Chondroitin Sulfate Glycosaminoglycan Research Articles

Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

Endothelial to mesenchymal transformation is induced by altered extracellular matrix in aortic valve endothelial cells

Alterations in shear stress, mechanical deformation, extracellular matrix (ECM) composition and exposure to inflammatory conditions are known to cause endothelial to mesenchymal transformation (EndMT). This change in endothelial phenotype has only recently been linked to adult pathologies such as cancer progression, organ fibrosis, and calcific aortic valve disease; and its function in adult physiology, especially in response to tissue mechanics, has not been rigorously investigated. EndMT is a response to mechanical and biochemical signals that results in the remodeling of underlying tissues. In diseased aortic valves, glycosaminoglycans (GAGs) are present in the collagen-rich valve fibrosa, and are deposited near calcified nodules. In this study, in vitro models of early and late-stage valve disease were developed by incorporating the GAGs chondroitin sulfate (CS), hyaluronic acid, and dermatan sulfate into 3D collagen hydrogels with or without exposure to TGF-β1 to simulate EndMT in response to microenvironmental changes. High levels of CS induced the highest rate of EndMT and led to the most collagen I and GAG production by mesenchymally transformed cells, which indicates a cell phenotype most likely to promote fibrotic disease. Mesenchymal transformation due to altered ECM was found to depend on cell-ECM bond strength and extracellular signal-regulated protein kinases 1/2 signaling. Determining the environmental conditions that induce and promote EndMT, and the subsequent behavior of mesenchymally transformed cells, will advance understanding on the role of endothelial cells in tissue regeneration or disease progression. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2729-2741, 2017.

Sustained Release of Transforming Growth Factor-β1 from Platelet-Rich Chondroitin Sulfate Glycosaminoglycan Gels.

Activated platelet-rich plasma (PRP), also referred to as platelet-rich fibrin (PRF), has been used to augment numerous techniques of cartilage repair in the knee but does not always result in superior quality of repair tissue. One possible reason that PRF does not consistently result in excellent cartilage regeneration is the transiency of growth factor provision with PRF. The objective of this study was to compare the release of transforming growth factor (TGF)-β1 from PRF and from PRP combined with a novel chondroitin sulfate glycosaminoglycan (CS-GAG) gel. PRP was prepared from nine healthy dogs and split into two aliquots: one activated with bovine thrombin and calcium chloride (CaCl2) to form PRF and the other aliquot was used to rehydrate a lyophilized CS-GAG gel. Both PRF and the CS-GAG gels were incubated in media for 13 days and media were collected, stored, and replaced every 48 hours and the concentration of TGF-β1 quantified in the media using an enzyme-linked immunosorbent assay. Concentrations of TGF-β1 in the media were up to three times greater with the CS-GAG gels and were significantly (p < 0.05) greater than with PRF on days 3, 5, 7, 9, and 13. Furthermore, TGF-β1 elution was still substantial at day 13 with the use of the CS-GAG gels. Additional in vitro work is warranted to characterize TGF-β1 elution from this CS-GAG gel with human PRP and to determine whether the use of these CS-GAG gels can augment cartilage repair in vivo.

An Oncofetal Glycosaminoglycan Modification Provides Therapeutic Access to Cisplatin-resistant Bladder Cancer

BackgroundAlthough cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. ObjectiveTo investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. Design, setting, and participantsAn ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC–treated MIBC patients. InterventionAn rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. Outcome measurements and statistical analysisAntineoplastic effects of targeting ofCS. Results and limitationsIn situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design. ConclusionsTargeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC. Patient summaryCisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin.

Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity.

Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments.

Structure and Properties of Cartilage Proteoglycans.

The most abundant cartilage proteoglycan is aggrecan, a bottlebrush shaped molecule that possesses over 100 glycosaminoglycan (chondroitin sulfate and keratan sulfate) chains. The side-chains are linear sulfated polysaccharides that are negatively charged under physiological conditions. Aggrecan interacts with hyaluronic acid (HA) to form large aggregates. Osmotic pressure measurements and rheological measurements are used to study the static and dynamic behavior of aggrecan assemblies at the macroscopic length scales. The microscopic properties of aggrecan solutions are determined by small angle neutron scattering (SANS), and static and dynamic light scattering (SLS and DLS). In dilute solutions aggrecan forms microgels with a diffuse boundary, composed of loosely connected clusters. The osmotic pressure of the aggrecan-HA system decreases with increasing HA content. DLS yields a relaxation rate that varies as q3, arising from internal modes in the microgel. The relaxation rate in the solutions of the aggrecan-HA complex is slightly greater than in the pure aggrecan solution.

Chondroitin Sulfate Glycosaminoglycan Matrices Promote Neural Stem Cell Maintenance and Neuroprotection Post-Traumatic Brain Injury.

There are currently no effective treatments for moderate-to-severe traumatic brain injuries (TBIs). The paracrine functions of undifferentiated neural stem cells (NSCs) are believed to play a significant role in stimulating the repair and regeneration of injured brain tissue. We therefore hypothesized that fibroblast growth factor (FGF2) enriching chondroitin sulfate glycosaminoglycan (CS-GAG) matrices can maintain the undifferentiated state of neural stem cells (NSCs) and facilitate brain tissue repair subacutely post-TBI. Rats subjected to a controlled cortical impactor (CCI) induced TBI were intraparenchymally injected with CS-GAG matrices alone or with CS-GAG matrices containing PKH26GL labeled allogeneic NSCs. Nissl staining of brain tissue 4 weeks post-TBI demonstrated the significantly enhanced (p < 0.05) tissue protection in CS-GAG treated animals when compared to TBI only control, and NSC only treated animals. CS-GAG-NSC treated animals demonstrated significantly enhanced (p < 0.05) FGF2 retention, and maintenance of PKH26GL labeled NSCs as indicated by enhanced Sox1+ and Ki67+ cell presence over other differentiated cell types. Lastly, all treatment groups and sham controls exhibited a significantly (p < 0.05) attenuated GFAP+ reactive astrocyte presence in the lesion site when compared to TBI only controls.

Interaction of Zika Virus Envelope Protein with Glycosaminoglycans.

In February 2016, the World Health Organization declared a Public Health Emergency of International Concern on Zika Virus (ZIKV), because of its association with severe fetal anomalies of congenitally infected humans. This has led to urgent efforts by academic, federal, and industry research groups to improve our understanding of the pathogenesis of ZIKV and to develop detection methods, therapeutic strategies, and vaccines. Although we still do not have the entire picture of the pathogenesis of ZIKV, extensive research has been conducted on related pathogenic flaviviruses (i.e., dengue virus, West Nile virus, and yellow fever virus). Binding to glycosaminoglycans (GAGs) through its envelope protein is the first step in successful host cell invasion of dengue virus. In this study, we examined ZIKV envelope protein (ZIKV E) binding to GAGs in a real time interaction study using surface plasmon resonance (SPR) to explore the role of GAGs in host cell entry of ZIKV into placenta and brain. ZIKV E strongly binds (KD = 443 nM) pharmaceutical heparin (HP), a highly sulfated GAG, and binds with lower avidity to less sulfated GAGs, suggesting that the ZIKV E-GAG interaction may be electrostatically driven. Using SPR competition assays with various chain length HP oligosaccharides (from 4 to 18 saccharide units in length), we observed that ZIKV E preferentially binds to longer HP oligosaccharides (with 8-18 saccharides). Next, we examined GAGs prepared from human placentas to determine if they bound ZIKV E, possibly mediating placental cell invasion of ZIKV. Compositional analysis of these GAGs as well as SPR binding studies showed that both chondroitin sulfate and heparan sulfate GAGs, present on the placenta, showed low-micromolar interactions with ZIKV E. Both porcine brain CS and HS also showed micromolar binding with ZIKV E. Moreover, heparan sulfate with a higher TriS content, the dominant repeating unit of HP, shows a high affinity for ZIKV E. These results suggest that GAGs may be utilized as attachment factors for host cell entry of Zika virus as they do in other pathogenic flaviviruses. They may also assist us in advancing our understanding of the pathogenesis of ZIKV and guide us in designing therapeutics to combat ZIKV with more insight.

Glycosaminoglycan Sequence Influence on Iduronate Ring Puckering

Iduronate (IdoA) is a major monosaccharide constituent of the linear polysaccharides, called glycosaminoglycans (GAGs), that form covalent conjugates with core proteins to make proteoglycans (PGs). PGs are known as important structural components of extracellular matrix, and are increasingly recognized for their roles in cell signaling. Toward advancing understanding of the atomic-resolution structural biology of PGs, we have performed all-atom explicit-solvent molecular dynamics (MD) simulations on IdoA as a monosaccharide and in the context of oligosaccharides corresponding to fragments of the GAG dermatan sulfate (DS). The stereochemistry of the carbon atoms in the pyranose ring of IdoA and the resulting spatial distribution of functional groups attached to the ring result in a delicate thermodynamic balance between preferred ring puckering conformation for IdoA. Therefore, Cremer-Pople ring pucker analysis was applied to understand the sequence influence on IdoA in the context of DS oligosaccharide vs. IdoA monosaccharide. These results are compared with analogous simulations for glucuronate (GlcA), which is a single-site epimer (at C6) of IdoA, and makes up the related GAG chondroitin sulfate (CS).

Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies.

The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time.

Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells

Background/Aims: Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis. Methods: We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis. Results: we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs. Conclusion: GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism.

Altering the Proteoglycan State of Transforming Growth Factor β Type III Receptor (TβRIII)/Betaglycan Modulates Canonical Wnt/β-Catenin Signaling

Hyperactive Wnt/β-catenin signaling is linked to cancer progression and developmental abnormalities, making identification of mechanisms controlling Wnt/β-catenin signaling vital. Transforming growth factor β type III receptor (TβRIII/betaglycan) is a transmembrane proteoglycan co-receptor that exists with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications in cells and has established roles in development and cancer. Our studies here demonstrate that TβRIII, independent of its TGFβ co-receptor function, regulates canonical Wnt3a signaling by controlling Wnt3a availability through its sulfated GAG chains. Our findings revealed, for the first time, opposing functions for the different GAG modifications on TβRIII suggesting that Wnt interactions with the TβRIII heparan sulfate chains result in inhibition of Wnt signaling, likely via Wnt sequestration, whereas the chondroitin sulfate GAG chains on TβRIII promote Wnt3a signaling. These studies identify a novel, dual role for TβRIII/betaglycan and define a key requirement for the balance between chondroitin sulfate and heparan sulfate chains in dictating ligand responses with implications for both development and cancer.

Highlights of This Issue

Despite therapeutic advances, glioblastoma burden inevitably progresses or relapses. There is accumulating evidence that glutathione and system xc–, a cystine transporter, mediates resistance of various cancers to cytotoxin-based therapies. In the current study, Polewski and colleagues discover that overexpression of SLC7A11, the catalytic subunit of system xc–, increases chemoresistance of glioma cells to temozolimide (TMZ) and alters cancer metabolism. This is the first report that links SLC7A11 overexpression with increased oxidative phosphorylation and ATP production to fulfill energy requirements while maintaining low ROS levels. Therefore, targeting SLC7A11 may be a promising therapeutic strategy for glioblastoma patients.Fibronectin and its fragments derived from bone-marrow mesenchymal stromal cells induce migratory and adhesive responses via the α5β1 integrin in PTEN-null prostate cancer cells, representing a seed-and-soil principle relevant to bone metastases, the lethal phenotype of prostate cancer. Here, α5 integrin knockdown downregulates the BCL-2 family of pro-apoptotic molecules and induces apoptosis. Chemical parsing indicates that potent BCL-XL inhibition specifically synergizes with PI3K/AKT inhibitors to induce synthetic lethality in PTEN-null prostate cancer cells. Given the importance of PTEN-loss in the progression of disease in prostate cancer, these findings are of significant translational relevance.Colon cancers classify into various molecular subtypes with diverse characteristics. In this study, McNew and colleagues determine sensitivity profiles to MEK and TAK1 kinase inhibition in a panel of colon cancer cell lines. MEK and TAK1 inhibition had strong apoptotic effects in cells displaying KRAS-dependent activation of pro-inflammatory signaling. Pro-inflammatory signaling in the context of KRAS/MEK and TAK1 dependency promotes tumor cell survival. Furthermore, a Wnt/NFkB transcriptional signature was derived that associates with KRAS dependency. Combined activation of Wnt/NFkB signaling associates with poor patient prognosis and could reveal subsets of colon cancers that are MEK- and TAK1-dependent.The ability of cells to sense and process information from the extracellular matrix (ECM) is of critical importance to understanding malignancy. Moreover, proteoglycans and their multitude of glycosaminoglycan (GAG) modifications are fundamental to the cells capacity to integrate this information. Clausen and colleagues, using a recombinant protein (rVAR2) derived from Plasmodium falciparum, demonstrate that the oncofetal chondroitin sulfate (ofCS) GAG regulates cancer cell motility through integrins and that blocking ofCS inhibits integrin signaling and metastatic phenotypes. Importantly, evidence is also provided that both human and murine metastatic lesions express high levels of ofCS; thus, suggesting ofCS as a universal cancer-specific marker and therapeutic target.

Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts.

Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA) and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna.

Versican accumulates in vascular lesions in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [(35)S]sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.

Abstract 3798: A glycan-binding malaria protein provides therapeutic access to cisplatin-resistant bladder cancer

Abstract Bladder cancer is a disease of compelling morbidity and mortality mainly due to its high recurrence rate. Cisplatin-based chemotherapy is an integral part of muscle invasive bladder cancer (MIBC) treatment. Although responses are common, a significant number of patients develop resistance to cisplatin and second line treatment options are not well established. No improvement of the MIBC treatment strategy has been achieved in the past two decades and therefore; new approaches to systemic therapy are urgently needed. The Plasmodium falciparum host-cell anchor protein VAR2CSA has been evolutionarily optimized to bind distinctly modified chondroitin sulfate (CS) glycosaminoglycan (GAG) chains expressed exclusively in the mammalian placenta. This is the underlying key event behind pregnancy-associated malaria outbreaks in endemic regions of the world. We have recently discovered that placental-type CS chains are re-expressed in the malignant compartment as a secondary oncofetal CS (ofCS) modification (Salanti et al. 2015, Cancer Cell). In the present study, we have analyzed the expression and role of ofCS in cisplatin-resistant bladder cancer and evaluated the potential of a VAR2-drug conjugate (VDC) to target cisplatin-resistant tumors. Using recombinant VAR2CSA protein (rVAR2) as an ofCS detection reagent, we analyzed a tissue microarray of 52 chemotherapy-naïve MIBC samples, with 36 matched post-chemotherapy cystectomy specimens from patients receiving neoadjuvant gemcitabine/cisplatin. In chemoresistant tumor specimens, of-CS expression was significantly upregulated in residual patient tumors after neoadjuvant chemotherapy (p = 0.001) and it was associated with advanced tumor stage (ypT3/4, p = 0.005) and poor overall survival (p = 0.04). Microarray analysis of primary human bladder tumors and subsequent in situ proximity ligation assay validation identified S100A9 and CD44 as the major ofCS-modified proteoglycans in chemoresistant bladder cancer. Binding of rVAR2 to ofCS chains on bladder cancer cells facilitated rapid internalization of the protein. Moreover, a rVAR2-drug conjugate (VDC) efficiently killed all bladder cancer cells in the low nanomolar IC50 concentration range in vitro and retarded growth of chemoresistant orthotopic MIBC xenografts in vivo. In summary, we demonstrate how a glycan-binding malaria protein can be utilized to gain therapeutic access to cisplatin-resistant bladder cancer. Thus, we provide a method to target cancer-specific glycan modifications for therapeutic intervention as a second line treatment in MIBC, not responding to cisplatin. Citation Format: HTOO ZARNI OO, Roland Seiler, Sherry S. Lee, Davide Tortora, Gunjan Kumar, Chris Wang, Thomas M. Clausen, Mette Ø. Agerbæk, Jamie R. Rich, John S. Babcook, Peter C. Black, Ali Salanti, Mads Daugaard. A glycan-binding malaria protein provides therapeutic access to cisplatin-resistant bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3798.

Specific ligands based on Tröger’s base derivatives for the recognition of glycosaminoglycans

Symmetric Tröger’s base derivatives with quinolinium substitution, 7 and 8, were prepared, and their structures were confirmed by 1H and 13C NMR, 2D NMR and NOE NMR techniques and elemental analysis. These compounds were studied as specific ligands for the recognition of glycosaminoglycans (heparin, heparan sulphate, chondroitin sulphate and hyaluronic acid). They exhibited high affinity and selectivity towards glycosaminoglycans, especially heparin. The conditional binding constants, linear ranges and detection limits were determined by UV–Vis spectroscopy. These values, especially at extremely low limits of detection, showed the high potential of the prepared ligands for the recognition and determination of glycosaminoglycans, especially heparin. For example, the minimum detectable heparin concentrations for 7 and 8 were 0.1 and 0.17 ng/mL (R2 = 0.988 and 0.9938) in media (1 mM NaCl (H2O:MeOH; 7:3, v/v), pH = 7.34), respectively.

Chondroitin sulphate glycosaminoglycans contribute to widespread inferior biomechanics in tendon after focal injury

Both mechanical and structural properties of tendon change after injury however the causal relationship between these properties is presently unclear. This study aimed to determine the extent of biomechanical change in post-injury tendon pathology and whether the sulphated glycosaminoglycans (glycosaminoglycans) present are a causal factor in these changes.Equine superficial digital flexor tendons (SDF tendons) were surgically-injured in vivo (n=6 injured, n=6 control). Six weeks later they were harvested and regionally dissected into twelve regions around the lesion (equal medial/lateral, proximal/distal). Glycosaminoglycans were removed by enzymatic (chondroitinase) treatment. Elastic modulus (modulus) and ultimate tensile strength (UTS) were measured under uniaxial tension to failure, and tendon glycosaminoglycan content was measured by spectrophotometry.Compared to healthy tendons, pathology induced by the injury decreased modulus (−38%; 95%CI −49% to −28%; P<0.001) and UTS (−38%; 95%CI −48% to −28%; P<0.001) and increased glycosaminoglycan content (+52%; 95%CI 39% – 64%; P<0.001) throughout the tendon. Chondroitinase-mediated glycosaminoglycan removal (50%; 95%CI 21–79%; P<0.001) in surgically-injured pathological tendons caused a significant increase in modulus (5.6MPa/µg removed; 95%CI 0.31–11; P=0.038) and UTS (1.0MPa per µg removed; 95%CI 0.043–2; P=0.041).These results demonstrate that the chondroitin/dermatan sulphate glycosaminoglycans that accumulate in pathological tendon post-injury are partly responsible for the altered biomechanical properties.

The Different Forms of Mucopolysaccharidosis with Neurological Involvement: A Case-Based Review

The mucopolysaccharidoses (MPSs) are a group of rare lysosomal storage disorders caused by deficiency of enzymes catalyzing the stepwise degradation of glycosaminoglycans dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin sulfate, and hyaluronic acid. There are seven groups of MPS, which are MPS-I (MPS-I-H or Hurler syndrome; MPS-I-S or Scheie syndrome; and MPS-I-HS or Hurler–Scheie syndrome), MPS-II (Hunter syndrome), MPS-III (Sanfilippo syndrome types A to D), MPS-IV (Morquio syndrome types A and B), MPS-VI (Maroteaux–Lamy syndrome), MPS-VII (Sly syndrome), and MPS-IX (Natowicz syndrome). All are inherited as autosomal recessive diseases, with the exception of Hunter syndrome, which follows an X-linked recessive inheritance pattern. The MPSs affect multiple organ systems (including bone, heart, and visceral organs), leading to organ failure. Involvement of central nervous system occurs only in the forms with heparan sulfate accumulation, that is, MPS-I, MPS-II, MPS-III, and MPS-VII. Therapy is available for MPS-I, MPS-II, MPS-IV, and MPS-VI. This review provides a case-based overview of the different forms of MPS with neurological involvement.

Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF.

Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events.