The isolation and characterization of glycosaminoglycans in normal human serum

The optimization of conditions for the isolation and characterization of human serum glycosaminoglycans (GAG) is described, together with studies of the accuracy and reproducibility of the method. The principle of the method is proteolytic digestion of serum using papain followed by precipitation of GAGs from the digested sample with cetyl pyridinium chloride (CPC). The uronic acid level and electrophoretic separations can be obtained from a 5 ml serum sample. The mean CPC-precipitable uronic acid level in pooled normal serum was 10.8 mg l(-1) serum. Using enzymatic and chemical analysis the major serum GAG was shown to be chondroitin sulphate (CS). Two distinct electrophoretic fractions were identified both consisting of CS but differing in their degree of sulphation. Dermatan sulphate, heparan sulphate and hyaluronic acid were not detected.     

The tilorone-induced mucopolysaccharidosis in rats. Biochemical investigations.

The tissues of rats chronically treated with tilorone exhibited a significant accumulation of acid glycosaminoglycans (GAGs): In the liver, the GAG concentration was found to be elevated by a factor of 38, in the spleen by a factor 15 and in the kidneys by a factor of 5. Furthermore, the renal excretion of GAGs was increased 32-fold as compared to control animals. Dermatan sulphate was predominant among the GAGs stored in the three organs; chondroitin sulphate and heparan sulphate were found in smaller amounts. GAG storage was accompanied by accumulation of the drug within the tissues: the molar ratio of tilorone per disaccharide unit of GAG was calculated to be one to two in each tissue. According to previous reports, tilorone-induced mucopolysacchariodosis is due to impaired lysosomal degradation of GAGs. The present results give support to the hypothesis that an interaction between the polyanionic GAGs and the dicationic drug may lead to GAG-drug complexes which cannot be digested by lysosomal enzymes.     

Role of glycosaminoglycans in inflammation

Glycosaminoglycans (GAGs) are large, polyanionic molecules expressed throughout the body, either in association with cell surfaces and extracellular matrices, or stored within intracellular compartments. The GAG heparin is synthesised by and stored exclusively in mast cells, which are strongly associated with allergy and inflammation and is co-released with histamine upon cellular degranulation. The closely related GAG heparan sulphate is expressed, as part of a proteoglycan, on cell surfaces. Most notably, heparan sulphate is associated with the surfaces of vascular endothelial cells, known to be pivotally involved in the control of inflammatory cell adhesion and extravasation. The physiological role of these molecules is not well understood but evidence suggests that they may be involved in limitation of the inflammatory response and, in particular, regulation of cell adhesion and trafficking.     

Effects of glycosaminoglycans on the in vitro colony formation of CD34+ megakaryocytic progenitor cells in human placental/umbilical cord blood

The in vitro effect of various glycosaminoglycans (GAGs) on the clonal growth of CD34+ megakaryocytic progenitor cells (CFU-Megs) isolated from human placental/umbilical cord blood (CB) was evaluated in human plasma containing semisolid culture stimulated by recombinant human thrombopoietin (TPO). The GAGs, including hyaluronic acid from human umbilical cords (HA-h), pig skins (HA-p) and rooster combs (HA-r), or keratan sulfate (KS), various chondroitin sulfates (CS-A, B, C, D, E), and heparan sulfate (HS), were tested. Each GAG alone did not affect the clonal growth of CFU-Meg. In the presence of TPO, adding of HA-p or HS (100 micrograms/ml) resulted in an approximately 1.3-fold increase, in the total number of colonies, due to an increase in large megakaryocyte colonies. In contrast, CS-E led to a marked decrease in CFU-Meg growth. At the end of the culture, the total number of cells increased 3.0-fold of the initial value of the control, but adding HA-p or HS showed an approximately 9.1-fold or 18.3-fold increase. Similarly, the total number of CFU-Meg detected in the harvested cells increased to 4.8-fold of the initial value, while, an approximately 18.3-fold or 38.8-fold increase was observed in the culture containing HA-p or HS, respectively. Flow cytometric analysis of the harvested cells showed no significant difference in the expression of surface antigens and DNA ploidy distribution of megakaryocytes between the control and GAG treatments. These results suggest that HA-p and HS promote the proliferation of immature CB CD34+ CFU-Meg in the presence of TPO.     

Glycosaminoglycans and the regulation of allergic inflammation

Glycosaminoglycans (GAGs) are large, polyanionic molecules expressed throughout the body. The GAG heparin, co-released with histamine, is synthesised by and stored exclusively in mast cells, whereas the closely related molecule heparan sulphate is expressed, as part of a proteoglycan, on cell surfaces and throughout tissue matrices. These molecules are increasingly thought to play a role in regulation of the inflammatory response and heparin like molecules are now being seriously considered to hold potential in the treatment of inflammatory diseases such as asthma. Heparin and related molecules have been found to exert anti-inflammatory effects in a wide range of in vitro assays, animal models and in human disease. The anti-inflammatory activities of heparin are independent of the well-established anticoagulant activity of heparin, suggesting that the separation of these properties could yield novel anti-inflammatory drugs, which may be useful in the future treatment of inflammatory diseases.     

Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds: differential cellular accumulation mechanisms for platinum

We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs) based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R(9) (a nonarginine peptide, R(9)) coupled to the TAMRA fluorescent label 5-(and 6-)carboxytetramethylrhodamine) in Chinese hamster ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of noncovalent TriplatinNC and, to a lesser degree, the covalently binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall, the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for the rational design of platinum antitumor drugs with distinct biological profiles in comparison to those of the clinically used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors.     

Glycosaminoglycan and chemokine/growth factor interactions

Heparin and glycosaminoglycans (GAGs) related structurally to heparin, notably heparan sulphate, bind to most, if not all, chemokines and many growth factors. The chemokine and growth factor interactions with GAGs localise the peptide mediators to specific sites in tissues and influence their stability and function. This chapter discusses the nature of these interactions and the effect on the function of a number of chemokines (PF-4, interleukin-8, RANTES and SDF-1) and growth factors (FGF, HGF, VEGF) in normal physiology and the disease setting. Novel therapeutic interventions that target chemokine and growth factor interactions with GAGs are also discussed.     

Pharmacological activities of a new glycosaminoglycan,acharan sulfate isolated from the giant African snail Achatina fulica

Acharan sulfate (AS) is a glycosaminoglycan (GAG) prepared from the giant African snail, Achatina fulica. In this study, some biological activities of AS were evaluated on the basis of structural similarities to heparin/heparan sulfate and the biological functions of GAGs. We demonstrated that it exhibited strong immunostimulating activities as measured by carbon clearance test in mice and in vivo phagocytosis. It also exhibited a significant hypoglycemic activity in epinephrine (EP)-induced hyperglycemia as well as antifatigue effects by weight-loaded forced swimming test. And it showed hypolipidemic activities in cholesterol-rich mixture induced hyperlipidemia in rats. The above results indicate that AS has diverse biological activities and suggest therapeutically important target molecules.     

Therapeutic applications of glycosaminoglycans

Complex polysaccharides, hyaluronic acid or hyaluronan (HA), keratan sulfate (KS), chondroitin sulfates (CSs) and heparin (Hep)/heparan sulfate (HS), are a class of ubiquitous molecules exhibiting a wide range of biological functions. They are widely distributed as glycosaminoglycans (GAGs) sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of improved enzymatic and analytical tools for the study of these complex sugars has produced a virtual explosion in the field of glycomics. In particular, the study of the GAG family of polysaccharides has shed considerable light on the way in which specific carbohydrate structures modulate cellular phenotypes. In addition to the well-known therapeutic applications of some of these macromolecules, such as HA and derivatives as structure modifying molecules and possessing gel-like properties able to provide functional support for tissues, Hep as an anticoagulant and antithrombotic drug and CS in the treatment of osteoarthritis (OA), this increased understanding of GAG structure-function relationship has led to the discovery of novel pharmaceuticals for the possible treatment of serious diseases, such as cancer. In this paper, the structure and the therapeutic applications of several complex natural polysaccharides, including HA, CS/DS, Hep and their derivatives, are presented and discussed also in the light of the many questions still left unanswered, such as improved preparation and GAG-based drugs with improved properties and new possible therapeutic applications.     

Intravesical glycosaminoglycan replenishment with chondroitin sulphate in chronic forms of cystitis. A multi-national, multi-centre, prospective observational clinical trial

Effectiveness, safety, and tolerability of instillation therapy with chondroitin sulphate (CAS 9082-07-9, Gepan instill) was investigated in a non-interventional study. 286 patients with clinically diagnosed chronic forms of cystitis, such as bladder pain syndromelinterstitial cystitis, radiation cystitis, overactive bladder syndrome and chronically-recurring cystitis were included. The course of symptoms was documented over 8 instillations at maximum, covering a period of approximately three months. All main symptoms of chronic cystitis declined consistently and statistically significantly (p < 0.0001). Both daytime and nighttime micturition frequencies as well as the score levels of urgency and pain declined significantly during the course of treatment. The functional bladder capacity as indicated by the volume of first morning voiding increased from 157.9 ml +/- 7.5 to 186.7 ml +/- 6.9 (mean +/- SE; p < 0.0001). The level of urgency decreased from 6.8 +/- 0.1 to 3.4 +/- 0.2 (mean +/- SE; numerical rating scale (11-point box scale); p < 0.0001) and nocturia decreased from 4.0 +/- 0.2 to 2.1 +/- 0.1 times (mean +/- SE; p < 0.0001). Chondroitin sulphate instillation was effective and well tolerated in the therapy of chronic forms of cystitis associated with a possible GAG layer deficit (GAG layer: mainly composed of the glycosaminoglycans chondroitin sulphate, dermatan sulphate and heparan sulphate), but the results need to be confirmed in a controlled study.     

Linear basic peptides for targeting interferon‐γ–glycosaminoglycan interactions: synthesis and inhibitory properties

Abstract: Glycosaminoglycans (GAGs) play an important role in inflammatory responses due to their ability to interact with cytokines and chemokines, resulting in the localization of these mediators to specific anatomical sites, where they function to direct leukocyte recruitment and activation. Targeting GAG–cytokine/chemokine interactions might may thus have therapeutic applications as anti‐inflammatory or immunomodulatory therapy in vivo. Peptides that mimic the heparin‐binding domains of cytokines may have a potential use as inhibitors of GAG–cytokine interactions. A linear octapeptide (MC‐2) derived from the conserved heparin‐binding region of interferon‐γ (IFN‐γ) was synthesized along with four analogs featuring a substitution of Phe for Leu in position 1 and varying number of positive charges on the octapeptide molecule. The relative abilities of the synthesized peptides to inhibit the interactions between IFN‐γ and GAGs were compared. From the results, it follows that the inhibitory potency of the octapeptide analogs was related to the number of positive charges in the molecule, while increased hydrophobicity had no significant effect.     

Determination of tamoxifen--DNA adducts in leukocytes from breast cancer patients treated with tamoxifen

Tamoxifen (TAM), a widely used antiestrogen for breast cancer therapy and chemoprevention, increases the incidence of endometrial cancer in women. The formation of DNA adducts induced by tamoxifen may initiate endometrial cancer. To evaluate the genotoxic risk of TAM, the formation of DNA adducts in leukocytes was examined. Blood samples were collected from 47 breast cancer patients (61.7 +/- 12.5 years) taking TAM (20 mg/day; average duration until sampling, approximately 37 months) and 20 untreated patients (58.2 +/- 12.3 years), and their leukocyte DNA was analyzed by 32P-postlabeling/HPLC analysis. This assay resolves synthetic standards, trans- and cis-diastereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM), alpha-(N2-deoxyguanosinyl)-N-desmethyltamoxifen 3'-monophosphate (dG3'P-N2-N-dMeTAM), and alpha-(N2-deoxyguanosinyl)tamoxifen N-oxide 3'-monophosphate', and is capable of determining TAM adducts quantitatively. The detection limit of this assay is 0.6 adducts/10(9) nucleotides. trans-dG3'P-N2-TAM (fr-2; one of the two trans-isomers) was detected in six of 47 breast cancer patients treated with TAM. Among them, trans-dG(3'P-N2-N-dMeTAM (fr-2) was also detected in two patients. The total amounts of TAM-DNA adducts in the positive patients were 2.6 +/- 3.0 adducts/10(9) nucleotides. No adducts were detected in the controls. The presence of TAM-DNA adducts in the leukocyte DNA samples was confirmed using several 32P-postlabeling/HPLC systems.     

The Structure of Glycosaminoglycans and their Interactions with Proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three‐dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti‐thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.     

Idursulfase in Hunter syndrome treatment

Hunter syndrome (mucopolysaccharidosis II, MPS II) is a rare X-linked lysosomal storage disorder caused by the deficiency of enzyme iduronate-2-sulfatase (I2S), which results in accumulation of undegraded dermatan and heparan sulfate in various tissues and organs. Enzyme replacement therapy with Elaprase (idursulfase, a recently approved orphan product) is the first treatment for Hunter syndrome. Results of the randomized, double-blind, placebo-controlled phase II/III clinical trial of idursulfase demonstrated that weekly infusions of idursulfase increase walking distance and improve pulmonary function as well as reduce organ size and urinary glycosaminoglycans (GAGs) excretion in MPS II patients. Idursulfase is generally well tolerated, although infusion reactions do occur. Clinical studies demonstrate that idursulfase may be the first successful symptomatic therapy that can benefit patients with MPS II by addressing the enzymatic defect.     

Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture

Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [(3)H]glucosamine, [(35)S]sodium sulfate or (35)S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high M(r) subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a K(av) ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1-0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately M(r) 45000 in biglycan and decorin, approximately M(r) 48000 in the small amount of HSPGs and approximately M(r) 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.     

Thermodynamic analysis of binding between drugs and glycosaminoglycans by isothermal titration calorimetry and fluorescence spectroscopy.

The thermodynamics of the interaction of positively charged drug molecules with negatively charged glycosaminoglycans (GAGs) is investigated by isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The drugs considered are propranolol hydrochloride, tacrine, and aminacrine, and the polymers used as model GAGs are dextran sulfate, chondroitin sulfate, and hyaluronic acid. The ITC results show that the interaction between drugs and GAGs is via direct binding and that GAGs bind to drugs at one set of sites. Large negative values of heat capacity change (DeltaC(p)) are observed upon binding of GAGs to drugs. Such negative DeltaC(p) is not expected for purely electrostatic interactions and suggests that hydrophobic and other interactions may be also involved in the binding process. These results are corroborated by fluorescence spectroscopy measurements, which show that specific drug/GAG complex formation is accompanied by a clear enhancement of the fluorescence intensity. The results highlight the importance of the formation of drug/GAG complexes as a primary step for the drug delivery process into cell membranes. It is concluded that the interactions are dependent on the nature of both GAG and drug and this is a fact to be taken into account when new drugs are designed.     

Lysosomal storage of sulfated glycosaminoglycans induced by two bis-aminomethyl anthrachinones

 Several immunomodulatory drugs, all of them symmetrically substituted dicationic amphiphilic compounds, are known to cause lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact animals and cultured fibroblasts. The storage is due to impaired GAG degradation. The standard compound is tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one). In the present study two bis-aminomethyl anthrachinones were examined for their ability to induce lysosomal GAG storage in cultured bovine corneal fibroblasts. For reference, a bis-aminoethoxy-anthrachinone compound (RMI-10.024) was included, which is known to be a potent inducer of lysosomal GAG storage. The present morphological, radiochemical, and biochemical results show that the bis-aminomethyl anthrachinone compounds investigated cause lysosomal storage of GAGs, although with significantly lower potencies than the bis-aminoethoxy anthrachinone. Dermatan sulfate contributed approximately 90% to the drug-induced increment of intracellular GAGs. The present results suggest that the length of the side chains, i. e., the distance between the aromatic ring system and the protonizable nitrogen of the side chains, and the position of the side chains relative to the aromatic ring system are important molecular features influencing the potency of inducing lysosomal GAG storage. Received: 14 July 1995 / Accepted: 17 October 1995     

Chemokines and breast cancer: a gateway to revolutionary targeted cancer treatments?

Breast cancer is an example of a solid tumour which is well treated in the early stages of disease by surgical excision, but once metastatic spread has occurred, medical therapies (chemotherapy and radiotherapy) are highly toxic, expensive and palliative. It is known that certain tumours exhibit specific patterns of metastasis, chemokines may provide a molecular answer to this mystery. Chemokines and their receptors play important roles in the various stages of tumour development and metastasis. Chemokines interact with their specific receptors as well as interacting with the glycosaminoglycan (GAG) component of proteoglycan. We discuss the basic metastatic process and the involvement of chemokines in breast cancer biology. Finally, we summarize potential therapeutic applications of chemokines and chemokine/glycosaminoglycan interactions including chemokine agonists, antagonists, anti-sense therapy, immunotherapy and soluble GAGs, as well as future perspectives in this field.     

Modulation of extracellular matrix metabolism in rabbit articular chondrocytes and human rheumatoid synovial cells by the non-steroidal anti-inflammatory drug etodolac. II: Glycosaminoglycan synthesis

The effect of Etodolac on glycosaminoglycan (GAG) synthesis by human synovial cells and rabbit articular chondrocytes in culture was studied at doses ranging from 0.01 to 10 micrograms/ml. In chondrocyte cultures, short-term exposure to Etodolac decreased the total amount of GAGs (mainly chondroitin sulfate) affecting essentially the cell layer-associated fraction. When cells were incubated with Interleukin-1 (IL-1), Etodolac still exerted its inhibiting effect on GAG synthesis. Long-term exposure of chondrocytes to Etodolac caused a diminution of the total GAG synthesis for the highest concentration studied (1 micrograms/ml). IL-1 alone or in combination with Etodolac decreased the synthesis of GAGs suggesting that pretreatment with Etodolac cannot prevent the action of IL-1. In synoviocyte cultures, the drug caused an inhibition of the GAG biosynthesis (mainly hyaluronic acid). In presence of IL-1, Etodolac at the highest concentration partially diminished the stimulatory effect of IL-1. A 8-day exposure of the cells to Etodolac led to a decrease of GAG synthesis. In the same experiment, IL-1 alone caused a slight increase of GAG production that was not abolished by Etodolac treatment.