RESUMO
AIMS/HYPOTHESIS: Pancreatic islet transplantation (PIT) offers a physiological treatment for type 1 diabetes, but the failure of islet engraftment hinders its application. The female hormone 17ß-oestradiol (E2) favours islet survival and stimulates angiogenesis, raising the possibility that E2 may enhance islet engraftment following PIT. METHODS: To explore this hypothesis, we used an insulin-deficient model with xenotransplantation of a marginal dose of human islets in nude mice rendered diabetic with streptozotocin. This was followed by 4 weeks of treatment with vehicle, E2, the non-feminising oestrogen 17α-oestradiol (17α-E2), the oestrogen receptor (ER) α agonist propyl-pyrazole-triol (PPT), the ERß agonist diarylpropionitrile (DPN) or the G protein-coupled oestrogen receptor (GPER) agonist G1. RESULTS: Treatment with E2, 17α-E2, PPT, DPN or G1 acutely improved blood glucose and eventually promoted islet engraftment, thus reversing diabetes. The effects of E2 were retained in the presence of immunosuppression and persisted after discontinuation of E2 treatment. E2 produced an acute decrease in graft hypoxic damage and suppressed beta cell apoptosis. E2 also acutely suppressed hyperglucagonaemia without altering insulin secretion, leading to normalisation of blood glucose. CONCLUSIONS/INTERPRETATION: During PIT, E2 synergistic actions contribute to enhancing human islet-graft survival, revascularisation and functional mass. This study identifies E2 as a short-term treatment to improve PIT.
Assuntos
Estrogênios/farmacologia , Transplante das Ilhotas Pancreáticas , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Ginsenosídeos/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Nitrilas/farmacologia , Propionatos/farmacologia , Sapogeninas/farmacologiaRESUMO
We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.
Assuntos
Anti-Inflamatórios , Anticoagulantes , Células Epiteliais/metabolismo , Técnicas de Transferência de Genes , Cavidade Peritoneal/citologia , Trombomodulina/genética , Animais , Anti-Inflamatórios/metabolismo , Anticoagulantes/metabolismo , Northern Blotting , Bovinos , Feminino , Expressão Gênica , Vetores Genéticos , Plasmídeos , Testes de Precipitina , Coelhos , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Trombomodulina/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In the immediately preceding paper, we demonstrated that the microvasculature supplying peritoneal lining tissues of mice bearing either of two transplantable ascites carcinomas was hyperpermeable to circulating macromolecules. Solid tumors have been shown to exhibit similar levels of microvascular hyperpermeability, leading to extravasation of plasma proteins, including fibrinogen which clots on extravasation to form an extravascular fibrin gel. To determine whether similar extravasation and clotting of plasma fibrinogen occurred in ascites tumors, we used 125I-labeled fibrinogen (125I-F) as a tracer to measure inflow of fibrinogen into the peritoneal cavities, and influx and accumulation of fibrinogen/fibrin in the peritoneal lining tissues (peritoneal wall, mesentery, and diaphragm) of mice bearing syngeneic TA3/St or MOT ascites tumors. The percentage of circulating 125I-F that extravasated into the peritoneal cavity was increased from 10- to 50-fold in mice bearing either ascites tumor. Influx into the peritoneal walls of ascites tumor-bearing mice was 3-7 times that of control mice and became maximal on day 8 (TA3/St) and day 15 (MOT). Accumulation of 125I-F in ascites fluid and peritoneal lining tissues was also increased substantially in mice bearing these ascites tumors, reaching maximal values on days 7-8 (TA3/St) and 19-29 (MOT) at levels 2- to 3-fold (peritoneal wall) and 33- to 148-fold (ascites fluid) above control levels. Significant amounts of the 125I-F that accumulated in the peritoneal lining tissues of ascites tumor-bearing animals were insoluble in 3 M urea, consistent with clotting of 125I-F to cross-linked fibrin. Autoradiographs of SDS-PAGE gels performed on extracts of peritoneal lining tissues of both ascites tumors revealed the characteristic signature of cross-linked fibrin, i.e., gamma-gamma dimers and alpha-polymers. Fibrin was also identified in peritoneal lining tissues of both ascites tumors by immunohistochemistry. Taken together, these data indicate that fibrinogen, like other circulating macromolecules, extravasates into the peritoneal cavity and peritoneal lining tissues of ascites tumor-bearing mice and does so with kinetics similar to those of other macromolecular tracers we have studied. Moreover, a portion of the fibrinogen that extravasated into peritoneal lining tissues clotted to form a cross-linked fibrin meshwork which trapped tumor cells and favored their attachment to the peritoneal surface. By analogy with solid tumors, such fibrin deposits may also be expected to have a role in initiating angiogenesis and the generation of mature tumor stroma.
Assuntos
Ascite/etiologia , Permeabilidade Capilar , Fibrinogênio/metabolismo , Cavidade Peritoneal/irrigação sanguínea , Peritônio/irrigação sanguínea , Animais , Ascite/metabolismo , Feminino , Fibrina/análise , Radioisótopos do Iodo/farmacocinética , Masculino , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Músculo Esquelético/metabolismo , Neoplasias Ovarianas/metabolismo , Peritônio/química , Peritônio/metabolismoRESUMO
Previous studies have shown that accumulation of tumor ascites fluid results in large part from increased permeability of peritoneal lining vessels (Nagy et al., Cancer Res., 49: 5449-5458, 1989; Nagy et al., Cancer Res., 53: 2631-2643, 1993). However, the specific microvessels rendered hyperpermeable have not been identified nor has the basis of peritoneal vascular hyperpermeability been established. To address these questions, TA3/St and MOT carcinomas, well-characterized transplantable murine tumors that grow in both solid and ascites form, were studied as model systems. Ascites tumor cells of either type were injected i.p. into syngeneic A/Jax and C3Heb/FeJ mice, and ascites fluid and plasma were collected at intervals thereafter up to 8 and 28 days, respectively. Beginning several days after tumor cell injection, small blood vessels located in tissues lining the peritoneal cavity (mesentery, peritoneal wall, and diaphragm) became hyperpermeable to several macromolecular tracers (125I-human serum albumin, FITC-dextran, colloidal carbon, and Monastral Blue B). Increased microvascular permeability correlated with the appearance in ascites fluid of vascular permeability factor (VPF), a tumor cell-secreted mediator that potently enhances vascular permeability to circulating macromolecules. VPF was measured in peritoneal fluid by both a functional bioassay and a sensitive immunofluorometric assay. The VPF concentration, total peritoneal VPF, ascites fluid volume, tumor cell number, and hyperpermeability of peritoneal lining microvessels were found to increase in parallel over time. The close correlation of peritoneal fluid VPF concentration with the development of hyperpermeable peritoneal microvessels in these two well-defined ascites tumors suggests that VPF secretion by tumor cells is responsible, in whole or in part, for initiating and maintaining the ascites pattern of tumor growth.