RESUMO
OBJECTIVE: A wide range of cardiac diseases is associated with inflammation. "Inflamed" heart tissue is infiltrated with pro-inflammatory macrophages which extensively secrete matrix metalloproteinase 9 (MMP9), a regulator of extracellular matrix turnover. As MMP9 is released from macrophages in a latent form, it requires activation. The present study addresses the role of cardiomyocytes in the course of this activation process. METHODS AND RESULTS: In mono- and co-cultures of pro-inflammatory rat macrophages (bone marrow-derived and peritoneal) and cardiomyocytes (H9C2 cell line) gelatin zymography demonstrated that activated macrophages robustly secreted latent pro-MMP9, whereas cardiomyocytes could not produce the enzyme. Co-culturing of the two cell species was critical for pro-MMP9 activation and was also accompanied by processing of cardiomyocyte-secreted pro-MMP2. A cascade of pro-MMP9 activation was initiated on macrophage membrane with pro-MMP2 cleavage. Namely, pro-inflammatory macrophages expressed an active membrane type 1 MMP (MT1MMP), which activated pro-MMP2, which in turn converted pro-MMP9. Downregulation of MT1MMP in macrophages by siRNA abolished activation of both pro-MMP2 and pro-MMP9 in co-culture. In addition, both cell species secreted MMP13 as a further pro-MMP9 activator. In co-culture, activation of pro-MMP13 occurred on membranes of macrophages and was enhanced in presence of active MMP2. Using incubations with recombinant MMPs and isolated macrophage membranes, we demonstrated that while both MMP2 and MMP13 individually had the ability to activate pro-MMP9, their combined action provided a synergistic effect. CONCLUSION: Activation of pro-MMP9 in a co-culture of pro-inflammatory macrophages and cardiomyocytes was the result of a complex interaction of several MMPs on the cell membrane and in the extracellular space. Both cell types contributed critically to pro-MMP9 processing.
Assuntos
Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Animais , Ratos , Células Cultivadas , Técnicas de Cocultura , Macrófagos/metabolismo , Metaloproteinase 13 da Matriz , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Emerging data suggest that type 1 diabetes affects not only the ß-cell-containing islets of Langerhans, but also the surrounding exocrine compartment. Using digital pathology, machine learning algorithms were applied to high-resolution, whole-slide images of human pancreata to determine whether the tissue composition in individuals with or at risk for type 1 diabetes differs from those without diabetes. Transplant-grade pancreata from organ donors were evaluated from 16 nondiabetic autoantibody-negative controls, 8 nondiabetic autoantibody-positive subjects with increased type 1 diabetes risk, and 19 persons with type 1 diabetes (0 to 12 years' duration). HALO image analysis algorithms were implemented to compare architecture of the main pancreatic duct as well as cell size, density, and area of acinar, endocrine, ductal, and other nonendocrine, nonexocrine tissues. Type 1 diabetes was found to affect exocrine area, acinar cell density, and size, whereas the type of difference correlated with the presence or absence of insulin-positive cells remaining in the pancreas. These changes were not observed before disease onset, as indicated by modeling cross-sectional data from pancreata of autoantibody-positive subjects and those diagnosed with type 1 diabetes. These data provide novel insights into anatomic differences in type 1 diabetes pancreata and demonstrate that machine learning can be adapted for the evaluation of disease processes from cross-sectional data sets.
Assuntos
Algoritmos , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/patologia , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Pâncreas/patologia , Adolescente , Autoanticorpos/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Humanos , Insulina/análise , Pâncreas/imunologia , Pâncreas/metabolismo , Doadores de TecidosRESUMO
Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.
Assuntos
Ilhotas Pancreáticas/citologia , Animais , Biomarcadores/análise , Diferenciação Celular , Linhagem da Célula/genética , Polaridade Celular , Proliferação de Células , Humanos , Resistência à Insulina , Ilhotas Pancreáticas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Via de Sinalização WntRESUMO
BACKGROUND: Mast cells (MCs) are best known as key effector cells of allergic reactions, but they also play an important role in host defense against pathogens. Despite increasing evidence for a critical effect of MCs on adaptive immunity, the underlying mechanisms are poorly understood. OBJECTIVE: Here we monitored MC intercellular communication with dendritic cells (DCs), MC activation, and degranulation and tracked the fate of exocytosed mast cell granules (MCGs) during skin inflammation. METHODS: Using a strategy to stain intracellular MCGs in vivo, we tracked the MCG fate after skin inflammation-induced MC degranulation. Furthermore, exogenous MCGs were applied to MC-deficient mice by means of intradermal injection. MCG effects on DC functionality and adaptive immune responses in vivo were assessed by combining intravital multiphoton microscopy with flow cytometry and functional assays. RESULTS: We demonstrate that dermal DCs engulf the intact granules exocytosed by MCs on skin inflammation. Subsequently, the engulfed MCGs are actively shuttled to skin-draining lymph nodes and finally degraded inside DCs within the lymphoid tissue. Most importantly, MCG uptake promotes DC maturation and migration to skin-draining lymph nodes, partially through MC-derived TNF, and boosts their T-cell priming efficiency. Surprisingly, exogenous MCGs alone are sufficient to induce a prominent DC activation and T-cell response. CONCLUSION: Our study highlights a unique feature of peripheral MCs to affect lymphoid tissue-borne adaptive immunity over distance by modifying DC functionality through delivery of granule-stored mediators.
Assuntos
Dermatite/metabolismo , Hipersensibilidade/metabolismo , Células de Langerhans/fisiologia , Mastócitos/fisiologia , Vesículas Secretórias/metabolismo , Pele/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , Dermatite/imunologia , Modelos Animais de Doenças , Endocitose , Humanos , Hipersensibilidade/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AIMS/HYPOTHESIS: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells. METHODS: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca2+ indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells. RESULTS: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity. CONCLUSIONS/INTERPRETATION: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
Assuntos
Trifosfato de Adenosina/metabolismo , Macrófagos/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Animais , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , CamundongosRESUMO
AIMS/HYPOTHESIS: Pancreatic beta cells maintain glucose homeostasis and beta cell dysfunction is a major risk factor in developing diabetes. Therefore, understanding the developmental regulatory networks that define a fully functional beta cell is important for elucidating the genetic origins of the disease. Aldehyde dehydrogenase activity has been associated with stem/progenitor cells and we have previously shown that Aldh1b1 is specifically expressed in pancreas progenitor pools. Here we address the hypothesis that Aldh1b1 may regulate the timing of the appearance and eventual functionality of beta cells. METHODS: We generated an Aldh1b1-knockout mouse line (Aldh1b1 (tm1lacZ)) and used this to study pancreatic development, beta cell functionality and glucose homeostasis in the absence of Aldh1b1 function. RESULTS: Differentiation in the developing pancreas of Aldh1b1 (tm1lacZ) null mice was accelerated. Transcriptome analyses of newborn and adult islets showed misregulation of key beta cell transcription factors and genes crucial for beta cell function. Functional analyses showed that glucose-stimulated insulin secretion was severely compromised in islets isolated from null mice. Several key features of beta cell functionality were affected, including control of oxidative stress, glucose sensing, stimulus-coupling secretion and secretory granule biogenesis. As a result of beta cell dysfunction, homozygous mice developed glucose intolerance and age-dependent hyperglycaemia. CONCLUSIONS/INTERPRETATION: These findings show that Aldh1b1 influences the timing of the transition from the pancreas endocrine progenitor to the committed beta cell and demonstrate that changes in the timing of this transition lead to beta cell dysfunction and thus constitute a diabetes risk factor later in life. Gene Expression Omnibus (GEO) accession: GSE58025.
Assuntos
Aldeído Desidrogenase/genética , Aldeído Desidrogenase/fisiologia , Células Secretoras de Insulina/metabolismo , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial , Alelos , Animais , Glicemia/análise , Diferenciação Celular , Glucose/metabolismo , Teste de Tolerância a Glucose , Glicogênio/metabolismo , Homeostase , Hiperglicemia/metabolismo , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Células-Tronco/citologia , TranscriptomaRESUMO
The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo.
Assuntos
Olho/metabolismo , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Leptina/metabolismo , Aloenxertos , Animais , Olho/citologia , Feminino , Ilhotas Pancreáticas/citologia , Leptina/genética , Camundongos , Camundongos ObesosRESUMO
The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Olho/inervação , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Animais , Proteínas de Fluorescência Verde/metabolismo , Iris/inervação , Iris/fisiologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras NervosasRESUMO
AIMS/HYPOTHESIS: Islet transplantation is used therapeutically in a minority of patients with type 1 diabetes. Successful outcomes are hampered by early islet beta cell loss. The adjuvant co-transplantation of mesenchymal stromal cells (MSCs) has the promise to improve islet transplant outcome. METHODS: We used a syngeneic marginal islet mass transplantation model in a mouse model of diabetes. Mice received islets or islets plus 250,000 MSCs. Kidney subcapsule, intra-hepatic and intra-ocular islet transplantation sites were used. Apoptosis, vascularisation, beta cell proliferation, MSC differentiation and laminin levels were determined by immunohistochemical analysis and image quantification post-transplant. RESULTS: Glucose homeostasis after the transplantation of syngeneic islets was improved by the co-transplantation of MSCs together with islets under the kidney capsule (p = 0.01) and by intravenous infusion of MSCs after intra-hepatic islet transplantation (p = 0.05). MSC co-transplantation resulted in reduced islet apoptosis, with reduced numbers of islet cells positive for cleaved caspase 3 being observed 14 days post-transplant. In kidney subcapsule, but not in intra-ocular islet transplant models, we observed increased re-vascularisation rates, but not increased blood vessel density in and around islets co-transplanted with MSCs compared with islets that were transplanted alone. Co-transplantation of MSCs did not increase beta cell proliferation, extracellular matrix protein laminin production or alpha cell numbers, and there was negligible MSC transdifferentiation into beta cells. CONCLUSIONS/INTERPRETATION: Co-transplantation of MSCs may lead to improved islet function and survival in the early post-transplantation period in humans receiving islet transplantation.
Assuntos
Diabetes Mellitus Experimental/patologia , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Animais , Glicemia , Proliferação de Células , Técnicas de Cocultura , Diabetes Mellitus Experimental/imunologia , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Transplante IsogênicoRESUMO
Over the last two decades, increased availability of human pancreatic tissues has allowed for major expansions in our understanding of islet biology in health and disease. Indeed, studies of fixed and frozen pancreatic tissues, as well as efforts using viable isolated islets obtained from organ donors, have provided significant insights toward our understanding of diabetes. However, the procedures associated with islet isolation result in distressed cells that have been removed from any surrounding influence. The pancreas tissue slice technology was developed as an in situ approach to overcome certain limitations associated with studies on isolated islets or fixed tissue. In this Perspective, we discuss the value of this novel platform and review how pancreas tissue slices, within a short time, have been integrated in numerous studies of rodent and human islet research. We show that pancreas tissue slices allow for investigations in a less perturbed organ tissue environment, ranging from cellular processes, over peri-islet modulations, to tissue interactions. Finally, we discuss the considerations and limitations of this technology in its future applications. We believe the pancreas tissue slices will help bridge the gap between studies on isolated islets and cells to the systemic conditions by providing new insight into physiological and pathophysiological processes at the organ level. ARTICLE HIGHLIGHTS: Human pancreas tissue slices represent a novel platform to study human islet biology in close to physiological conditions. Complementary to established technologies, such as isolated islets, single cells, and histological sections, pancreas tissue slices help bridge our understanding of islet physiology and pathophysiology from single cell to intact organ. Diverse sources of viable human pancreas tissue, each with distinct characteristics to be considered, are available to use in tissue slices for the study of diabetes pathogenesis.
Assuntos
Diabetes Mellitus , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Humanos , Pâncreas , Doadores de TecidosRESUMO
Insulin-producing ß-cells in pancreatic islets are regulated by systemic cues and, locally, by adjacent islet hormone-producing 'non-ß-cells' (namely α-cells, δ-cells and γ-cells). Yet whether the non-ß-cells are required for accurate insulin secretion is unclear. Here, we studied mice in which adult islets are exclusively composed of ß-cells and human pseudoislets containing only primary ß-cells. Mice lacking non-ß-cells had optimal blood glucose regulation, enhanced glucose tolerance, insulin sensitivity and restricted body weight gain under a high-fat diet. The insulin secretion dynamics in islets composed of only ß-cells was comparable to that in intact islets. Similarly, human ß-cell pseudoislets retained the glucose-regulated mitochondrial respiration, insulin secretion and exendin-4 responses of entire islets. The findings indicate that non-ß-cells are dispensable for blood glucose homeostasis and ß-cell function. These results support efforts aimed at developing diabetes treatments by generating ß-like clusters devoid of non-ß-cells, such as from pluripotent stem cells differentiated in vitro or by reprograming non-ß-cells into insulin producers in situ.
RESUMO
Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.
Assuntos
Diabetes Mellitus Tipo 1 , Insulina , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Insulina/metabolismo , Feminino , Secreção de Insulina/efeitos dos fármacos , Adulto , Pessoa de Meia-Idade , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Glucagon/metabolismo , Glucose/metabolismo , Autoanticorpos/imunologia , Amilases/metabolismoRESUMO
Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.
RESUMO
As a major cause of morbidity and mortality globally, hypertension remains a serious threat to global public health. Despite the availability of many antihypertensive medications, several hypertensive individuals are resistant to standard treatments, and are unable to control their blood pressure. Regulation of the renin-angiotensin-aldosterone system (RAAS) controlling blood pressure, activation of the immune system triggering inflammation and production of reactive oxygen species, leading to oxidative stress and redox-sensitive signaling, have been implicated in the pathogenesis of hypertension. Thus, besides standard antihypertensive medications, which lower arterial pressure, antioxidant medications were tested to improve antihypertensive treatment. We review and discuss the role of oxidative stress in the pathophysiology of hypertension and the potential use of antioxidants in the management of hypertension and its associated organ damage.
Assuntos
Proteínas de Fluorescência Verde/genética , Insulina/genética , Ilhotas Pancreáticas/fisiologia , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Regulação da Expressão Gênica/genética , Glucose/farmacologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/embriologia , Sus scrofa , SuínosRESUMO
Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic ß-cells. ß-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between ß-cells via gap junctions. Here, we identify the importance of signaling between ß-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult ß-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between ß-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating ß-cells.
Assuntos
Sinalização do Cálcio , Insulinas , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo , Insulinas/metabolismo , Difosfato de Adenosina/metabolismoRESUMO
Under physiological conditions and in the pathogenesis of diabetes mellitus systemic influences play a substantial role for function and survival of cells of the islet of Langerhans. Therefore, in vivo studies to understand islet biology are indispensible and imaging techniques are increasingly used for this purpose. Among the diverse imaging modalities currently only laser scanning microscopy (LSM) allows resolution and visualization of individual cells and cellular processes. To overcome limited tissue penetration and working distance of LSM and enable in vivo investigations of islet cell physiology, various experimental approaches have been developed. Especially, the recently developed imaging platforms have significantly improved the possibility to study islets at a cellular level in vivo, and provided novel insight into islet biology in health and disease. The various approaches, their applications, and reported results, as well as their limitations are reviewed in this article.
Assuntos
Diagnóstico por Imagem/métodos , Transplante das Ilhotas Pancreáticas , Humanos , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Microscopia ConfocalRESUMO
Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.
Assuntos
Comunicação Celular , Diabetes Mellitus Tipo 2/patologia , Sistema Imunitário/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/fisiologia , Animais , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Sistema Imunitário/citologia , Ilhotas Pancreáticas/citologia , CamundongosRESUMO
Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Células-Tronco Pluripotentes , Animais , Humanos , Camundongos , Mutação , Organoides , Ductos Pancreáticos , Neoplasias Pancreáticas/genética , ProteômicaRESUMO
Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.