RESUMEN
Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Macrófagos/metabolismo , Proteína smad7/metabolismo , Regulación hacia Arriba , Animales , Movimiento Celular , Núcleo Celular/metabolismo , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Inflamación/metabolismo , Inflamación/patología , Ligadura , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. METHODS: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. RESULTS: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. CONCLUSIONS/INTERPRETATION: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Conductos Pancreáticos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Epiteliales/citología , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Islotes Pancreáticos/citología , Trasplante de Islotes Pancreáticos , Ratones , Neovascularización Fisiológica , ARN Interferente Pequeño/metabolismo , Factor de Transcripción SOX9/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Transdiferenciación Celular/fisiología , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Conductos Pancreáticos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Células Secretoras de Insulina/citología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Conductos Pancreáticos/citología , Succinimidas/farmacologíaRESUMEN
VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.
Asunto(s)
Hipoglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Apoptosis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proliferación Celular , Células Cultivadas , Expresión Génica , Células Secretoras de Insulina/fisiología , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Ratones , Páncreas/irrigación sanguínea , Páncreas/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
BACKGROUND: Peer support programs have evolved to train physicians to provide outreach and emotional first aid to their colleagues when they experience the inevitable challenge of a serious adverse event, whether or not it is related to a medical error. Most pediatric surgeons have experienced the trauma of a medical error, yet, in a survey of APSA membership, almost half said that no one reached out to them, and few were satisfied with their institution's response to the error. Thus, the APSA Wellness Committee developed an APSA-based peer support program to meet this need. METHODS: Peer supporters were nominated by fellow APSA members, and the group was vetted to ensure diversity in demographics, practice setting, and seniority. Formal virtual training was conducted before the program went live in 2020. Trained supporters were surveyed 6 months after the program launched to evaluate their experiences with providing peer support. RESULTS: 15 referrals were made in the first year, 60 % of which were self-initiated. Most referrals were for distress related to adverse events or toxic work environments (33 % each). While only about 25 % of trained supporters had provided formal support through the APSA program, more than 80 % reported using the skills to support colleagues and trainees within their own institutions. CONCLUSION: Our experience in the first year of the APSA peer support program demonstrates the feasibility of building and maintaining a national program to provide emotional first aid by a professional society to expand the safety net for surgeons who are suffering.
Asunto(s)
Grupo Paritario , Humanos , Errores Médicos/prevención & control , Errores Médicos/psicología , Sociedades Médicas , Cirujanos/psicología , Cirujanos/educación , Agotamiento Profesional/prevención & control , Agotamiento Profesional/psicología , Pediatría/educación , Apoyo Social , Estados Unidos , Evaluación de Programas y Proyectos de Salud , Encuestas y CuestionariosRESUMEN
Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFß receptor signaling in ß-cells has been shown to increase ß-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFß receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by ß-cells, and significantly increased ß-cell number by increasing ß-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human ß-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in ß-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFß receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing ß-cell number and function.
Asunto(s)
Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Animales , Benzamidas/farmacología , Glucemia/metabolismo , Western Blotting , Péptido C/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dioxoles/farmacología , Femenino , Humanos , Insulina/sangre , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratones Endogámicos NOD , Ratones SCID , Microscopía Confocal , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Trasplante HeterólogoRESUMEN
A key question in diabetes research is whether new ß-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into ß-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-ß receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new ß-cells in normal young developing mice as well as in adult TGF-ß signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to ß-cells in the postnatal pancreas but also importantly implicates TGF-ß signaling in this process.
Asunto(s)
Transdiferenciación Celular , Células Secretoras de Insulina/citología , Insulina/biosíntesis , Islotes Pancreáticos/citología , Conductos Pancreáticos/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Adolescente , Factores de Edad , Animales , Cadáver , Preescolar , Femenino , Humanos , Lactante , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/fisiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Mutantes , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Pancreatectomía , Conductos Pancreáticos/crecimiento & desarrollo , Conductos Pancreáticos/fisiología , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Regeneración , Proteína Fluorescente RojaRESUMEN
Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.