Divergent antioxidant capacity of human islet cell subsets: A potential cause of beta-cell vulnerability in diabetes and islet transplantation.

BACKGROUND: Type 1 and Type 2 diabetes mellitus (T1DM and T2DM) are caused by beta(ß)-cell loss and functional impairment. Identification of mechanisms of ß-cell death and therapeutic interventions to enhance ß-cell survival are essential for prevention and treatment of diabetes. Oxidative stress is a common feature of both T1DM and T2DM; elevated biomarkers of oxidative stress are detected in blood, urine and tissues including pancreas of patients with DM. Islet transplantation is a promising treatment for diabetes. However, exposure to stress (chemical and mechanical) and ischemia-reperfusion during isolation and transplantation causes islet loss by generation of reactive oxygen species (ROS). Human intracellular antioxidant enzymes and related molecules are essential defenses against ROS. Antioxidant enzyme levels including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) have been shown to be low in islet cells. However, little is known about the expression and function of antioxidant enzymes within islet cell subsets. We evaluated the expression of the key antioxidant enzymes in ß- and alpha(α)-cell and accessed effects of oxidative stress, islet isolation and transplantation on ß/α-cell ratio and viability in human islets. METHODS: Human pancreata from T1DM, T2DM and non-diabetic deceased donors were obtained and analyzed by confocal microscopy. Isolated islets were (I) transplanted in the renal sub-capsular space of streptozotocin-induced diabetic nude mice (in vivo bioassay), or (II) exposed to oxidative (H2O2) and nitrosative (NO donor) stress for 24 hrs in vitro. The ratio, % viability and death of ß- and α-cells, and DNA damage (8OHdG) were measured. RESULTS AND CONCLUSIONS: Catalase and GPX expression was much lower in ß- than α-cells. The ß/α-cell ratio fells significantly following islet isolation and transplantation. Exposure to oxidative stress caused a significantly lower survival and viability, with higher DNA damage in ß- than α-cells. These findings identified the weakness of ß-cell antioxidant capacity as a main cause of vulnerability to oxidative stress. Potential strategies to enhance ß-cell antioxidant capacity might be effective in prevention/treatment of diabetes.

Influence of in vitro and in vivo oxygen modulation on ß cell differentiation from human embryonic stem cells.

The possibility of using human embryonic stem (hES) cell-derived ß cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional ß cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells. We have previously determined that oxygenation is a powerful driver of murine PP differentiation along the endocrine lineage of the pancreas. We hypothesized that targeting physiological oxygen partial pressure (pO2) levels seen in mature islets would help the differentiation of PP cells along the ß-cell lineage. This hypothesis was tested both in vivo (by exposing PP-transplanted immunodeficient mice to a daily hyperbaric oxygen regimen) and in vitro (by allowing PP cells to mature in a perfluorocarbon-based culture device designed to carefully adjust pO2 to a desired range). Our results show that oxygen modulation does indeed contribute to enhanced maturation of PP cells, as evidenced by improved engraftment, segregation of α and ß cells, body weight maintenance, and rate of diabetes reversal in vivo, and by elevated expression of pancreatic endocrine makers, ß-cell differentiation yield, and insulin production in vitro. Our studies confirm the importance of oxygen modulation as a key variable to consider in the design of ß-cell differentiation protocols and open the door to future strategies for the transplantation of fully mature ß cells.

Effect of the purinergic inhibitor oxidized ATP in a model of islet allograft rejection.

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R⁺CD4⁺ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function.

Inhibition of C-jun N-terminal kinase improves insulin sensitivity but worsens albuminuria in experimental diabetes.

C-jun N-terminal kinase (JNK) regulates both the development of insulin resistance and inflammation. Podocytes of the widely used db/db mouse model of diabetic nephropathy lose their ability to respond to insulin as albuminuria develops, in comparison to control db/+ mice. Here we tested whether JNK inhibition or its gene deletion would prevent albuminuria in experimental diabetes. Phosphorylated/total JNK was significantly increased in vivo in glomeruli of db/db compared to db/+ mice. Treatment of podocytes isolated from these two strains of mice with tumor necrosis factor-alpha caused greater phosphorylation of JNK in those obtained from diabetic animals. When db/db mice were treated with a cell-permeable TAT-JNK inhibitor peptide, their insulin sensitivity and glycemia significantly improved compared to controls. We induced diabetes in JNK1 knockout mice with streptozotocin and found that they had significantly better insulin sensitivity compared to diabetic wild-type or JNK2 knockout mice. Albuminuria was, however, worse in all mice treated with the JNK inhibitor and in diabetic JNK2 knockout mice compared to controls. Nephrin expression was also reduced in JNK inhibitor-treated mice compared to controls. A similar degree of mesangial expansion was found in all diabetic mice. Our study shows that targeting JNK to improve systemic insulin sensitivity does not necessarily prevent diabetic nephropathy.

Characterization of pancreatic ductal cells in human islet preparations.

Substantial amounts of nonendocrine cells are implanted as part of human islet grafts, and a possible influence of nonendocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for nonendocrine cells due to lack of available methods. The aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDCs) for clinical islet transplantation and to characterize them regarding phenotype, viability, and function. We assessed 161 human islet preparations using laser scanning cytometry (LSC/iCys) for phenotypic analysis of nonendocrine cells and flow cytometry (FACS) for PDC viability. PDC and beta-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce proinflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF) relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDCs, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than beta-cells (PDC vs beta-cell: 75.5+/-13.9 and 62.7+/-18.7%; P<0.0001). Although beta-cell viability was independent of its density, that of PDCs was higher as the density from which they were recovered increased. There was no correlation between PDCs and beta-cell viability (R(2)=0.0078). PDCs sorted from high-density fractions produced significantly higher amounts of proinflammatory mediators and VEGF, but not TF. We conclude that PDCs isolated from different fractions had different viability and functions. The precise characterization and assessment of these cells in addition to beta-cells in human islet cell products may be of assistance in understanding their contribution to islet engraftment and in developing strategies to enhance islet graft function.

Islet Cell Transplantation: In Vivo and in Vitro Functional Assessment of Nonhuman Primate Pancreatic Islets.

Transplantation of pancreatic islets of Langerhans as a therapeutic approach for treatment of type I diabetes offers an alternative to subcutaneous insulin injections. Normalization of blood glucose levels by transplanted islets may prevent the development of diabetes-related complications. Problems related to rejection, recurrence of autoimmunity, and local inflammation upon transplantation of islets into the liver need to be solved before the implementation of islet cell transplantation can be viewed as a justifiable procedure in a large cohort of patients. Islet cell isolation has been quite successful in small animals, but the translation of this approach to nonhuman primates has been less rewarding. One of the main problems encountered in nonhuman primate models is the difficulty of isolating an adequate number of functional islets for transplantation. The aim of the present study was to develop a method for isolating a sufficient number of viable islets from nonhuman primates to allow for reversal of diabetes. By implementing minor modifications in the automated method for human islet isolation we were able to obtain viable, functional islets that responded normally to glucose stimulation in vitro. These islets were also able to reverse diabetes in immunocompromised nude mice, rendered diabetic by streptozotocin. This method of islet cell isolation has enabled us to proceed with protocols of allogeneic islet cell transplantation in preclinical, nonhuman primate models.