Poster Presentation The 13th International Congress of the Immunology of Diabetes Society 2013

Heparan sulfate (HS) deficiency in isolated islets is due to oxidative damage and not heparanase (Hpse)-mediated degradation (#185)

Charmaine Simeonovic 1 , Fui Jiun Choong 1 , Sarah Popp 1 , Debra Brown 1 , Lora Jensen 1 , Craig Freeman 1 , Ghassan Magzhal 2 , Roland Stocker 2 , Christopher Parish 1
  1. Department of Immunology, The John Curtin School of Medical Research, Canberra, ACT, Australia
  2. Vascular Biology Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

Background: The glycosaminoglycan HS is covalently attached to the core proteins of HS proteoglycans (HSPGs). HS is strongly expressed in islet beta cells in situ, plays a critical role in beta cell survival and is degraded by leukocyte-derived Hpse during T1D development in NOD mice (1).
Objective: This study examined the mechanism responsible for depleting intra-islet HS during islet isolation.
Methods: HSPGs (collagen type XVIII, syndecan-1) and HS in B6 and B6.Hpse-KO (lacking the HS-degrading enzyme Hpse) islets pre- and post-isolation were examined by immunohistochemistry and Alcian blue histochemistry, respectively. Donor mice were treated with hydroethidine (80 nmol/kg i.v.) to identify specific oxidants/reactive oxygen species (ROS) in extracts of isolated islets by liquid chromatography/mass spectroscopy. Isolated beta cells ± culture with 50 μg/ml heparin were examined for intracellular ROS using dihydrodichlorofluorescein diacetate (H2DCFDA) and flow cytometry. Additional donor mice were treated with the antioxidant butylated hydroxyanisole (BHA, 120 mg/kg i.p.) and the antioxidant dimethylthiourea (DMTU, 50 mM) was included in the islet isolation process.
Results: Islets pre- and post-isolation showed intense expression of collagen type XVIII and syndecan-1 core proteins. In contrast, B6 and B6.Hpse-KO isolated islets showed ∼80% loss of intra-islet HS. The non-specific oxidation product, ethidium, was identified in islet extracts. DCF-derived fluorescence (identifying ROS) was detected in control isolated beta cells but not following HS replacement with heparin. Antioxidant treatment significantly improved the HS content of isolated islets.
Conclusions: These findings suggest that loss of islet HS during islet isolation is due to degradation by excessive oxidant levels and not to Hpse or loss of HSPG core proteins. The antioxidant properties of HS may therefore depend on the oxidant levels. This study suggests that oxidant-mediated depolymerisation may represent a supplementary mechanism for HS loss in islet beta cells during T1D. 

  1. Ziolkowski et al (2012) Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes. J. Clin. Invest. 122: 132-141.