Roles for Islet Heparan Sulfate and heparanase in Islet transplantation

Abstract

Previously, it has been shown that islets are surrounded by a continuous basement membrane (BM) containing perlecan, a heparan sulfate proteoglycans (HSPGs). Islets also express extraordinarily high levels of HS, which acts as an antioxidant and is critical for beta cell survival. Islet HS is lost following islet isolation. The onset of Type 1 diabetes in the NOD mice correlated with the production of catalytic active heparanase, an HS-degrading endoglycosidase by the insulitis mononuclear cells (MNCs), accompanied by the loss of islet HS. This thesis entails a study to investigate the mechanisms contributing to the loss of HS during islet isolation and to assess the status of islet HS following transplantation. Additionally, the role of heparanase in the rejection of islet allografts is examined. This project provided evidence that islets express various forms of HSPGs, e.g., type XVIII collagen, syndecan-1 and CD44, which possibly contribute to the extraordinarily high levels of intra-islet HS. Islets lose their BM and HS but retain the core proteins of HSPGs following isolation. In addition, we identified reactive oxygen species as the primary factor contributing to the loss of islet HS during the islet isolation process. Combined treatment with antioxidant butylated hydroxyanisole, BHA (120 mg/kg i.p) and N,N-dimethylthiourea, DMTU (50 millimolar), as well as addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, DIDS (200 micromolar), a chloride ion channel blocker which is anti-oxidative and anti-apoptotic, significantly increase intra-islet HS content by 2-2.7-fold, compared to non-treated islets following isolation. Islet BM and HS recover by 10 days after isotransplantation. In allografts, islet BM recovery is initiated early post-transplant. However, islet BM matrix proteins are later diverted to form enlarged peri-islet blood vessels that may facilitate leukocyte recruitment to the graft site during rejection. Likewise, islet HS shows signs of recovery in allografts early post-transplant. Subsequent loss of Islet HS correlates with the expression of high levels of heparanase by the infiltrating MNCs during the rejection process. Adoptive transfer of activated anti-H-2k heparanase knockout (B6.Hpse-KO) effector T cells delays the rejection of established islet allografts in immunoincompetent B6.Rag1-/- transplant recipients, compared to the transfer of activated wild-type C57BL/6 effector T cells. We propose that heparanase produced by the MNCs degrades the subendothelial BM HS and intra-graft HS, to facilitate their entry to the graft site. Heparanase could also degrade the newly restored peri-islet BM. In contrast, islets which lack their islet BM, would be expected to be highly susceptible to MNC invasion. Once islet cell invasion is achieved, the degradation of intra-islet HS by the local production of heparanase would contribute to beta cell death. This study has identified a potential strategy for improving the quality of isolated islets for transplantation and alternative approaches for preventing islet transplant rejection. These important findings may help to establish clinical islet transplantation as a more routine therapy for patients with established Type 1 diabetes. Ultimately islet transplantation has the potential to prevent the development of serious secondary vascular complications of Type 1 diabetes, resulting from imperfect control of blood glucose levels by insulin therapy. Show more