Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply.

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO to a supraphysiological level that maintains all islets viable and functional.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes.

Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression.

Vessel Network Architecture of Adult Human Islets Promotes Distinct Cell-Cell Interactions In Situ and Is Altered After Transplantation.

Islet-cell hormone release is modulated by signals from endothelial and endocrine cells within the islet. However, models of intraislet vascularization and paracrine cell signaling are mostly based on the rodent pancreas. We assessed the architecture and endocrine cell interaction of the vascular network in unperturbed human islets in situ and their potential to re-establish their endogenous vascular network after transplantation in vivo.

Production of high-quality islets from goettingen minipigs: Choice of organ preservation solution, donor pool, and optimal cold ischemia time.

Background: The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT).

P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells.

Background: Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression.

Diabetes induction by total pancreatectomy in minipigs with simultaneous splenectomy: a feasible approach for advanced diabetes research.

Background: Safe and reliable diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents and also widely used in large animal models of non-human primates and minipigs. However, uncertain efficacy, the potential of beta-cell regeneration, and relevant side effects are debatable aspects particularly in large animals.

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed.

Transplantation of human islets without immunosuppression.

Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression.

The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients.

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11beta-hydroxysteroid dehydrogenase.

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis.

Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry.

The size composition of human islet preparations has been attributed to functional potency, islet survival and transplantation outcomes. In the early post-transplantation phase islets are supplied with oxygen by diffusion only and are at risk of critical hypoxia. The high rate of early islet graft dysfunction is in part attributed to this condition.

Impaired insulin turnover in islets from type 2 diabetic patients.

Failure of pancreatic β-cells contributes to the development of type 2 diabetes. Besides evidence of reduced glucose-stimulated insulin secretion and β-cell mass, little information is available about the molecular deficits of human diabetic islets. Islets were isolated from macroscopically normal pancreatic tissue from 8 patients with type 2 diabetes and 17 matched non-diabetic patients who underwent pancreatic surgery.

Tamoxifen-independent recombination in the RIP-CreER mouse.

Background: The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Islet versus pancreas transplantation in type 1 diabetes: competitive or complementary?

Whole organ pancreas and pancreatic islet transplantation are currently the only forms of clinically available β-cell replacement. Both therapeutic options can provide good glycemic control and prevention or stabilization of diabetic complications, but at the price of permanent immunosuppression. Therefore, the indication for transplantation of type 1 diabetes patients must be balanced carefully and should be restricted to a subgroup of patients with extreme lability of metabolic control and frequent hypoglycemia despite optimal medical therapy.

The influence of oxygen supply on metabolism of neural cells cultured on a gas-permeable PTFE foil.

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen-permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro.

Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets.

Therapeutic strategies for transplantation of pancreatic islet cells are urgently needed to expand beta-cell mass by stimulating islet cell proliferation and/or prolonging islet cell survival. Control of the islets by different growth factors provides a potential venue for augmenting beta-cell mass. In the present study, we show the expression of the biologically active splice variant-1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor in rat insulinoma (INS-1) cells as well as in rat and human pancreatic islets.

Effects of thrombin on RPE cells are mediated by transactivation of growth factor receptors.

Purpose: To determine the expression of blood coagulation factors and thrombin receptors in retinal pigment epithelial (RPE) cells and whether the effects of thrombin on the chemotaxis and the secretion of VEGF are mediated by transactivation of growth factor receptors.

Methods: Gene expression in acutely isolated and cultured human RPE cells was evaluated by RT-PCR. Alterations in gene expression and secretion of VEGF were determined by real-time RT-PCR and ELISA, respectively.

Apocynin-induced vasodilation involves Rho kinase inhibition but not NADPH oxidase inhibition.

Aims: The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro, it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all.

Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency.

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet beta-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+(i)) (Fura Red), stimulated with glucose, and analyzed on a FACS Vantage flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides.

Positive feedback regulation between MMP-9 and VEGF in human RPE cells.

Purpose: The proteolytic activity of matrix metalloproteinases (MMPs) is involved in pathologic angiogenesis in the eye. However, it is unknown whether MMPs may stimulate the production of the major angiogenic factor, vascular endothelial growth factor (VEGF). The authors investigated whether MMP-2 and MMP-9 alter the expression of VEGF by retinal pigment epithelial (RPE) cells.

Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats.

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival. A number of clinical and experimental studies suggest that tumor-induced lymphangiogenesis driven by vascular endothelial growth factor (VEGF)-C- and/or VEGF-D-induced activation of VEGF receptor (VEGFR)-3 may promote metastasis to regional lymph nodes. Here we show that constitutive VEGF-C and VEGF-D expression by tumor cells of diverse origin grown in tissue culture does not correlate with metastatic potential in vivo.