Oxidative stress produced during pancreatic islet isolation leads to significant β-cell damage. Homeostatic cytokines secreted subsequently to islet transplantation damage β-cells by generating oxygen free radicals. In this study, exendin-4, a glucagon-like peptide-1 analog improved islet transplantation outcome by increasing the survival of diabetic recipient mice from 58% to 100%. We hypothesized that this beneficial effect was due to the ability of exendin-4 to reduce oxidative stress. Further experiments showed that it significantly reduced the apoptotic rate of cultured β-cells subjected to hypoxia or to IL-1β. Reduction of apoptotic events was confirmed in pancreatic islet grafts of exendin-4-treated mice. Exendin-4 enhanced Akt phosphorylation of β-cells and insulin released from them. It even augmented insulin secretion from islets cultivated at hypoxic conditions. Exposure to hypoxia led to a decrease in the activation of Akt, which was reversed when β-cells were pretreated with exendin-4. Moreover, exendin-4 increased the activity of redox enzymes in a hypoxia-treated β-cell line and reduced reactive oxygen species production in isolated pancreatic islets. Recovery from diabetes in mice transplanted with hypoxic islets was more efficient when they received exendin-4. In conclusion, exendin-4 rescued islets from oxidative stress caused by hypoxia or due to cytokine exposure. It improved the outcome of syngenic and xenogenic islet transplantation.

Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus.

Intraportal islet transplantation suffers from low efficiency caused by substantial islet mass loss after transplantation. How this process is regulated is still unclear. Here, we show that NF-j B activation was detectable in islet grafts shortly after transplantation of porcine islets to diabetic NMRI nu/nu mice, and systemic NF-j B inhibition in transplanted animals significantly prolonged islet graft survival. Proinflammatory cytokines alone did not cause evident cell death in pancreatic islet within 24 h, while the combination of cytokines with hypoxia resulted in a strong induction of cell death that could be blocked dose-dependently by a selective IKK-b inhibitor. Under hypoxia, NF-j B activity impaired expression of antiapoptotic gene BCL-xL, c-FLIP and survivin. NF-j B activation in isolated islets was reduced by hypoxia in a time-dependent manner, accordingly, NF-j B activation in transplanted islets diminished by time. Our data indicate that, while NF-j B has an antiapoptotic role under normoxia, low oxygen conditions decrease its activity and transform it to a proapoptotic transcription factor in pancreatic islets. We conclude that NF-j B inhibition represents a potential strategy to improve islet transplantation efficiency.Key words: Islet transplantation, transplantation efficiency, engraftment, NF-kappaB, hypoxia, innate immunity Abbreviations: IKK-b , inhibitor of kappaB kinase beta; NF-j B, nuclear factor kappa B; T1D, type 1 diabetes; IEQ, islet equivalent; PI, Propidium Iodide; RPL13A, ribosomal protein L13a; Ct, threshold cycle; HIF-1a , hypoxia inducible factor 1, alpha subunit.

Islet transplantation provides an approach to compensate for loss of insulin-producing cells in patients with type 1 diabetes. However, the intraportal route of transplantation is associated with instant inflammatory reactions to the graft and subsequent islet destruction as well. Although matrix metalloprotease (MMP)-2 and -9 are involved in both remodeling of extracellular matrix and leukocyte migration, their influence on the outcome of islet transplantation has not been characterized. We observed comparable MMP-2 mRNA expressions in control and transplanted groups of mice, whereas MMP-9 mRNA and protein expression levels increased after islet transplantation. Immunostaining for CD11b (Mac-1)-expressing leukocytes (macrophage, neutrophils) and Ly6G (neutrophils) revealed substantially reduced inflammatory cell migration into islet-transplanted liver in MMP-9 knockout recipients. Moreover, gelatinase inhibition resulted in a significant increase in the insulin content of transplanted pancreatic islets and reduced macrophage and neutrophil influx compared with the control group. These results indicate that the increase of MMP-9 expression and activity after islet transplantation is directly related to enhanced leukocyte migration and that early islet graft survival can be improved by inhibiting MMP-9 (gelatinase B) activity.

Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet‐specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti‐GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators.