RESUMEN
Type 1 diabetes is due to destruction of pancreatic ß-cells. Lysine deacetylase inhibitors (KDACi) protect ß-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing ß-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1ß + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.
Asunto(s)
Cromatina/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Células Secretoras de Insulina/citología , Animales , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Factor de Transcripción GATA3/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Procesamiento Proteico-Postraduccional , Ratas , Factores de Tiempo , VorinostatRESUMEN
Diabetes is a prevalent chronic disease affecting millions of people globally. To address this health challenge, advanced beta cell therapy using biomaterials-based macroscale, microscale, and nanoscale encapsulation devices must tackle various obstacles. First, overcoming foreign body responses is a major focus of research. Strategies such as immunomodulatory materials and physical immunoshielding are investigated to reduce the immune response and improve the longevity of the encapsulated cells. Furthermore, oxygenating strategies, such as the use of oxygen-releasing biomaterials, are developed to improve oxygen diffusion and promote cell survival. Finally, yet importantly, promoting vascularization through the use of angiogenic growth factors and the incorporation of pre-vascularized materials are also explored to enhance nutrient and oxygen supply to the encapsulated cells. This review seeks to specifically highlight the emerging research strategies developed to overcome these challenges using micro and nanoscale biomaterial encapsulation devices. Continuously improving and refining these strategies make an advance toward realizing the improved therapeutic potential of the encapsulated beta cells.
Asunto(s)
Materiales Biocompatibles , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 1/terapia , Animales , Materiales Biocompatibles/química , Encapsulación Celular/métodos , Oxígeno/químicaRESUMEN
Type 1 diabetes mellitus (T1DM) is a chronic disease affecting millions worldwide. Insulin therapy is currently the golden standard for treating T1DM; however, it does not restore the normal glycaemic balance entirely, which increases the risk of secondary complications. Beta-cell therapy may be a possible way of curing T1DM and has already shown promising results in the clinic. However, low retention rates, poor cell survival, and limited therapeutic potential are ongoing challenges, thus increasing the need for better cell encapsulation devices. This study aimed to develop a mechanically reinforced vascular endothelial growth factor (VEGF)-delivering encapsulation device suitable for beta cell encapsulation and transplantation. Poly(l-lactide-co-ε-caprolactone) (PLCL)/gelatin methacryloyl (GelMA)/alginate coaxial nanofibres were produced using electrospinning and embedded in an alginate hydrogel. The encapsulation device was physically and biologically characterised and was found to be suitable for INS-1E beta cell encapsulation, vascularization, and transplantation in terms of its biocompatibility, porosity, swelling ratio and mechanical properties. Lastly, VEGF was incorporated into the hydrogel and the release kinetics and functional studies revealed a sustained release of bioactive VEGF for at least 14 days, making the modified alginate system a promising candidate for improving the beta cell survival after transplantation.
Asunto(s)
Alginatos , Gelatina , Hidrogeles , Células Secretoras de Insulina , Factor A de Crecimiento Endotelial Vascular , Hidrogeles/química , Alginatos/química , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Gelatina/química , Animales , Poliésteres/química , Ratas , Supervivencia Celular/efectos de los fármacos , Humanos , Diabetes Mellitus Tipo 1/terapia , Metacrilatos/química , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/farmacología , Inductores de la Angiogénesis/administración & dosificación , Propiedades de SuperficieRESUMEN
BACKGROUND: The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. METHODS: Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. RESULTS: Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. CONCLUSIONS: OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE(2).
Asunto(s)
Colon/metabolismo , Neoplasias Colorrectales/metabolismo , AMP Cíclico/metabolismo , Mucosa Intestinal/metabolismo , Transportadores de Anión Orgánico/metabolismo , Anciano , Anciano de 80 o más Años , Membrana Basal/metabolismo , Neoplasias Colorrectales/patología , Dinoprostona/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Células Caliciformes/metabolismo , Aparato de Golgi/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Diabetes is a serious chronic disease, which globally affects more than 400 million patients. Beta cell therapy has potential to serve as an effective cure to type 1 diabetes and several studies have already shown promising results in this regard. One of the major obstacles in cell therapy, however, is the hypoxic environment that therapeutic cells are subjected to immediately after the transplantation. In this study, a new approach is presented, based on hydrogels composed of thiolated hyaluronic acid (tHA), 8-arm-Poly(ethylene glycol)-Acrylate (PEGA), and calcium peroxide (CPO) as an oxygen releasing system. Hydrogels containing 0, 7.5, and 30% CPO were prepared, and the presence of CPO was confirmed via FTIR and Alizarin Red within the network. Oxygen release kinetics were monitored over time, and the results revealed that the hydrogels containing 30% CPO could release oxygen for at least 30 h. All three combinations were found to be injectable and suitable for beta cell therapy based on their mechanical and rheological properties. Additionally, to investigate the functionality of the system, insulin secreting INS-1E reporter cell clusters were encapsulated, and their viability was evaluated, which showed that CPO incorporation enhanced cell survival for at least three days.
Asunto(s)
Hidrogeles , Células Secretoras de Insulina , Supervivencia Celular , Humanos , Oxígeno , PolietilenglicolesRESUMEN
Foreign body reaction (FBR) to implanted biomaterials and medical devices is common and can compromise the function of implants or cause complications. For example, in cell encapsulation, cellular overgrowth (CO) and fibrosis around the cellular constructs can reduce the mass transfer of oxygen, nutrients and metabolic wastes, undermining cell function and leading to transplant failure. Therefore, materials that mitigate FBR or CO will have broad applications in biomedicine. Here we report a group of zwitterionic, sulfobetaine (SB) and carboxybetaine (CB) modifications of alginates that reproducibly mitigate the CO of implanted alginate microcapsules in mice, dogs and pigs. Using the modified alginates (SB-alginates), we also demonstrate improved outcome of islet encapsulation in a chemically-induced diabetic mouse model. These zwitterion-modified alginates may contribute to the development of cell encapsulation therapies for type 1 diabetes and other hormone-deficient diseases.
Asunto(s)
Alginatos/química , Betaína/análogos & derivados , Encapsulación Celular/métodos , Diabetes Mellitus Tipo 1/terapia , Reacción a Cuerpo Extraño/prevención & control , Animales , Betaína/química , Ácido Carbónico , Proliferación Celular , Diabetes Mellitus Experimental , Perros , Fibrosis , Trasplante de Islotes Pancreáticos/métodos , Ratones , Ratas , PorcinosRESUMEN
OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Diabetes Mellitus Experimental/terapia , Evaluación Preclínica de Medicamentos/métodos , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Ratones , Ratones SCIDRESUMEN
Human T2D is characterized by a low-grade systemic inflammation, loss of ß-cells, and diminished insulin production. Local islet immunity is still poorly understood, and hence, we evaluated macrophage subpopulations in pancreatic islets in the well-established murine model of T2D, the db/db mouse. Already at 8 weeks of disease, on average, 12 macrophages were observed in the diabetic islets, whereas only two were recorded in the nondiabetic littermates. On a detailed level, the islet resident macrophages increased fourfold compared with nondiabetic littermates, whereas a pronounced recruitment (eightfold) of a novel subset of macrophages (CD68+F4/80-) was observed. The majority of the CD68+F4/80+ but only 40% of the CD68+F4/80- islet macrophages expressed CD11b. Both islet-derived macrophage subsets expressed moderate MHC-II, high galectin-3, and low CD80/CD86 levels, suggesting the cells to be macrophages rather than DCs. On a functional level, the vast majority of the macrophages in the diabetic islets was of the proinflammatory, M1-like phenotype. The systemic immunity in diabetic animals was characterized by a low-grade inflammation with elevated cytokine levels and increase of splenic cytokine, producing CD68+F4/80- macrophages. In late-stage diabetes, the cytokine signature changed toward a TGF-ß-dominated profile, coinciding with a significant increase of galectin-3-positive macrophages in the spleen. In summary, our results show that proinflammatory M1-like galectin-3+ CD80/CD86(low) macrophages invade diabetic islets. Moreover, the innate immunity matures in a diabetes-dependent manner from an initial proinflammatory toward a profibrotic phenotype, supporting the concept that T2D is an inflammatory disease.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Islotes Pancreáticos/inmunología , Macrófagos/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Glucemia , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Insulina/biosíntesis , Insulina/deficiencia , Islotes Pancreáticos/patología , Macrófagos/metabolismo , Masculino , Ratones , FenotipoRESUMEN
Type 2 diabetes (T2D) is evolving into a global disease and patients have a systemic low-grade inflammation, yet the role of this inflammation is still not established. One plausible mechanism is enhanced expression and activity of the innate immune system. Therefore, we evaluated the expression and the function of the toll-like receptor 4 (TLR4) on pancreatic ß-cells in primary mouse islets and on the murine ß-cell line MIN6 in the presence or absence of macrophages. Diabetic islets have 40% fewer TLR4 positive ß-cells, but twice the number of TLR4 positive macrophages as compared to healthy islets. Healthy and diabetic islets respond to a TLR4 challenge with enhanced production of cytokines (5-10-fold), while the TLR4 negative ß-cell line MIN6 fails to produce cytokines. TLR4 stimulation induces ß-cell dysfunction in mouse islets, measured as reduced glucose stimulated insulin secretion. Diabetic macrophages from 4-months old mice have acquired a transient enhanced capacity to produce cytokines when stimulated with LPS. Interestingly, this is lost in 6-months old diabetic mice. TLR4 activation alone does not induce apoptosis in islets or MIN-6 cells. In contrast, macrophages mediate TLR4-dependent cell-contact dependent (3-fold) as well as cell-contact independent (2-fold) apoptosis of both islets and MIN-6 cells. Importantly, diabetic macrophages have a significantly enhanced capacity to induce ß-cell apoptosis compared to healthy macrophages. Taken together, the TLR4 responsiveness is elevated in the diabetic islets and mainly mediated by newly recruited macrophages. The TLR4 positive macrophages, in both a cell-contact dependent and independent manner, induce apoptosis of ß-cells in a TLR4 dependent fashion and TLR4 activation directly induces ß-cell dysfunction. Thus, targeting either the TLR4 pathway or the macrophages provides a novel attractive treatment regime for T2D.
Asunto(s)
Apoptosis/fisiología , Diabetes Mellitus Tipo 2/fisiopatología , Islotes Pancreáticos/metabolismo , Macrófagos/metabolismo , Receptor Toll-Like 4/metabolismo , Análisis de Varianza , Animales , Citocinas/metabolismo , Femenino , Citometría de Flujo , Inmunoensayo , Insulina/sangre , Mediciones Luminiscentes , Masculino , Ratones , Receptor Toll-Like 4/inmunologíaRESUMEN
AIM: An insufficient functional ß-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore ß-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in ß-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced ß-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to ß cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net ß-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes. MATERIALS AND METHODS: miRNA expression was manipulated using Lentiviral transduction of the ß-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. RESULTS: Overexpression of miR-21 decreased net ß-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net ß-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. CONCLUSION: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase ß-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce ß-cell death and dysfunction.
Asunto(s)
Apoptosis/genética , Proliferación Celular/genética , Células Secretoras de Insulina/citología , MicroARNs/genética , Animales , Apoptosis/efectos de los fármacos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Óxido Nítrico/metabolismo , Ratas , TransfecciónRESUMEN
Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Transporte de Catión/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/farmacología , Hierro/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Diabetes Mellitus Experimental , Dieta Alta en Grasa , Intolerancia a la Glucosa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Noqueados , Modelos Biológicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.