Islet Heparan Sulfate but Not Heparan Sulfate Proteoglycan Core Protein Is Lost During Islet Isolation and Undergoes Recovery Post-Islet Transplantation

Abstract

Islet beta cells in situ express intracellular heparan sulfate (HS), a property previously shown in vitro to be important for their survival. We report that HS levels inside islet beta cells correlate with the novel intracel-lular localization of the HSPG core proteins for collagen type XVIII (Col18), a conventional extracellular matrix component. Syndecan-1 (Sdc1) and CD44 core proteins were similarly localized inside beta cells. During isolation, mouse islets selectively lose HS to 11-27% of normal levels but retain their HSPG core proteins. Intra-islet HS failed to recover substantially during culture for 4 days and was not reconstituted in vitro using HS mimetics. In contrast, significant recovery of intra-islet HS to $40-50% of normal levels occurred by 5-10 days after isotransplantation. Loss of islet HS during the isolation procedure is independent of heparanase (a HS-degrading endoglycosidase) and due, in part, to oxidative damage. Treatment with antioxidants reduced islet cell death by $60% and increased the HS content of isolated islets by $twofold compared to untreated islets, preserving intra-islet HS to $60% of the normal HS content of islets in situ. These findings suggest that the preservation of islet HS during the islet isolation process may optimize islet survival posttransplant. Abbreviations: AEC, 3-amino-9-ethylcarbazole; A.U., arbitrary units; B6.Hpse-KO, heparanase knockout C57BL/6; BHA, butylated hydroxyanisole; BM, basement membrane; Col18, collagen type XVIII; DMTU, dimethylthiourea; ECM, extracellular matrix; EXT1-2, exostosin 1-2; EXTL1-3, exostosin-like 1-3; FITC, fluo-rescein isothiocyanate; GMFR, geometric mean fluo-rescence ratio; Gpc2, glypican-2; H&E, haematoxylin and eosin; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MFI, mean fluorescence intensity; MNCs, mononuclear cells; Ndst2, N-deacetylase/N-sulfotransferase 2; NOD/Lt, nonobese diabetic; PSN, penicillin-streptomycin-neomycin; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; Sdc1, syndecan-1; SEM, standard error of the mean

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation

“…HSPGs, including perlecan, are composed of HS chains attached to individual core proteins . During mouse islet isolation, HS is lost and fails to recover during culture , yet, importantly, antioxidant treatment preserved HS during islet isolation, resulting in a significant improvement in islet survival . This suggests that loss of perlecan during human islet isolation, as demonstrated in our study, may substantially affect islet viability and function; indeed, the extensive loss of perlecan was evident in all islet preparations studied, with the mean viability being 77 ± 2%.…”

“…Notably, our study found a positive correlation between islet yield and laminin content following 24‐h culture, suggesting that substantial loss of laminin and other components of the BM may affect islet survival pretransplantation. Heparan sulfate (HS) has recently been shown to play a role in β cell survival and differentiation and in protection against reactive oxygen species–induced damage . HSPGs, including perlecan, are composed of HS chains attached to individual core proteins .…”

“…The human islet BM contains numerous laminin isoforms as well as perlecan, the predominant HSPG , which links laminins to collagen IV . Laminins and HSPGs are implicated in multiple signaling pathways, conferring downstream signals for optimal cell survival and function .…”

Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends

“…The human islet BM contains a variety of laminin isoforms 16 . Laminins are known to be involved in numerous cell survival and function signaling pathways 14 , 15 , 18 , 19 . The laminin-α5 isoform is present in both layers of the duplex BM 16 and so is an ideal marker to study BM integrity, along with collagen IV due to its role as the major structural BM component.…”

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

“…Interaction of collagen IV with its receptors also contributes to β-cell differentiation, maturation, and survival[83-85]. Other BM components such as fibronectin and heparin sulfate also play roles in β-cell migration, growth, differentiation and survival[1,86-88]. …”