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Bleomycin is a clinically used anticancer drug, but it induces lung fibrosis in certain cancer patients with unknown mechanism. Glycosaminoglycans (GAGs) are required for lung morphogenesis during animal development. In current study, GAG disaccharides including heparan sulfate (HS) and chondroitin sulfate (CS) from bleomycin-induced and control lung tissues in lung fibrosis mouse model were tagged with 1-phenyl-3-methyl-5-pyrazolone (PMP) and deuterated PMP, respectively. The differentially isotope-tagged disaccharides were quantitatively compared by LC-MS. At day 10, the amount of CS disaccharides (U0a0, U0a6, and U0a4) and non-sulfated HS disaccharide (U0A0) were increased by 1.3-, 1.6-, 11.7-, and 2.2-fold, respectively, whereas the amount of CS disaccharide (U0a2), hyaluranan disaccharide (UβA0), and six HS disaccharides (U0A6, U2A0, U0H6, U0S0, U2S0, and U2S6) were decreased from1.1- to 14.3-fold compared to that of the controls. At day 15, under-sulfation of both HS and CS disaccharides was persisted. At day 30, the CS disaccharide compositions were recovered to that of the control levels whereas the HS were still remarkably under-sulfated. In conclusion, GAGs, especially HS, from fibrotic lungs induced by a single injection of bleomycin were significantly under-sulfated up to 30 days, suggesting GAGs might be another class of defective signaling molecules involved in bleomycin-induced lung fibrosis.

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.

Glycosaminoglycans (GAGs) are heterogeneous biomacromolecules made by all animal cells with overlapping molecular weight and high negative charge densities, which make thorough separation of different types of GAGs and elimination of all GAG-binding proteins difficult. Even with the constant challenge of quality control, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin glycosaminoglycans (GAGs) have been used as nutraceuticals and modern drugs for many years worldwide. Testing galactosamine in heparin has been added to the USP monograph after contaminated heparin event, but the general monosaccharide composition analysis has not been developed for GAG quality control purposes. Using a PCR-facilitated hydrolysis assay, the hydrolyzed GAG saccharides were labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) and quantified by high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Glucosamine was found in both chondroitin sulfate and dermatan sulfate whereas galactosamine was observed in both heparan sulfate and heparin, indicating the cross contamination among different types of GAGs. Moreover, fucose was detected in chondroitin sulfate, dermatan sulfate, and heparan sulfate, and both fucose and mannose were detected in chondroitin sulfate, suggesting the co-presence of other types of glycans or novel fucosylated GAG structures. Furthermore, both the amount and structure of acid-resistant disaccharides provide distinguishable features for each type of GAGs at the same hydrolysis condition. Thus, monosaccharide analysis provides a practical and quantitative way for GAG quality control.

Releasing all monosaccharides during acid hydrolysis for composition analysis of polysaccharides has been a time consuming process. In current study, an efficient (10 μL sample + 10 μL acid), sensitive, and quick monosaccharide composition analysis of polysaccharides was accomplished by using microwave-assisted HCl hydrolysis (10 min) of the polysaccharides followed by high-performance anion-exchange chromatography (HPAEC) combined with pulsed amperometric detection (PAD) analysis. Compared to the conventional hydrolysis procedure, this method is an efficient approach for monosaccharide composition analysis of acidic, basic, and neutral polysaccharides and particularly suited to polysaccharides that are difficult to hydrolyse fully such as chitosan, heparin and chondroitin sulfates.

Heparin is the first glycosaminoglycan ever identified. All the heparin-like glycosaminoglycans that are also isolated from animal tissues or any polysaccharides that mimic the biological activities of heparin are called heparinoids. Heparin is the mostly sulfated glycosaminoglycan made by mast cells and an essential anticoagulant drug in modern medicine. Heparin inhibits both thrombin generation and thrombin activity, releases tissue factor pathway inhibitor, and possesses anti-inflammatory, anti-viral, anti-angiogenesis, anti-neoplastic, and anti-metastatic properties though high affinity interactions with a variety of proteins in the blood circulation. The multi-pharmacological effects of heparin are both sequence- and sulfation degree dependent. Less sulfated heparinoids have been indicated to have more physiological functions than heparin. Since the anticoagulant heparin is associated with severe side effects, such as bleeding and heparin-induced thrombocytopenia and thrombosis, it is expected that the less sulfated heparinoids might serve as alternative drugs for patients who cannot use heparin. The crude heparin isolated from animal tissues contains ~50% heparin and ~50% less sulfated heparinoids. Indeed, the less sulfated waste heparinoids 1 during heparin production is chemically degraded and developed into the clinical drug Danaparoid and the more sulfated waste heparinoids 2 during heparin production is chemically degraded and developed into the clinical drug Sulodexide. Moreover, clinical studies indicate that Danaparoid and Sulodexide have the expected pharmacological activities. We will provide an update on the chemical characteristics and clinical use of the heparinoids Danaparoid and Sulodexide. In addition, the potential clinical applications of Danaparoid and Sulodexide in other therapeutic area will also be discussed.

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.

The low-molecular-weight fucosylated chondroitin sulfate (LFCS) was prepared from native fucosylated chondroitin sulfate (FCS), which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC) was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF), increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and downregulated the matrix metalloproteinases (MMPs) level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

Thrombin generation is thought to be mediated predominantly by the tissue factor or ‘‘extrinsic’’ coagulation pathway. An alternate pathway to thrombin generation (the ‘‘intrinsic’’ pathway or contact system) has been observed when blood or plasma comes in contact with artificial surfaces. Here we present evidence for a new route to thrombin formation that begins with the activation of the contact system protein prekallikrein by oversulfated heparin (OS-HB). Kallikrein, instead of activated factor X, cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form fibrin clots. Moreover, we show that OS-HB by-products induce kallikrein- and thrombin-like activities in normal human plasma and in human plasma devoid of coagulation factor X or downstream contact system components factor IX or factor XI. Oversulfated heparin by-product-induced thrombin generation may have had a role in the adverse reactions associated with the recent clinical use of contaminated heparin.

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection., Author Summary Dengue virus (DENV) is a mosquito-transmitted virus that infects humans and has become a global emerging infectious disease threat. Four serotypes of DENV exist, and the most severe cases are associated with secondary infection with a different virus serotype. Clinical deterioration is characterized by bleeding and selective vascular leakage from endothelium in specific tissue sites. An increased understanding of how DENV proteins contribute to this phenotype is vital to developing novel vaccines and identifying individuals at risk for severe disease. DENV nonstructural protein-1 (NS1) is one such protein: during infection, it is secreted and accumulates in the supernatant and on the surface of cells. In this study, we demonstrate that soluble DENV NS1 attaches to subsets of cells, including some but not all endothelial cells, primarily via an interaction with specific glycosaminoglycans (heparan sulfate and chondroitin sulfate E). This was confirmed in tissue binding studies as DENV NS1 bound to lung and liver but not intestine or brain endothelium. Our findings suggest that the selective vascular leakage that occurs in severe DENV infection may be related to the relative ability of endothelial cells in different tissues to bind soluble NS1 and to be targeted by cross-reactive anti-NS1 antibodies during secondary infection.