Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

ABSTRACT Despite recent successful control efforts, malaria remains a leading global health burden. Alarmingly, resistance to current antimalarials is increasing and the development of new drug families is needed to maintain malaria control. Current antimalarials target the intraerythrocytic developmental stage of the Plasmodium falciparum life cycle. However, the invasive extracellular parasite form, the merozoite, is also an attractive target for drug development. We have previously demonstrated that heparin-like molecules, including those with low molecular weights and low anticoagulant activities, are potent and specific inhibitors of merozoite invasion and blood-stage replication. Here we tested a large panel of heparin-like molecules and sulfated polysaccharides together with various modified chemical forms for their inhibitory activity against P. falciparum merozoite invasion. We identified chemical modifications that improve inhibitory activity and identified several additional sulfated polysaccharides with strong inhibitory activity. These studies have important implications for the further development of heparin-like molecules as antimalarial drugs and for understanding merozoite invasion.

The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of > 2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

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 intracellular 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.

Cigarette smoke induces injury and neutrophilic inflammation in the airways of smokers. The stability and activity of inflammatory effectors, IL8 and neutrophil elastase (NE), can be prolonged by binding to airway heparan sulfate (HS)/syndecan-1, posing risk for developing chronic obstructive pulmonary disease(COPD). We hypothesize that antagonizing HS/syndecan-1 binding of the inflammatory effectors could reduce smoking-related neutrophil-mediated airway inflammation. Analysis of bronchoalveolar lavage fluid(BALF) of COPD patients found both total and unopposed NE levels to be significantly higher among smokers with COPD than non-COPD subjects. Similar NE burden was observed in smoke-exposed rats compared to sham air controls. We chose sulfated-maltoheptaose(SM), a heparin-mimetic, to antagonize HS/sydecan-1 binding of the inflammatory mediators in airway fluids and lung tissues of the smoke-exposed rat model. Airway treatment with SM resulted in displacement of CINC-1 and NE from complexation with bronchio-epithelial HS/syndecan-1, dissipating the chemokine gradient for neutrophil flux across to the bronchial lumen. Following SM displacement of NE from shed HS/syndecan-1 in bronchial fluids, NE became accessible to inhibition by α1-antitrypsin endogenous in test samples. The antagonistic actions of SM against syndecan-1 binding of NE and CINC-1 in smoke-exposed airways suggest new therapeutic opportunities for modulating airway inflammation in smokers with SM delivery.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

Heparanase (Hpse) is the only known mammalian endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS), found attached to the core proteins of heparan sulfate proteoglycans (HSPGs). Hpse plays a homeostatic role in regulating the turnover of cell-associated HS and also degrades extracellular HS in basement membranes (BMs) and the extracellular matrix (ECM), where HSPGs function as a barrier to cell migration. Secreted Hpse is harnessed by leukocytes to facilitate their migration from the blood to sites of inflammation. In the non-obese diabetic (NOD) model of autoimmune Type 1 diabetes (T1D), Hpse is also used by insulitis leukocytes to solubilize the islet BM to enable intra-islet entry of leukocytes and to degrade intracellular HS, an essential component for the survival of insulin-producing islet beta cells. Treatment of pre-diabetic adult NOD mice with the Hpse inhibitor PI-88 significantly reduced the incidence of T1D by ~50% and preserved islet HS. Hpse therefore acts as a novel immune effector mechanism in T1D. Our studies have identified T1D as a Hpse-dependent disease and Hpse inhibitors as novel therapeutics for preventing T1D progression and possibly the development of T1D vascular complications.

Event Abstract Back to Event New insights into Type 1 diabetes development and therapy Christopher R. Parish1*, Andrew Ziolkowski1, Zuopeng Wu1, Sarah Popp1, Craig Freeman1 and Charmaine J. Simeonovic1 1 John Curtin School of Medical Research, Australian National University, Department of Immunology, Australia Type 1 diabetes (T1D) is an autoimmune disease in which the insulin-producing beta cells in the pancreas are destroyed by the immune system, although the molecular basis of this autoimmune destruction is unclear. We have recently reported that mouse islets express extraordinarily high levels of the glycosaminoglycan heparan sulfate (HS) intracellularly and are also surrounded by a basement membrane (BM) rich in HS. In fact, beta cells are exquisitely dependent on intracellular HS for their survival, with in vitro addition of HS or related molecules maintaining beta cell viability and rendering the cells highly resistant to damage by reactive oxygen species. Furthermore, we have found in vivo that destructive insulitis is associated with high level expression by leukocytes of enzymatically active heparanase, an endoglycosidase that degrades HS. In addition, in vivo treatment with several heparanase inhibitors significantly protected NOD mice from clinical diabetes. Based on these data we propose that initially leukocyte-derived heparanase degrades HS in the islet BM, allowing leukocyte entry into islets. Thereafter, heparanase-mediated degradation of beta cell-associated HS results in a novel mechanism of beta cell death. Recent studies using an acute T1D model in heparanase knock out mice has confirmed the importance of heparanase in T1D development, with heparanase expression by several different cell types being important. Thus heparanase inhibition, possibly combined with HS replacement in beta cells, has great therapeutic promise for treating T1D patients at an early stage of the disease and, potentially, for the prevention of T1D in “at risk” individuals. Acknowledgements JDRF, Diabetes Australia and National Health and Medical Research Council of Australia (NHMRC) for grant support References Ziolkowski, A., Popp, S., Freeman, C., Parish, C.R. and Simeonovic, C.J. (2012) Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes. J. Clin. Invest. 122, 132-141. Keywords: type 1 diabetes, Autoimmunity, Leukocyte migration, Heparan sulfate, Heparanase Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Parish CR, Ziolkowski A, Wu Z, Popp S, Freeman C and Simeonovic CJ (2013). New insights into Type 1 diabetes development and therapy. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00770 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Christopher R Parish, John Curtin School of Medical Research, Australian National University, Department of Immunology, Canberra, ACT, 2601, Australia, christopher.parish@anu.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Christopher R Parish Andrew Ziolkowski Zuopeng Wu Sarah Popp Craig Freeman Charmaine J Simeonovic Google Christopher R Parish Andrew Ziolkowski Zuopeng Wu Sarah Popp Craig Freeman Charmaine J Simeonovic Google Scholar Christopher R Parish Andrew Ziolkowski Zuopeng Wu Sarah Popp Craig Freeman Charmaine J Simeonovic PubMed Christopher R Parish Andrew Ziolkowski Zuopeng Wu Sarah Popp Craig Freeman Charmaine J Simeonovic Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

Recent findings on the role of circulating histone proteins in mediating acute lung injury prompted us to investigate whether there is a specific mechanism for accumulation of histones in the lungs. Binding sites for polycations are already known in the vasculature of the lungs, and we postulated that these could also be involved in histone accumulation, since histones have a high content of positively charged amino acids. Using a histone-coated colloid of a radiolabelled nanocomposite to track histone biodistribution with imaging techniques, it was found that histones bind avidly in the lungs of rabbits after intravenous injection. Blocking experiments with competing polycations in vivo characterised histone lung binding as dependent on a charge interaction with microvessel polyanions. Pretreatment of rabbits with a specific heparinase confirmed that the lung binding sites consist of heparan sulphate in the endothelial glycocalyx. A range of heparan sulphate analogues was accordingly shown to prevent histone accumulation in the lungs by neutralising histones in blood. These findings provide a rational basis for the design of polyanions that can prevent accumulation of cytotoxic histones in the lungs and thereby intervene at an early key step in the development of acute lung injury.

Proteoglycans (PGs) are major components of the cell surface and extracellular matrix and play critical roles in development and maintenance of the central nervous system (CNS). PGs are a family of proteins, all of which contain a core protein to which glycosaminoglycan side chains are covalently attached. PGs possess diverse physiological roles, particularly in neural development, and are also implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). The main functions of PGs in the CNS are reviewed as are the roles of PGs in brain injury and in the development or treatment of AD. © 2013 IUBMB Life, 65(2)108–120, 2013.

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.

Alzheimer's disease is associated with abnormal accumulation of Aβ, which is produced from the β‐amyloid precursor protein ( APP ) by the β‐site APP ‐cleaving enzyme ( BACE 1) and γ‐secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE 1 and ADAM 10. In this study, we examined the effects of glycosaminoglycans ( GAG s) on APP processing and Aβ production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP ( SW )Tg2576 mice and the level of APP , proteolytic products of APP and APP ‐cleavage enzymes were measured. The effect of GAG s on APP processing was both size‐ and sulfation‐dependent. 6‐O‐Sulfation was important for the effect on APP processing as heparin lacking 6‐O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAG s on APP processing and that certain GAG s have a greater effect on Aβ production than others. This suggests that it might be possible to alter the structure of GAG s to achieve more specific inhibitors of APP processing that can cross the blood–brain barrier.