Boosting islet heparan sulfate or inhibiting heparanase prevents development of disease.
Scientists suggest that either boosting pancreatic islet levels of heparan sulfate (HS) or administering an inhibitor of the heparanase enzyme that is responsible for its breakdown could provide a new approach to preventing development or slowing progression of type 1 diabetes. A team at The Australian National University’s John Curtin School of Medical Research initially showed that HS is expressed at very high levels in the mouse islet and is essential for β-cell survival.
Their subsequent in vivo studies in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) found that infiltrating mononuclear cells associated with the destruction of islets produced catalytically active heparanase that led to loss of islet HS. Treating animals with an inhibitor of heparanase protected the animals from developing T1D and led to preservation of islet HS.
Charmaine J. Simeonovic, M.D., and colleagues, report their findings in The Journal of Clinical Investigation, in a paper titled “Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes.”
NOD mice develop T1D spontaneously, and represent a widely recognized model for human T1D. The disease in these animals progresses slowly and initially involves nondestructive insulitis (NDI), characterized by the accumulation of mononuclear cells (MNCs) around the islet periphery, followed by autoimmune destruction of insulin-producing pancreatic β cells, when the insulitis MNCs become destructive.
Pancreatic islet β cells themselves display properties that render them vulnerable to inflammatory attack, the authors report. The cells secrete chemokines that could play a role in inflammation and also secrete low levels of free radical scavenger enzymes that could increase their sensitivity to free radical-mediated damage.
What hasn’t yet been explored at length is whether islets and β cells have natural defense and survival mechanisms against immune attack. The university team’s previous work had demonstrated that NOD mouse islets are surrounded by a basement membrane (BM) that contains the heparin sulphate proteoglycan (HSPG) perlecan to which the glycosaminoglycan heparin sulphate (HS) is covalently attached as side chains. Studies have suggested that when localized in BMs, HSPGs can obstruct cell migration, indicating that islet BM containing perlecan could represent a defense mechanism to hold back invasion by destructive insulitis (DI) MNCs.
In order to get past this barrier and migrate to sites of peri-islet insulitis in the pancreas, leukocytes must produce a range of enzymes capable of breaking down the matrix components of subvascular endothelial BM. One of these, heparanase (encoded by HPSE), is the only known mammalian endoglycosidase that can efficiently degrade the HS side chains of HSPGs, the team continues. Indeed, their own previous research found that MNC entry into islets at the start of the destructive autoimmune attack in NOD mice corresponds with degradation of the islet BM.
The researchers designed experiments to investigate whether HS plays a role in protecting islets against MNC infiltration. Immunohistochemical analyses and staining first confirmed that in situ, the islets of healthy non-NOD mice, young NOD mice, and immunoincompetent NOD/SCID mice exhibited incredibly high levels of intracellular HS. Conversely, when single β cells were cultured in vitro, within a couple of days they began to demonstrate a 2–3-fold higher level of cell surface HS and a significant decrease in intraceullar HS. This loss of intracellular HS occurred alongside a substantial increase in β-cell death. “These findings suggested that β cells may require intracellular HS for their survival,” the authors note.
Interestingly, β-cell death in culture could be prevented by adding the highly sulphated HS analogue, heparin, to the culture medium: This addition boosted cell viability from 37% to 95%. Similar results were obtained when highly sulphated HS or a highly sulphated HS mimetic, PI-88, were added to the culture medium. Progen Pharmaceuticals’ PI-88 (Muparfostat) is a noncleavable HS mimetic that acts as a competitive inhibitor of heparanase and is in clinical trials as an anticancer drug.
“All three compounds decreased the absolute number of dead β cells by 6- to 14-fold compared with controls, despite comparable total cell numbers in the cultures,” the authors write. Flow cytometry analysis and confocal microscopy confirmed that β-cell protection correlated with the intracellular localization of heparin in about 86% of the β cells. Cultured β cells treated using either heparin, highly sulfated HS, or PI-88 were in addition markedly resistant to treatment with the reactive oxygen species (ROS) hydrogen peroxide.
Notably, treating isolated islets with heparanase for 24 hours resulted in peripheral islet damage with increased intra-islet cell apoptosis, the team continues. When they examined the islets of NOD mice at different stages of disease, they found that islets lacking insulitis or with NDI exhibited widespread and well-preserved HS, whereas islets damaged by DI showed extensive disruption of islet HS. RT-PCR and immunohistochemical analyses further indicated that the infiltrated DI MNCs strongly expressed Hpse and that insulitis MNCs gain the ability to generate enzymatically active heparanase at the same time as they become able to mediate destructive disease.
Critically, treating prediabetic female MOD mice using PI-88, at an age when they were expected to exhibit both NDI and DI, significantly slowed the development of T1D by 10 weeks. By age 36 weeks, the number of diabetic mice among the PI-88 cohort was just half that of the control group. When the islets of both control and PI-88-treated mice were compared, the PI-88-treated animals exhibited a much greater proportion of islets with no insulitis, a reduced proportion of islets with DI, and much better preservation/restoration of islet-associated HS. “These results support the notion that heparanase critically contributes to T1D development by inhibiting islet invasion by insulitis MNCs and subsequent loss of intra-islet HS,” the investigators suggest.
“We report here that pancreatic β cells have a uniquely abundant intracellular store of HS, which plays a role in β-cell survival that we believe to be previously unrecognized,” they conclude. “Overall, our data are completely consistent with the idea that T1D is heparanase dependent,” they conclude. “DI MNCs produce catalytically active heparanase that—by solubilizing the islet BM HS—allows intra-islet MNC invasion, induces β-cell death, and initiates autoimmune diabetes.
“Our findings unveil a critical role for islet HS in β-cell survival and highlight the potential for new therapeutic approaches for rescuing β-cell function at the time of T1D onset by HS replacement therapy as well as for blocking progressive loss of islet-associated HS during disease development with heparanase inhibitors, both of which could be achieved using appropriately designed HS mimetics.”
