Cowley M.J.,Peter Wills Bioinformatics Center |
Cowley M.J.,Garvan Institute of Medical Research |
Weinberg A.,Peter Wills Bioinformatics Center |
Weinberg A.,Garvan Institute of Medical Research |
And 16 more authors.
In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-kBdependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-kB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-kβ target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-kB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG-/- recipients. Thus, human islets can provide a reservoir of NF-kB-dependent inflammatory factors that have the potential to contribute to the antiislet- graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-kB, and transplanted them into allogenic recipients. These NF-kB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-kB-activated islets were rejected with a faster tempo as compared to non-NFkB- activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention. © 2012 Cognizant Comm. Corp. Source
Campbell P.D.,St. Vincents Institute |
Campbell P.D.,University of Melbourne |
Weinberg A.,Garvan Institute of Medical Research |
Chee J.,St. Vincents Institute |
And 11 more authors.
Human islets are subjected to a number of stresses before and during their isolation that may influence their survival and engraftment after transplantation. Apoptosis is likely to be activated in response to these stresses. Apoptosis due to intrinsic stresses is regulated by pro- and antiapoptotic members of the Bcl-2 family. While the role of the Bcl-2 family in apoptosis of rodent islets is becoming increasingly understood, little is known about which of these molecules are expressed or required for apoptosis of human islets. This study investigated the expression of the Bcl-2 family of molecules in isolated human islets. RNA and protein lysates were extracted from human islets immediately postisolation. At the same time, standard quality control assays including viability staining and β-cell content were performed on each islet preparation. Microarrays, RT-PCR, and Western blotting were performed on islet RNA and protein. The prosurvival molecules Bcl-xl and Mcl-1, but not Bcl-2, were highly expressed. The multidomain proapoptotic effector molecule Bax was expressed at higher levels than Bak. Proapoptotic BH3-only molecules were expressed at low levels, with Bid being the most abundant. The proapoptotic molecules BNIP3, BNIP3L, and Beclin-1 were all highly expressed, indicating exposure of islets to oxygen and nutrient deprivation during isolation. Our data provide a comprehensive analysis of expression levels of pro- and antiapoptotic Bcl-2 family members in isolated human islets. Knowledge of which molecules are expressed will guide future research to understand the apoptotic pathways activated during isolation or after transplantation. This is crucial for the design of methods to achieve improved transplantation outcomes. © 2012 Cognizant Comm. Corp. Source
Cantley J.,Garvan Institute |
Cantley J.,University of New South Wales |
Walters S.N.,University of New South Wales |
Walters S.N.,Garvan Institute |
And 14 more authors.
We examined whether hypoxic exposure prior to the event of transplantation would have a positive or negative effect upon later islet graft function. Mouse islets exposed to hypoxic culture were transplanted into syngeneic recipients. Islet graft function, β-cell physiology, as well as molecular changes were examined. Expression of hypoxia-response genes in human islets pre- and posttransplant was examined by microarray. Hypoxiapreexposed murine islet grafts provided poor glycemic control in their syngeneic recipients, marked by persistent hyperglycemia and pronounced glucose intolerance with failed first- and second-phase glucose-stimulated insulin secretion in vivo. Mechanistically, hypoxic preexposure stabilized HIF-1α with a concomitant increase in hypoxic-response genes including LDHA, and a molecular gene set, which would favor glycolysis and lactate production and impair glucose sensing. Indeed, static incubation studies showed that hypoxia-exposed islets exhibited dysregulated glucose responsiveness with elevated basal insulin secretion. Isolated human islets, prior to transplantation, express a characteristic hypoxia-response gene expression signature, including high levels of LDHA, which is maintained posttransplant. Hypoxic preexposure of an islet graft drives a HIFdependent switch to glycolysis with subsequent poor glycemic control and loss of GSIS. Early intervention to reverse or prevent these hypoxia-induced metabolic gene changes may improve clinical islet transplantation. © 2013 Cognizant Comm. Corp. Source