(s)
Carbidopa/administración & dosificación , Dihidroxifenilalanina/análogos & derivados , Insulinoma/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Tomografía de Emisión de Positrones/métodos , Animales , Línea Celular Tumoral , Dihidroxifenilalanina/farmacocinética , Femenino , Aumento de la Imagen/métodos , Insulinoma/diagnóstico por imagen , Tasa de Depuración Metabólica/efectos de los fármacos , Ratones Desnudos , Especificidad de Órganos/efectos de los fármacos , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular/efectos de los fármacosRESUMEN
INTRODUCTION: Herein we have evaluated the uptake of O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) in insulinoma in comparison with those of 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-FDOPA) providing first data from both murine xenograft model and one patient with proved endogenous hyperinsulinemic hypoglycemia. METHODS: Dynamic 18F-FET and carbidopa-assisted 18F-FDOPA PET were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F murine beta cells and on a 30-year-old man with type-1 multiple endocrine neoplasia and hyperinsulinemic hypoglycemia defined by a positive fasting test. RESULTS: Seven and three nude mice bearing a RIN-m5F insulinoma xenograft were respectively studied by 18F-FET and 18F-FDOPA µPET. Insulinoma xenograft was detected in all the imaged animals. Xenograft was characterized by an early but moderate increase of 18F-FET uptake followed by a slight decline of uptake intensity during the 20 min dynamic acquisition. Tumoral radiotracer peak intensity and the highest tumor-to-background contrast were reached about 5 minutes after 18F-FET iv. injection (mean SUV: 1.21 ± 0.10). The biodistribution of 18F-FET and 18F-FDOPA and their dynamic tumoral uptake profile and intensity were similar. In the examined patient, 18F-FDOPA and 18F-FET PET/CT showed one concordant focal area of well-defined increased uptake in the pancreatic tail corresponding to 11 mm histologically proved insulinoma. The SUVmax tumor to liver ratio was 1.5, 1.1 for 18F-FDOPA, 1.1, 1 for 18F-FET at early (0-5 min post injection) and delayed (5-20 min post injection) PET/CT acquisition, respectively. Despite the relatively low tumoral uptake intensity, insulinoma was clearly identified due to the low background in the pancreas. At the contrary, no 18F-FDOPA or 18F-FET tumoral uptake was revealed on whole-body PET/CT images performed about 30 min after radiotracer administration. Note of worth, the dynamic uptake pattern of 18F-FET and 18F-FDOPA were similar between human insulinoma and mice xenograft tumor. CONCLUSION: 18F-FET PET compared equally to 18F-FDOPA PET in a preclinical RIN-m5F murine model of insulinoma and in one patient with insulinoma-related hypoglycemia. However, in both cases, the tumoral uptake intensity was moderate and the tumor was only visible until 20 min after radiotracer injection. Hence, caution should be taken before asserting the translational relevance of our results in the clinical practices. However, the structural analogies between 18F-FET and 18F-FDOPA as well as the limited pancreatic uptake of 18F-FET in human, encourage evaluating 18F-FET as diagnostic radiotracer for insulinoma detection in further prospective studies involving large cohorts of patients.
Asunto(s)
Transformación Celular Neoplásica , Insulinoma/metabolismo , Insulinoma/patología , Tirosina/análogos & derivados , Adulto , Animales , Transporte Biológico , Línea Celular Tumoral , Femenino , Humanos , Insulinoma/diagnóstico por imagen , Masculino , Ratones , Ratones Desnudos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tirosina/metabolismoRESUMEN
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.