Suppression of Fibrotic Reactions of Chitosan-Alginate Microcapsules Containing Porcine Islets by Dexamethasone Surface Coating

Background@#The microencapsulation is an ideal solution to overcome immune rejection without immunosuppressive treatment. Poor biocompatibility and small molecular antigens secreted from encapsulated islets induce fibrosis infiltration. Therefore, the aims of this study were to improve the biocompatibility of microcapsules by dexamethasone coating and to verify its effect after xenogeneic transplantation in a streptozotocin-induced diabetes mice. @*Methods@#Dexamethasone 21-phosphate (Dexa) was dissolved in 1% chitosan and was cross-linked with the alginate microcapsule surface. Insulin secretion and viability assays were performed 14 days after microencapsulation. Dexa-containing chitosan-coated alginate (Dexa-chitosan) or alginate microencapsulated porcine islets were transplanted into diabetic mice. The fibrosis infiltration score was calculated from the harvested microcapsules. The harvested microcapsules were stained with trichrome and for insulin and macrophages. @*Results@#No significant differences in glucose-stimulated insulin secretion and islet viability were noted among naked, alginate, and Dexa-chitosan microencapsulated islets. After transplantation of microencapsulated porcine islets, nonfasting blood glucose were normalized in both the Dexa-chitosan and alginate groups until 231 days. The average glucose after transplantation were lower in the Dexa-chitosan group than the alginate group. Pericapsular fibrosis and inflammatory cell infiltration of microcapsules were significantly reduced in Dexa-chitosan compared with alginate microcapsules. Dithizone and insulin were positive in Dexa-chitosan capsules. Although fibrosis and macrophage infiltration was noted on the surface, some alginate microcapsules were stained with insulin. @*Conclusion@#Dexa coating on microcapsules significantly suppressed the fibrotic reaction on the capsule surface after transplantation of xenogenic islets containing microcapsules without any harmful effects on the function and survival of the islets.

Reversal of Hypoglycemia Unawareness with a Single-donor, Marginal Dose Allogeneic Islet Transplantation in Korea: A Case Report

Pancreatic islet transplantation is a physiologically advantageous and minimally invasive procedure for the treatment of type 1 diabetes mellitus. Here, we describe the first reported case of successful allogeneic islet transplantation alone, using single-donor, marginal-dose islets in a Korean patient. A 59-yr-old patient with type 1 diabetes mellitus, who suffered from recurrent severe hypoglycemia, received 4,163 islet equivalents/kg from a single brain-death donor. Isolated islets were infused intraportally without any complications. The immunosuppressive regimen was based on the Edmonton protocol, but the maintenance dosage was reduced because of mucositis and leukopenia. Although insulin independence was not achieved, the patient showed stabilized blood glucose concentration, reduced insulin dosage and reversal of hypoglycemic unawareness, even with marginal dose of islets and reduced immunosuppressant. Islet transplantation may successfully improve endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus.

Adenoviruses Expressing PDX-1, BETA2/NeuroD and MafA Induces the Transdifferentiation of Porcine Neonatal Pancreas Cell Clusters and Adult Pig Pancreatic Cells into Beta-Cells

BACKGROUND: A limitation in the number of insulin-producing pancreatic beta-cells is a special feature of diabetes. The identification of alternative sources for the induction of insulin-producing surrogate beta-cells is a matter of profound importance. PDX-1/VP16, BETA2/NeuroD, and MafA overexpression have been shown to influence the differentiation and proliferation of pancreatic stem cells. However, few studies have been conducted using adult animal pancreatic stem cells. METHODS: Adult pig pancreatic cells were prepared from the non-endocrine fraction of adult pig pancreata. Porcine neonatal pancreas cell clusters (NPCCs) were prepared from neonatal pigs aged 1-2 days. The dispersed pancreatic cells were infected with PDX-1/VP16, BETA2/NeuroD, and MafA adenoviruses. After infection, these cells were transplanted under the kidney capsules of normoglycemic nude mice. RESULTS: The adenovirus-mediated overexpression of PDX-1, BETA2/NeuroD and MafA induced insulin gene expression in NPCCs, but not in adult pig pancreatic cells. Immunocytochemistry revealed that the number of insulin-positive cells in NPCCs and adult pig pancreatic cells was approximately 2.6- and 1.1-fold greater than those in the green fluorescent protein control group, respectively. At four weeks after transplantation, the relative volume of insulin-positive cells in the grafts increased in the NPCCs, but not in the adult porcine pancreatic cells. CONCLUSION: These data indicate that PDX-1, BETA2/NeuroD, and MafA facilitate the beta-cell differentiation of NPCCs, but not adult pig pancreatic cells. Therefore PDX-1, BETA2/NeuroD, and MafA-induced NPCCs can be considered good sources for the induction of pancreatic beta-cells, and may also have some utility in the treatment of diabetes.

Effects of Anti-Vascular Endothelial Growth Factor (VEGF) on Pancreatic Islets in Mouse Model of Type 2 Diabetes Mellitus / 당뇨병

BACKGROUND: Vascular endothelial growth factor (VEGF) is associated with the development of diabetic complications. However, it is unknown whether systemic VEGF treatment has any effects on the pancreatic islets in an animal model of type 2 diabetes mellitus. METHODS: Anti-VEGF peptide (synthetic ATWLPPR, VEGF receptor type 2 antagonist) was injected into db/db mice for 12 weeks. We analyzed pancreatic islet morphology and quantified beta-cell mass. Endothelial cell proliferation and the severity of islet fibrosis were also measured. VEGF expression in isolated islets was determined using Western blot analysis. RESULTS: When anti-VEGF was administered, db/db mice exhibited more severe hyperglycemia and associated delayed weight gain than non-treated db/db mice. Pancreas weight and pancreatic beta-cell mass were also significantly decreased in the anti-VEGF-treated group. VEGF and VEGF receptor proteins (types 1 and 2) were expressed in the pancreatic islets, and their expression was significantly increased in the db/db group compared with the db/dm group. However, the elevated VEGF expression was significantly reduced by anti-VEGF treatment compared with the db/db group. The anti-VEGF-treated group had more prominent islet fibrosis and islet destruction than db/db mice. Intra-islet endothelial cell proliferation was also remarkably reduced by the anti-VEGF peptide. CONCLUSION: Inhibition of VEGF action by the VEGF receptor 2 antagonist not only suppressed the proliferation of intra-islet endothelial cells but also accelerated pancreatic islet destruction and aggravated hyperglycemia in a type 2 diabetes mouse model. Therefore, the potential effects of anti-VEGF treatment on pancreatic beta cell damage should be considered.

The Effects of Exendin-4 on IRS-2 Expression and Phosphorylation in INS-1 Cells / 당뇨병

BACKGROUND: Insulin receptor substrate 2 (IRS-2) is a key regulator of beta cell proliferation and apoptosis. This study was aimed to investigate effect of the glucolipotoxicity on apoptosis in INS-1 cell, and the effect of Exendin-4, a GLP-1 receptor agonist, on IRS-2 expression in the glucolipotoxicity induced INS-1 cell. The goal was to discover the new action mechanism and function of Exendin-4 in beta cell apoptosis. METHOD: INS-1 cells were cultured in glucolipotoxic condition for 2, 4 or 6 days and were categorized as G groups. Another group in which 50 nM Exendin-4 was added to INS-1 cells, cultured in glucolipotoxic condition, were named as Ex-4 groups. We investigated the expression of IRS-2 by RT-PCR, phosphorylated IRS-2 and phosphorylated Akt protein levels by western blot. We measured the apoptosis ratio of INS-1 cell in glucolipotoxic condition by TUNEL staining in both groups. RESULT: IRS-2 expression of INS-1 cells decreased with correlation to the time of exposure to glucolipotoxic condition. pIRS-2 and pAkt protein levels decreased in the similar pattern in glucolipotoxicity group. However, this effect of glucolipotoxicity on INS-1 cell was inhibited by the Exendin-4 treatment. In the Ex-4 groups, IRS-2 expression, pIRS-2 and pAkt protein levels remained at the similar level to low glucose condition state. Also, apoptosis induced by glucolipotoxicity was suppressed by Exendin-4 treatment significantly. CONCLUSION: We showed that the long-term treatment of Exendin-4 inhibited the apoptosis of beta cells significantly in glucolipotoxic condition and that this effect of Exendin-4 was related with IRS-2 and Akt among the beta cell's intracellular signal transduction pathway.