Heparan Sulfate Organizes Neuronal Synapses through Neurexin Partnerships.

Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development.

Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Dystroglycan-HSPG interactions provide synaptic plasticity and specificity.

AIM: This study examined the roles of the laminin and proteoglycan receptor dystroglycan (DG) in extracellular matrix stabilization and cellular mechanosensory processes conveyed through communication between the extracellular matrix (ECM) and cytoskeleton facilitated by DG. Specific functional attributes of HS-proteoglycans (HSPGs) are conveyed through interactions with DG and provide synaptic specificity through diverse interactions with an extensive range of cell attachment and adaptor proteins which convey synaptic plasticity. HSPG-DG interactions are important in phototransduction and neurotransduction and facilitate retinal bipolar-photoreceptor neuronal signaling in vision. Besides synaptic stabilization, HSPG-DG interactions also stabilize basement membranes and the ECM and have specific roles in the assembly and function of the neuromuscular junction. This provides neuromuscular control of muscle systems that control conscious body movement as well as essential autonomic control of diaphragm, intercostal and abdominal muscles and muscle systems in the face, mouth and pharynx which assist in breathing processes. DG is thus a multifunctional cell regulatory glycoprotein receptor and regulates a diverse range of biological and physiological processes throughout the human body. The unique glycosylation of the αDG domain is responsible for its diverse interactions with ECM components in cell-ECM signaling. Cytoskeletal cell regulatory switches assembled by the ßDG domain in its role as a nuclear scaffolding protein respond to such ECM cues to regulate cellular behavior and tissue homeostasis thus DG has fascinating and diverse roles in health and disease.

Investigation of sample handling steps for accurate heparan sulphate disaccharide analysis using HPLC-MS.

Heparan sulphates (HSs), a specific class of glycosaminoglycans (GAGs), are important participants of cellular signalling. Analytical characterization of GAGs requires a complex sample preparation workflow. Although a detailed stability and recovery study is available for the chondroitin sulphate GAG class, the literature concerning HS is incomplete in this regard. Therefore, our aim was to systematically investigate various parameters that could potentially influence the stability and recovery of HS samples when performing disaccharide analysis using high-performance liquid chromatography-mass spectrometry. First, effects concerning vacuum evaporation and freezing were investigated. Next, the storage stability of the HS disaccharides was analysed under several conditions such as temperature, pH, digestion buffers, injection solvents and storage vessels. We have identified several critical parameters influencing the stability and recovery of HS disaccharides. We concluded that major sample loss is expected when Tris-HCl is used as digestion buffer, followed by vacuum evaporation at elevated temperatures, or samples are stored under alkaline conditions. Following the practical considerations of this paper can contribute to increasing the reliability of future analytical measurements.

Repurposing of drugs targeting heparan sulphate binding site of dengue virus envelope protein: an in silico competitive binding study.

Dengue virus, an arbovirus, leads to millions of infections every year ultimately leading to a high rate of mortality. Highly effective and specific therapeutic option is not available till date to combat viral infection. One of the first stages in the virus lifecycle encompasses the viral entry into the host cell which is mediated by the interaction between heparan sulphate and the Dengue virus envelope protein in turn leading to the interaction between the envelope protein receptor binding domain and host cell receptors. The heparan sulphate binding site on the envelope protein was established using literature survey and the result validated using ColDock simulations. We have performed virtual screening against the heparan sulphate binding site using DrugBank database and short-listed probable inhibitors based on binding energy calculation following Molecular Dynamics (MD) simulations in this study. Two compounds (PubChem IDS 448062 and 656615) were selected for further analyses on which RAMD simulations were performed to quantitate the binding stability of both the molecules in the protein binding pocket which ultimately led to the selection of ZK-806450 molecule as the final selected compound. Competitive binding MD simulation against dengue virus envelope protein was performed for this molecule and heparan sulphate in order to ascertain the efficiency of binding of this molecule to the dengue virus envelope protein in the presence of its natural ligand molecule and found that this molecule has a higher affinity for the dengue virus envelope protein GAG binding site than heparan sulphate. This study may help in the use of this inhibitor molecule to combat dengue virus infection in foreseeable future and open a new avenue for drug repurposing methodology using competitive binding MD simulation.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

The role of glycosaminoglycan modification in Hedgehog regulated tissue morphogenesis.

Patterns of gene expression, cell growth and cell-type specification during development are often regulated by morphogens. Morphogens are signalling molecules produced by groups of source cells located tens to hundreds of micrometers distant from the responding tissue and are thought to regulate the fate of receiving cells in a direct, concentration-dependent manner. The mechanisms that underlie scalable yet robust morphogen spread to form the activity gradient, however, are not well understood and are currently intensely debated. Here, based on two recent publications, we review two in vivo derived concepts of regulated gradient formation of the morphogen Hedgehog (Hh). In the first concept, Hh disperses on the apical side of developing epithelial surfaces using the same mechanistic adaptations of molecular transport that DNA-binding proteins in the nucleus use. In the second concept, Hh is actively conveyed to target cells via long filopodial extensions, called cytonemes. Both concepts require the expression of a family of sugar-modified proteins in the gradient field called heparan sulphate proteoglycans as a prerequisite for Hh dispersal, yet propose different - direct versus indirect - roles of these essential extracellular modulators.

Ηeparan sulphate in infectious and non-infectious exacerbations of COPD.

BACKGROUND AND OBJECTIVE: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are associated with worsening health outcomes and effective treatment of each episode is essential. In this study, we aimed to investigate if plasma levels of heparan sulphate (HS) are associated with the aetiology of AECOPD. METHODS: COPD patients (N = 1189), GOLD grade II-IV, from a discovery cohort (N = 638) and from a validation cohort (N = 551), were included in the study. HS and heparanase (HSPE-1) were measured longitudinally in plasma at stable state, at AECOPD and at 4 weeks follow-up. RESULTS: Plasma HS was higher in patients with COPD as compared with non-COPD controls and was significantly increased at AECOPD as compared to stable state (p < 0.001) in the discovery and in the validation cohorts. Four distinct exacerbation groups were classified based on aetiology (no-infection/bacterial-infection/viral-infection/bacterial and viral coinfection) in the validation cohort. The fold-increase of HS from stable state to AECOPD was associated with the aetiology of exacerbation and was higher in cases with bacterial and viral coinfections. HSPE-1 was also significantly increased at AECOPD, however, there was no association of HSPE-1 levels with the aetiology of these events. The probability of having an infection at AECOPD was raised as HS levels increased from stable state to AECOPD. This probability was higher for bacterial infections than viral infections. CONCLUSION: The results of our study indicate that circulating levels of HS are increased at AECOPD and this increase may be associated with the aetiology of these events.

An ultra-rare case of immunoskeletal dysplasia with neurodevelopmental abnormalities in an Indian patient with homozygous c.953C > T variant in EXTL3 gene: a case report.

BACKGROUND: Immunoskeletal dysplasia with neurodevelopmental abnormalities (ISDNA) is an ultra-rare genetic condition that belongs to the group of spondyloepimetaphyseal dysplasias. It is caused due to presence of biallelic variants in the EXTL3 gene. The encoded exostosin like glycosyltransferase 3 (EXTL3) protein plays a key role in heparan sulfate synthesis. The skeletal and nervous systems are prominently affected in ISDNA with variability in immunological manifestations. Here, we report the 15th case of ISDNA (third patient of an Indian ancestry) in the world, along with a review of literature. CASE PRESENTATION: A 15-month-old female child with clinical indications of global developmental delay, short stature, coarse facial features, and hypotonia was referred to our clinic. Spondyloepimetaphyseal dysplasias associated with extra-skeletal manifestations was suspected based on clinic-radiological correlation. Whole exome sequencing analysis revealed the presence of a homozygous known pathogenic variant c.953C > T (p. Pro318Leu) in exon 3 of the EXTL3 gene, thereby confirming diagnosis of ISDNA. CONCLUSION: We present an ultra-rare case of ISDNA- third patient of Indian ancestry and only the 15th reported case in the literature. On review of all cases in the literature, we find that the affected individuals show abnormalities primarily in three systems namely- skeletal, nervous and immune system. Notably, patients harbouring the same variant in EXTL3 gene show phenotypic variability especially with respect to presence or absence of immunological manifestations, suggesting a role of unknown modifiers. Hence, it is currently not possible to correlate the variant position in the EXTL3 gene with disease severity.

Glycosaminoglycans from Litopenaeus vannamei Inhibit the Alzheimer's Disease ß Secretase, BACE1.

Only palliative therapeutic options exist for the treatment of Alzheimer's Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer's Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer's disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal ß-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered.

What Are the Potential Roles of Nuclear Perlecan and Other Heparan Sulphate Proteoglycans in the Normal and Malignant Phenotype.

The recent discovery of nuclear and perinuclear perlecan in annulus fibrosus and nucleus pulposus cells and its known matrix stabilizing properties in tissues introduces the possibility that perlecan may also have intracellular stabilizing or regulatory roles through interactions with nuclear envelope or cytoskeletal proteins or roles in nucleosomal-chromatin organization that may regulate transcriptional factors and modulate gene expression. The nucleus is a mechano-sensor organelle, and sophisticated dynamic mechanoresponsive cytoskeletal and nuclear envelope components support and protect the nucleus, allowing it to perceive and respond to mechano-stimulation. This review speculates on the potential roles of perlecan in the nucleus based on what is already known about nuclear heparan sulphate proteoglycans. Perlecan is frequently found in the nuclei of tumour cells; however, its specific role in these diseased tissues is largely unknown. The aim of this review is to highlight probable roles for this intriguing interactive regulatory proteoglycan in the nucleus of normal and malignant cell types.

Pingyangmycin inhibits glycosaminoglycan sulphation in both cancer cells and tumour tissues.

Pingyangmycin is a clinically used anticancer drug and induces lung fibrosis in certain cancer patients. We previously reported that the negatively charged cell surface glycosaminoglycans are involved in the cellular uptake of the positively charged pingyangmycin. However, it is unknown if pingyangmycin affects glycosaminoglycan structures. Seven cell lines and a Lewis lung carcinoma-injected C57BL/6 mouse model were used to understand the cytotoxicity of pingyangmycin and its effect on glycosaminoglycan biosynthesis. Stable isotope labelling coupled with LC/MS method was used to quantify glycosaminoglycan disaccharide compositions from pingyangmycin-treated and untreated cell and tumour samples. Pingyangmycin reduced both chondroitin sulphate and heparan sulphate sulphation in cancer cells and in tumours. The effect was persistent at different pingyangmycin concentrations and at different exposure times. Moreover, the cytotoxicity of pingyangmycin was decreased in the presence of soluble glycosaminoglycans, in the glycosaminoglycan-deficient cell line CHO745, and in the presence of chlorate. A flow cytometry-based cell surface FGF/FGFR/glycosaminoglycan binding assay also showed that pingyangmycin changed cell surface glycosaminoglycan structures. Changes in the structures of glycosaminoglycans may be related to fibrosis induced by pingyangmycin in certain cancer patients.

A role of heparan sulphate proteoglycan in the cellular uptake of lipocalins ß-lactoglobulin and allergen Fel d 4.

Lipocalins, small extracellular hydrophobic molecule carriers, can be internalized by a variety of different cells. However, to date receptors have only been identified for human lipocalins. Here, we specifically investigated uptake mechanisms for lipocalins ß-lactoglobulin and Fel d 4 in HeLa and Chinese hamster ovary (CHO) cells. We provide evidence that cell surface heparan sulphate proteoglycan is essential for internalization of these lipocalins. In HeLa cells, lipocalin uptake was inhibited by competition with soluble heparin, enzymatic digestion of cellular heparan sulphate by heparinase and inhibition of its biosynthesis by sodium chlorate. Biochemical studies by heparin affinity chromatography and colocalization studies further supported a role of heparan sulphate proteoglycan in lipocalin uptake. Finally, lipocalin uptake was blocked in CHO mutant cells defective in glycosaminoglycan biosynthesis whereas in wild-type cells it was clearly detectable. Thus, cell surface heparan sulphate proteoglycan represents a novel component absolutely participating in the cellular uptake of some lipocalins.

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

New classes of potent heparanase inhibitors from ligand-based virtual screening.

Heparanase is a validated target in cancer therapy and a potential target for several inflammatory pathologies. A ligand-based virtual screening of commercial libraries was performed to expand the chemical space of small-molecule inhibitors. The screening was based on similarity with known inhibitors and was performed in several runs, starting from literature compounds and progressing through newly discovered inhibitors. Among the fifty-five tested compounds, nineteen had IC50 values lower than 5 µM and some showed remarkable potencies. Importantly, tere- and isophthalamides derivatives belong to new structural classes of heparanase inhibitors and some of them showed enzyme affinities (61 and 63, IC50 = 0.32 and 0.12 µM, respectively) similar to those of the most potent small-molecule inhibitors reported so far. Docking studies provided a comprehensive binding hypothesis shared by compounds with significant structural diversity. The most potent inhibitors reduced cell invasiveness and inhibited the expression of proangiogenic factors in tumour cell lines.

The role of innate immunity in mucopolysaccharide diseases.

Mucopolysaccharidoses are lysosomal storage disorders characterised by accumulation of abnormal pathological glycosaminoglycans, cellular dysfunction and widespread inflammation, resulting in progressive cognitive and motor decline. Lysosomes are important mediators of immune cell function, and therefore accumulation of glycosaminoglycans (GAGs) and other abnormal substrates could affect immune function and directly impact on disease pathogenesis. This review summarises current knowledge with regard to inflammation in mucopolysaccharidosis, with an emphasis on the brain and outlines a potential role for GAGs in induction of inflammation. We propose a model by which the accumulation of GAGs and other factors may impact on innate immune signalling with particular focus on the Toll-like receptor 4 pathway. Innate immunity appears to have a dominating role in mucopolysaccharidosis; however, furthering understanding of innate immune signalling would have significant impact on highlighting novel anti-inflammatory therapeutics for use in mucopolysaccharide diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".

Evaluation of biomarkers for Sanfilippo syndrome.

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Early disease course is unaltered in mucopolysaccharidosis type IIIA (MPS IIIA) mice lacking α-synuclein.

BACKGROUND: Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is an inherited paediatric-onset neurodegenerative disorder caused by the lysosomal deficiency of sulphamidase with subsequent accumulation of heparan sulphate. The pathological mechanisms responsible for clinical disease are unknown; however, intraneuronal accumulation of aggregation-prone proteins such as α-synuclein, phosphorylated tau and amyloid precursor protein suggests inefficient intracellular trafficking and lysosomal degradation. AIM: To investigate the contribution the accumulating α-synuclein plays in early symptom emergence that is, impaired cognition, reduced anxiety and motor deficits, first detectable between 3-5 months of age. METHODS: We have crossed congenic MPS IIIA mice with α-synuclein-deficient (Sncatm1Rosl /J) mice and evaluated phenotype and brain disease lesions. RESULTS: In a battery of behavioural tests performed on mice aged 12-22 weeks, we were unable to differentiate α-synuclein-deficient MPS IIIA mice from those with one or both copies of the α-synuclein gene; all three affected genotypes were significantly impaired in test performance when compared to wild-type littermates. Histological studies revealed that the rate, location and nature of deposition of other proteinaceous lesions, the disruption to endolysosomal protein expression and the inflammatory response seen in the brain of α-synuclein-deficient MPS IIIA mice reflected that seen in MPS IIIA mice homo- or heterozygous for α-synuclein. CONCLUSION: Deletion and/or deficiency of α-synuclein does not influence clinical and neuropathological disease progression in murine MPS IIIA, demonstrating that in and of itself, this protein does not initiate the cognitive and motor symptoms that occur in the first 5 months of life in MPS IIIA mice.

Antitumor activity of sulforaphane in mice model of skin cancer via blocking sulfatase-2.

Although there are many treatment options for skin cancer, the chemotherapeutic agents for skin cancer are linked with many adverse effects as well as the development of multidrug resistance. Sulforaphane is an isothiocyanate, which is found in cruciferous vegetables. Consumption of sulforaphane-rich diet has been linked to inhibition of UV-exposed skin carcinogenesis. Therefore, the goal of this study was to determine the ability of sulforaphane to reduce skin cancer in mice through inhibition of sulfatase-2 enzyme. Epicutaneous application of 7,12-dimethylbenz (a) anthracene was performed on the shaved dorsal skin of mice followed by croton oil. Sulforaphane (9 µmol/mouse/day) was administered to mice orally. Skin was removed from the dorsal area for assessment of sulfatase-2, glypican-3, heparan sulphate proteoglycans (HSPGs), nuclear factor (NF)κB, nuclear factor E2-related factor 2 (Nrf2), tumor necrosis factor (TNF)-α, IL-1ß and caspase-3. In addition, skin sections were stained with haematoxylin/eosin, Mallory and cytokeratin immunostaining. We found that, sulforaphane blocked sulfatase-2 activity, leading to significant elevation in HSPGs as well as significant reduction in glypican-3. In addition, sulforaphane significantly activated Nrf2 and reduced both the gene and protein expression of NFκB, TNF-α, IL-1ß and caspase-3. In parallel, stained sections obtained from skin cancer mice treated with sulforaphane showed significant reduction in hyperkeratosis, acanthosis and epithelial dysplasia. The collective results indicate that sulforaphane suppresses skin cancer via blocking sulfatase-2 with subsequent elevation in HSPGs and reduction in glypican-3. Moreover, sulforaphane attenuated skin cancer-induced activation of inflammatory and apoptotic pathways.

Heparan sulphate and hyaluronic acid components of the glycocalyx do not play a role in flow-mediated dilation of the iliac in the anaesthetized pig.

The shear-stress sensor function of vascular glycocalyx heparan sulphate and hyaluronic acid was investigated in vivo by assessing flow-mediated dilation before and after their removal. Heparinase III exposure (100 mU·mL-1 for 20 min;n = 6) did not significantly affect flow-mediated dilation of the iliac, from 0.42 ± 0.08 mm (mean ± SEM) to 0.34 ± 0.07 mm after (P = 0.12; paired Student's t test) for a statistically similar increase in shear stress; 18.24 ± 4.2 N·m-2 for the control and 15.8 ± 3.6 N·m-2 for the heparinase III experiment (P = 0.18). Hyaluronidase exposure (0.14-1.4 mg·mL-1 for 20 min; n = 8) also did not significantly reduce flow-mediated dilation of the iliac, which averaged 0.39 ± 0.08 mm before and 0.38 ± 0.09 mm after (P = 0.11) for a statistically similar increase in shear stress; 11.90 ± 3.20 N·m-2 for the control and 9.8 ± 3.33 N·m-2 for the hyaluronidase experiment (P = 0.88). Removal of both heparan sulphate and hyaluronic acid was confirmed using immunohistochemistry. Neither the heparan sulphate nor the hyaluronic acid components of the glycocalyx mediate shear-stress-induced vasodilation in conduit arteries in vivo.