Dynamics of pancreatic islet basement membrane proteins during isolation and transplantation (#165)
Although advances in immunosuppression have improved outcomes in clinical transplantation of islets for the treatment of insulin-dependent diabetes, numerous significant hurdles remain. Among these, the changes to islet architecture produced during harvest of cells impair their survival and function early after transplantation. Therefore we investigated changes in the composition of islet basement membrane (BM) proteins before harvest, in culture and after transplantation beneath the kidney capsule to better to understand their roles in islet integrity and function following transplantation. Using immunofluorescence and specific antibodies against BM proteins laminins, collagen type IV, nidogen-2 and perlecan, we (i) compared expression of proteins in intact mouse (CBA and C57BL/6) islets with that after dissociation by collagenase, in standard culture and following transplantation (3 - 10 days); (ii) ascertained the potential for BM restoration in islets in vitro and following iso- and allograft transplantation; and (iii) identified a potential mechanism of BM recovery. Islet vasculature was assessed with an endothelial marker, PECAM-1, and flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet beta cells. Collagenase digestion markedly decreased islet BM, which did not recover over 4 days in vitro culture. After transplantation, there was a transient absence of a peri-islet BM and intra-islet vascular endothelium, suggesting a period of increased risk to islet cells. Although complete restoration of the islet BM occurred after isotransplantation, allotransplantation resulted only in incomplete recovery. Complete re-vascularisation of islets occurred in isografts, however allografts lacked intra-islet capillaries, consistent with an association between islet vascularisation and the restoration of the BM architecture. Flow cytometry indicated that beta cells were not the source of BM proteins. Furthermore, in contrast to isografts, the allografts had increased peri-islet vasculature and dilatation, and reduced PECAM-1 staining. Peri-islet capillaries often contained nucleated DAPI-positive cells, consistent with the delivery of mononuclear cells to the engrafted islets. Absence of BM barrier function may increase islet susceptibility to mononuclear cell invasion during rejection. Furthermore, we identified critical roles for vascular endothelial cell migration and subendothelial BM remodelling in the reassembly of the peri-islet BM, and in the restoration of the intra-islet vasculature. We postulate that prolonged matrix detachment from both peri- and intra- islet BMs negatively impacts on the survival of islet beta cells, and supplements mononuclear cell mediated destruction of islet allografts in the absence of adequate anti-rejection therapy. These findings strongly support the critical need for the islet BMs to be preserved during islet isolation.