Recent Advances in the Structural Study of Functional Chondroitin Sulfate and Dermatan Sulfate in Health and Disease

Chondroitin sulfate (CS) dermatan sulfate (DS), and CS/DS hybrid chains are biologically active like heparan sulfate, and structurally the most complex species of the glycosaminoglycan family along with heparan sulfate. They exist at the cell surface and extracellular matrix in the form of proteoglycans. They function as regulators of functional proteins such as growth factors, cytokines, chemokines, adhesion molecules, and lipoproteins through interactions with the ligands of these proteins via specific saccharide domains. Structural alterations have been often implicated in pathological conditions, such as cancer and atherosclerosis. Recent microsequencing of CS/DS oligosaccharides that bind growth factors, such as pleiotrophin, and various monoclonal antibodies against CS/DS, have revealed a considerable number of unique oligosaccharide sequences. This review focuses on recent advances in the study of the structure-function relation of CS, DS and their hybrid chains in physiological and pathological conditions.     

Synthesis of Helical Polyanilines Using Chondroitin Sulfate as a Molecular Template

Summary: Water‐processable conducting polyaniline derivatives (PANIs), such as polyaniline (PANI), poly(o‐methylaniline) and poly(o‐methoxyaniline), were synthesized by in situ chemical polymerization of the corresponding achiral monomer in the presence of the novel chiral polymeric template, chondroitin sulfate. The resulting conducting PANIs were doped and complexed with chondroitin sulfate (CHS) to form intertwined intermacromolecular complexes of PANIs‐CHS, which was confirmed by UV‐vis and FTIR spectroscopy. The induction of the excess one‐handed helical structure into PANIs was confirmed by circular dichroism (CD) spectroscopy with the characteristic bisignate bands at approximately 400 and 450 nm for the emeraldine salt. The effect of ionic strength was investigated on the formation of the intertwined PANI‐CHS complex and the induction of chirality into PANI by adding NaCl to the reaction solution. The CD spectral change of PANI‐CHS in the cycle of dedoping and redoping was also studied, and it was found that intermacromolecular complexation helps to stabilize the chiral conformation in PANI against racemization. Interestingly enough, it was found that optically active PANI‐CHS exhibited pH‐sensitive chiroptical properties. A study of the conductivity, electrochemical activity and the chiroptical properties in different redox states of PANIs provided proof for their use as chiral electrode and electroactively chiral material.

Structure of the complexes of PANIs‐CHS.     

Chondroitin sulfate-binding peptides block chondroitin 6-sulfate inhibition of cortical neurite growth

Chondroitin sulfate (CS) expression is increased in the glial scar following spinal cord injury demonstrating the importance understanding the role of CS in the central nervous system (CNS). There have been conflicting studies on the effects of the most abundant types of CS, chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), found in the CNS. In this study, the effects of C4S and C6S on rat embryonic day 18 cortical neurons were investigated. C4S had no effect on neuron behavior whereas C6S inhibited neurite outgrowth at higher concentrations (>10 μg/ml). Two C6S-binding peptides (C6S-1 and C6S-2) were tested for their ability to block the inhibitory activity of C6S on neurite outgrowth. Neurons cultured with C6S and C6S-binding peptide at higher peptide concentrations had neurite lengths similar to neurons cultured without C6S. Therefore, the C6S-binding peptides were effective at blocking the inhibitory activity of C6S. The C6S-1 peptide had a higher binding affinity than the C6S-2 peptide and was consequently more effective at blocking C6S inhibition of neurite growth. To date, this is the first study to employ an alternative strategy from enzymatic digestion of CS chains to increase neurite outgrowth. These studies warrant further investigation of the use of C6S-binding peptides to increase nerve regeneration following spinal cord injury.     

The Activities of Heparan Sulfate and its Analogue Heparin are Dictated by Biosynthesis,Sequence, and Conformation

The glycosaminoglycan heparan sulfate (HS), is expressed on the surface of virtually all mammalian cells and is implicated in many crucial biological activities. The activities of HS and its close structural analogue heparin are mediated through interactions with proteins. However, the relationship between structure and activity is not simple, because the structure and conformation of HS and heparin are complex. This review surveys some of the relevant findings in HS/heparin chemistry, biochemistry, and biology.     

Sulfate as a pivotal factor in regulation of Serratia sp. strain S2B pigment biosynthesis

In the present work, we investigated the prodiginine family as secondary metabolite members. Bacterial strain S2B, with the ability to produce red pigment, was isolated from the Sarcheshmeh copper mine in Iran. 16S rDNA gene sequencing revealed that the strain was placed in the Serratia genus. Pigment production was optimized using low-cost culture medium and the effects of various physicochemical factors were studied via statistical approaches. Purification of the produced pigment by silica gel column chromatography showed a strong red pigment fraction and a weaker orange band. Mass spectrometry, FT-IR spectroscopy and ¹H NMR analysis revealed that the red pigment was prodigiosin and the orange band was a prodigiosin-like analog, with molecular weights of 323 and 317 Da, respectively. Genotoxicity and cytotoxicity studies confirmed their membership in the prodiginine family. Analysis of the production pattern of the pigments in the presence of different concentrations of ammonium salts revealed the role of sulfate as an important factor in regulation of the pigment biosynthesis pathway. Overall, the data showed that regulation of the pigment biosynthesis pathway in Serratia sp. strain S2B was affected by inorganic micronutrients, particularly the sulfate ions.     

Fibroblast Growth Factor-21 as a Therapeutic Agent for Metabolic Diseases

Fibroblast growth factor (FGF)-21 is a unique member of the FGF family, with several molecular characteristics that differ from classical FGFs and exhibiting a pharmacologic profile that includes a variety of metabolic responses in vitro and when tested in vivo in animal models. FGF21 represents a novel and attractive therapeutic agent for type 2 diabetes mellitus, because of its ability to modulate disease phenotype in preclinical settings without inducing any apparent adverse effects. Although FGF21 was discovered relatively recently, the understanding of its biology and therapeutic utility is rapidly evolving. A number of key metabolically linked molecules and pathways have been suggested to be involved in the mechanism of action of FGF21, depending on the specific target tissue/organ. Further research into these mechanisms should lead to important advances in the understanding of FGF21 biology and pave the way for novel therapeutic strategies. The specifics of FGF21 activities both in cell culture and in vivo, its potential as a target for diabetes, and insights into the molecular mechanisms of FGF21 metabolic actions will be discussed in this review.     

Stimulation of Chondroitin Sulfate Proteoglycan Production by Chondrocytes in Monolayer

Chondrocytes in monolayer undergo morphological and biochemical changes which culminate in the establishment of cartilage nodules in vitro. Chondroitin sulfate or heparin, added to the culture media of these cells, stimulates the production of chondroitin sulfate proteoglycan over the entire period of culture with a maximum effect during the log phase of growth. In addition, a lag of 2–3 hours is required before an increase in sulfate incorporation into polysaccharide is observed. The responsiveness of chondrocytes is influenced by several factors, such as cell density, conditioned media and enzyme treatment. Furthermore, puromycin abolishes the endogenous as well as the stimulated synthesis, demonstrating the necessity for core protein synthesis in both synthetic processes. Addition of j3-D-xylosides (which presumably act as initiators of chondroitin sulfate polysaccharide synthesis) and chondroitin sulfate, concurrently, stimulate sulfate incorporation to levels higher than either agent alone, indicating that these compounds act by different mechanisms.     

Inhibition of Binding of Malaria-Infected Erythrocytes by a Tetradecasaccharide Fraction from Chondroitin Sulfate A

Adherence of parasite-infected erythrocytes (IEs) to the microvascular endothelium of various organs, a process known as sequestration, is a feature of Plasmodium falciparum malaria. This event is mediated by specific adhesive interactions between parasite proteins, expressed on the surface of IEs, and host molecules. P. falciparum IEs can bind to purified chondroitin sulfate A (CS-A), to the proteoglycan thrombomodulin through CS-A side chains, and to CS-A present on the surface of brain and lung endothelial cells and placental syncytiotrophoblasts. In order to identify structural characteristics of CS-A important for binding, oligosaccharide fragments ranging in size from 2 to 20 monosaccharide units were isolated from CS-A and CS-C, following controlled chondroitin lyase digestion, and used as competitive inhibitors of IE binding to immobilized ligands. Inhibition of binding to CS-A was highly dependent on molecular size: a CS-A tetradecasaccharide fraction was the minimum length able to almost completely inhibit binding. The effect was dose dependent and similar to that of the parent polysaccharide, and the same degree of inhibition was not found with the CS-C oligosaccharides. There was no effect on binding of IEs to other ligands, e.g., CD36 and intercellular adhesion molecule 1. Hexadeca- and octadecasaccharide fractions of CS-A were required for maximum inhibition of binding to thrombomodulin. Analyses of oligosaccharide fractions and polysaccharides by electrospray mass spectrometry and high-performance liquid chromatography suggest that the differences between the activities of CS-A and CS-C oligosaccharides can be attributed to differences in sulfate content and sulfation pattern and that iduronic acid is not involved in IE binding.     

Fursultiamine, a vitamin B1 derivative, enhances chondroprotective effects of glucosamine hydrochloride and chondroitin sulfate in rabbit experimental osteoarthritis

Object and design: The therapeutic effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) in combination with fursultiamine, a vitamin B1 derivative, on the development of cartilage lesions was investigated in an animal model of osteoarthritis (OA).Methods: The OA model was created by partial medial meniscectomy of the right knee joint (day 0). The rabbits were placed into three experimental groups: operated (OA) rabbits that received placebo treatment, OA rabbits that received GH (1000 mg/kg) + CS (800 mg/kg), and OA rabbits that received GH + CS + fursultiamine (100 mg/kg). Each treatment was initiated on day 3 and continued for 8 weeks. Macroscopic and histologic analyses were performed on the cartilage. The level of MMP-1 in OA cartilage chondrocytes was evaluated by immunohistochemistry.Results: Only the group receiving combined treatment with GH + CS + fursultiamine showed a significant reduction in the severity of macroscopic and histologic lesions on tibial plateau, which is the weight bearing cartilage surface of the tibia, compared with placebo-treated OA rabbits. This treatment group also revealed a small, but significant, decrease in the body weight gain of the rabbits. In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in superficial layer stained positive for MMP-1 compared with unoperated control. Rabbits treated with the GH + CS + fursultiamine revealed a significant reduction in the level of MMP-1.Conclusion: These results suggest that the chondroprotective effect of GH + CS is enhanced by the addition of fursultiamine in experimental OA. This effect was associated with a reduction in the level of MMP-1, which are known to play an important role in the pathophysiology of OA lesions.Received 23 October 2004; returned for revision 7 January 2005; accepted by M. Katori 11 February 2005     

Effect of Decorin and Dermatan Sulfate on the Mechanical Properties of a Neocartilage

Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.     

壳聚糖与硫酸软骨素共混膜性质的研究

以壳聚糖和硫酸软骨素按一定比例制备出共混膜，研究了膜片的透光性、含水量、渗透性、力学性质、表面结构、生物降解性、生物相容性等性质。结果表明该共混膜具有较好的透光性、通透性、生物降解性和生物相容性，膜表面较粗糙。以此共混膜为载体培养兔角膜基质细胞，发现细胞在此共混膜上生长良好。制备膜片随着加入CaSO4量的增加，膜的通透性也随之增加。     

The Therapeutic Potential of Intracellular Antibodies

Antibody engineering techniques permit the exploitation of the highly specific and diverse nature of the humoral immune system. Intracellular antibodies, consisting minimally of the antigen-binding domains of the parent immunoglobulin protein, may be used to manipulate microbial and cellular target molecules. Applications of this technology described to date include inhibition of replication of an infectious virus, downregulation of growth factor receptors, and inactivation of oncoproteins in cell culture systems. The ability to functionally manipulate virtually any target molecule using intracellular antibodies is clearly beneficial to basic science and molecular medicine.     

Soluble Recombinant Neutral Endopeptidase (CD10) as a Potential Antiinflammatory Agent

Several endogenous peptides, including bradykinin and sustance P, have potent inflammatory effects in the joint. Levels of these peptides are regulated by plasma and cell-associated peptide degrading enzymes. One of these peptidases, neutral endopeptidase-24.11 (NEP-24.11), is expressed constitutively and in high density on human synovial cells and is presumed to play a critical role in local regulation of peptide levels in the joint. We examined the role of endogenous NEP-24.11 in regulating bradykinin-mediated effects in an articular model, and investigated the ability of soluble, recombinant human NEP-24.11 to augment the effects of the endogenous enzyme. Our studies demonstrate that endogenous synovial NEP-24.11 does not significantly modulate inflammatory peptide effects on cells when competing with colocalizing peptide receptors expressed in high density. Administration of excess, soluble recombinant NEP-24.11 can overcome this problem, however. Furthermore, the activity of the recombinant enzyme was not compromised in the presence of oxidants or inflammatory joint fluids. Recombinant NEP-24.11 holds promise as a novel therapeutic strategy for the treatment of inflammatory arthritis.     

Distribution of Hyaluronan and Dermatan/Chondroitin Sulfate Proteoglycans in Human Aortic Dissection

Aortic dissections (AD) are characterized by the separation of the artery into two sheets, possibly due to fragility of the vessel wall. A mucoid histological pattern, imparted to the tissues mainly by hyaluronan and proteoglycans, can be seen in “cysts” and, in chronic cases, in a band of repair tissue. We studied the localization of hyaluronan, versican, decorin and biglycan in situ in aortas of 21 patients with recent AD, 8 with chronic AD and in 15 control cases. None of these substances was increased in the areas of mucoid “cysts” that possibly contain anomalous material. Similar distributions were seen in normal and dissected aortas: versican and hyaluronan were more prominent in the external half of the medial layer where the dissection usually occurs. Since these molecules play a role in resistance to compression, disorders not detected by our method may be involved in aortic dissection. Hyaluronan was seen adjacent to fibrin at the dissection tear, probably as an early wound repair phenomenon. Biglycan, hyaluronan and mostly versican are seen during advanced repairing. The mucoid deposits may represent various compounds which reflect different disorders in vascular biology.     

The Emerging Role of Leptin Antagonist as Potential Therapeutic Option for Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic relapsing immune-mediated inflammatory disorder that affects millions of people around the world. Leptin is a satiety hormone produced primarily by adipose tissue and acts both centrally and peripherally. Leptin has been shown to play a major role in regulating metabolism, which increases during IBD progression. Leptin mediates several physiological functions including elevated blood pressure, tumorogenesis, cardiovascular pathologies and enhanced immune response in many autoimmune diseases. Recent development of a leptin mutant antagonist that blocks leptin activity raises great hope and opens up new possibilities for therapy in many autoimmune diseases including IBD. To this end, preliminary data from an ongoing study in our laboratory on pegylated leptin antagonist mutant L39A/D40A/F41A (PEG-MLA) treatment shows an inhibition of chronic colitis in IL-10^?/? mice. PEG-MLA effectively attenuates the overall clinical scores, reverses colitis-associated pathogenesis including a decrease in body weight, and decreases systemic leptin level. PEG-MLA induces both central and peripheral leptin deficiency by mediating the cellular immune response. In summary, after blocking leptin activity, the correlative outcome between leptin-mediated cellular immune response, systemic leptin levels, and amount of adipose tissue together may provide new strategies for therapeutic intervention in autoimmune diseases, especially for intestinal inflammation.     

Chondroitin sulphate proteoglycans: extracellular matrix proteins that regulate immunity of the central nervous system

The extracellular matrix (ECM) is a complex network of scaffolding molecules that also plays an important role in cell signalling, migration and tissue structure. In the central nervous system (CNS), the ECM is integral to the efficient development/guidance and survival of neurons and axons. However, changes in distribution of the ECM in the CNS may significantly enhance pathology in CNS disease or following injury. One group of ECM proteins that is important for CNS homeostasis is the chondroitin sulphate proteoglycans (CSPGs). Up-regulation of these molecules has been demonstrated to be both desirable and detrimental following CNS injury. Taking cues from arthritis, where there is a strong anti-CSPG immune response, there is evidence that suggests that CSPGs may influence immunity during CNS pathological conditions. This review focuses on the role of CSPGs in CNS pathologies as well as in immunity, both from a viewpoint of how they may inhibit repair and exacerbate damage in the CNS, and how they are involved in activation and function of peripheral immune cells, particularly in multiple sclerosis. Lastly, we address how CSPGs may be manipulated to improve disease outcomes.     

Hyaluronan and Chondroitin Sulfate Proteoglycans in the Supramolecular Organization of the Mammalian Vitreous Body

The mammalian vitreous gel is a specialized type of highly hydrated extracellular matrix, which is composed of interwoven networks of uronic acid-containing polyanionic macromolecules, (i.e., hyaluronan, versican, and IX collagen) and collagen fibrils. Hyaluronan comprises the vast majority of the uronic acid-containing molecules, which contributes to structure and function of vitreous in at least two ways: its unique biophysical and hydrodynamic properties influence the vitreous homeostasis and biomechanics; it is also a template for assembly of other extracellular macromolecules, for example, versican. The other uronic acid-containing molecules namely versican and IX collagen—two chondroitin sulfate (CS) proteoglycans—occur in the vitreous without significant quantitative variations among different mammalians but with some marked variations on the molecular size and sulfation pattern of their chondroitin sulfate side chains. The contribution of versican and IX collagen (through their protein and their CS side chains) to the supramolecular organization of the vitreous gel is poorly understood. However, versican having the ability to bind hyaluronan via its N-terminal and other binding partners via its C-terminal region can play a crucial role on the structural stability and functionality of the vitreous.     

The Therapeutic Potential of Epigenetics in Autoimmune Diseases

Autoimmune diseases now include over 100 conditions and are estimated to affect over 20 million people in the United States or 5% of the world population with numerous geographical differences coined as geoepidemiology. Further, concordance rates in monozygotic twins are significantly higher compared to dizygotic sets while being significantly below 50% for most autoimmune diseases. These lines of evidence suggest that additional mechanisms are needed to link the individual susceptibility with the proposed chemical and infectious factors in the environment. Epigenetics may well constitute this missing link to include DNA methylation, histone changes, and microRNA which contribute to the epigenome characterizing specific diseases. Importantly, these epigenetic changes may be ideal targets for new personalized treatments as suggested by data in cancer. A number of chemical and physical factors, along with proposed infectious agents or aging, are involved in the etiopathogenesis of autoimmune diseases through epigenetic changes. The most prominent evidence on the association between environment and autoimmunity has been reported in systemic lupus erythematosus, but similar mechanisms were proposed in rheumatoid arthritis, systemic sclerosis, and type 1 diabetes.