Loss of intra-islet heparan sulfate represents a novel marker for the progression of type 1 diabetes in humans

Abstract

Background and aims: Human type 1 diabetes (T1D) is an autoimmune disease and multiple destructive mechanisms are likely. In addition, a residual beta cell mass can exist at diagnosis, highlighting the potential for rescue by novel therapies. We have previously demonstrated unusually high levels of the polysaccharide heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) inside mouse beta cells, a critical role for intracellular HS in beta cell survival, the progressive loss of islet HS during T1D progression in NOD mice and expression of the HS-degrading enzyme heparanase (Hpse) by islet-infiltrating leukocytes. Treatment of NOD mice with PI-88, a Hpse inhibitor/HS mimetic, reduced the incidence of T1D by 50% and preserved intra-islet HS, suggesting that Hpsemediated loss of beta cell HS contributes to T1D disease. In this study we examined the clinical relevance of HS for the viability of human beta cells and as a target for destruction in human T1D. Materials and methods: HS, HSPG core proteins (collagen type XVIII (Col18), syndecan-1 (Sdc1)), insulin, glucagon and Hpse were stained by immunohistochemistry/ immunofluorescence in paraffin sections (post-antigen retrieval) of normal (n=8) and T1D (n=8) human pancreases with insulin+ve islets, obtained from the JDRF Network for Pancreas Organ Donors with Diabetes (nPOD, USA); the stained islet area was quantified using Image J software. 10E4 anti-HS mAb was used to localise highly sulfated HS and HP130 mAb identified Hpse. Isolated human islets were dispersed into single cells (using Accutase) for flow cytometry analysis of HS/HSPGs in beta cells and of beta cell viability after culture with HS mimetics (heparin or PI-88) ± acute treatment with 30% hydrogen peroxide. Beta cells were identified using Newport Green (NG) and damaged cells were stained using 7AAD or Sytox Green. Results: Localisation of HS, Col18 and Sdc1 in normal human pancreas correlated with insulin-containing beta cells. For insulin-positive T1D islets, the insulin-stained islet area was 85% of normal islets. However, the area stained for HS, Col18 and Sdc1 was significantly reduced to 41% (P<0.0001), 55% (P<0.0001) and 42% (P<0.0001) of normal islets, respectively. Insulitis leukocytes showed cell surface staining for Hpse. Flow cytometry analyses showed that 84.1±3.3% of human islet cells were beta cells and contained intracellular HS and HSPG core protein. Uptake of HS mimetics during culture of human beta cells significantly improved beta cell viability by 1.6-fold (P<0.001) i.e., as HS replacers, significantly reduced the proportion of damaged/non-viable beta cells to 25-31% of controls (P<0.001) and significantly reduced hydrogen peroxide-induced death by 3-fold (P<0.001). Conclusion: These findings suggest that in human T1D, the loss of beta cell HS could be mediated by leukocyte-derived Hpse and result in increased susceptibility to oxidative damage. Dual activity Hpse inhibitors/HS replacers could therefore represent a novel class of T1D therapeutic. Show more