Glycosaminoglycan levels and structure in a mucopolysaccharidosis IIIA mice and the effect of a highly secreted sulfamidase engineered to cross the blood-brain barrier.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A) is a neurodegenerative lysosomal storage disorder caused by the deficiency of sulphamidase enzyme (SGSH) leading to accumulation of heparan sulfate (HS). We quantitatively and structurally characterize primary stored HS and other glycosaminoglycans (GAGs) possibly accumulated through a secondary storage in brain, liver, kidney and lung of MPS IIIA mouse model. This analysis was also performed in MPS IIIA mice upon the intravenous treatment with an engineered human sulphamidase (chimeric hSGSH) capable to increase its secretion from the liver and to cross the blood-brain barrier. MPS IIIA animals showed a huge accumulation of HS, from ~15 up to ~24-times higher than wild type and also of hyaluronic acid (HA) (from 2.5 up to ~5.0-times more) and chondroitin sulfate (CS)/dermatan sulfate (DS) (from ~2 up to ~5-times more) in all studied organs. We also observed a significant increase in the overall HS charge density and in particular of 2-O-sulfation in MPS IIIA mice organs. 8 months after a systemic treatment with an engineered SGSH, the enzyme was highly efficient in the reduction of all accumulated GAGs in liver, brain and lung up to values of wild type mice. On the contrary, even if reduced, GAGs levels still remained significantly elevated in kidney. Overall data obtained by this detailed analysis of GAGs in the different organs of affected and treated animals with chimeric hSGSH may have implications for the evaluation of an effective therapeutic option of MPS IIIA and for the reduction of related neuropathology.

Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

Is osteopontin involved in cutaneous fibroblast activation? Its hypothetical role in scleroderma pathogenesis.

Osteopontin (OPN) is an extracellular matrix protein implicated in bone remodeling, but it presents also pro-inflammatory and pro-fibrotic properties. OPN expression also occurs upon exposure of cells to classical mediators of acute inflammation such as tumor necrosis growth factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), as well as fibrogenic cytokines such as transforming growth factor beta (TGF-beta), although a detailed understanding of these regulatory pathways is still unknown. Plasma OPN levels in both limited and diffuse systemic sclerosis patients (lSSc and dSSc) were statistically higher compared to those of control subjects. Immunohistology demonstrated that high TGF-beta levels, alpha smooth muscle actin (alphaSMA) levels and consequently high OPN levels were found in the affected skin of sclerodermic patients (lSSc and dSSc) compared to levels found in healthy skin. In order to better understand how OPN interferes with the fibrotic process, healthy skin fibroblasts were treated for 24 and 48 hours with bleomycin and with endothelin-1 (ET-1) plus TGF-beta in order to induce the fibrogenesis. After 48 hours of stimulation, healthy treated fibroblasts showed statistically increased alphaSMA levels (index of differentiation into myofibroblasts) and simultaneously statistically increased OPN levels compared to healthy untreated ones. This study demonstrates that OPN levels increase simultaneously with the increasing of alphaSMA levels, therefore it is reasonable to hypothesize that OPN interferes in the pathogenesis of Systemic Sclerosis in the early stage of fibroblast differentiation process.

Macitentan slows down the dermal fibrotic process in systemic sclerosis: in vitro findings.

Systemic sclerosis (or scleroderma) is an autoimmune disease characterized by skin and internal organ fibrosis, caused by microvascular dysfunction. The microvascular damage seems to be a consequence of an endothelial autoimmune response, followed by activation of the inflammatory cascade and massive deposition of collagen. Endothelin-1 (ET-1) contributes to the inflammatory and fibrotic processes by increasing the concentration of pro-inflammatory and pro-fibrotic cytokines, and it is considered one of the most relevant mediators of vascular damage in scleroderma. It is indeed found in very high concentration in serum of sclerodermic patients. Moreover, in these pathological conditions there is an increased expression of ET-1 receptors (ETA and ETB), which mediate the detrimental action of ET-1, and often a change of ETA/ETB ratio. The aim of the present study is to evaluate the in vitro effect of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist, and its major metabolite (ACT-132577) on alpha smooth muscle actin (alphaSMA) expression, evaluated on dermal fibroblasts from healthy subjects and on dermal fibroblasts from lesional and non-lesional skin from sclerodermic patients. The combination of macitentan and its major metabolite reduced the levels of αSMA after 48 h in sclerodermic fibroblasts from lesional skin. No relevant changes in αSMA levels were found in fibroblasts from non-lesional skin, whose behavior is similar to that of dermal fibroblasts from healthy patients.

Expression of RXFP1 in skin of scleroderma patients and control subjects.

OBJECTIVES: Relaxin (RLX) is involved in extracellular matrix and collagen remodelling. The therapeutic role of the circulating isoform RLX-2 as an anti-fibrotic factor in systemic sclerosis (SSc) has been investigated. Several RLX family peptide receptors (RXFPs) are recognized in humans: RLX-2 is a ligand for RXFP1/LGR7 and RXFP2/LGR8. The aim of this study was to define the pattern of expression of LGR7 in different types of human skin cells and to compare normal skin with lesional and unaffected skin from patients with limited SSc (lSSc). METHOD: We analysed RXFP1 immunolocalization on skin biopsies and cultured fibroblasts from lSSc patients and control subjects. Western blot analysis was carried out on fibroblast lysates. RESULTS: RXFP1 showed cytoplasmic localization on skin cells from control subjects and non-lesional skin from lSSc patients: keratinocytes, gland epithelial cells, endothelium, smooth muscle cells, and fibroblasts. Immunogold electron microscopy confirmed a diffuse epithelial cytoplasmic localization of RXFP1. A substantially lower RXFP1 expression was observed in scleroderma skin, with a lack of staining in most cells. Occasional weak reactivity was observed in cultured scleroderma fibroblasts, while control fibroblasts showed a diffuse cytoplasmic immunoreactivity of RXFP1, confirmed by Western blot analysis. CONCLUSIONS: The decreased cellular expression of RLX-2 receptor RXFP1 in scleroderma skin might represent a pro-fibrotic factor and contribute to the substantial inefficacy of RLX treatment in SSc, as reported in the literature. The pathophysiology of the decrease in RXFP1 may be linked to high RLX-2 serum levels previously detected in SSc, but it has yet to be elucidated.

Chondroitin sulfate effect on induced arthritis in rats.

OBJECTIVE: Rodent models of osteoarthritis and rheumatoid arthritis are useful tools to study these disease processes. Adjuvant arthritis (AAR) is a model of polyarthritis widely used for preclinical testing of antiarthritis substances. We report the effect of two different doses of highly purified chondroitin sulfate (CS) pharmaceutical grade in the AAR animal model after oral administration. DESIGN: AAR was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experiments included healthy animals, untreated arthritic animals, arthritic animals having been administered 300 or 900 mg/kg of CS daily, 14 days before AAR induction until the end of the experiment (day 28), arthritic animals having been administered 300 or 900 mg/kg of CS daily, from day 1 until the end of the experiment. RESULTS: CS was capable of significantly reducing the severity of arthritis along with oxidative stress, a consequence of chronic inflammatory processes occurring in AAR. The CS pre-treatment regimen was effective throughout the whole subacute phase, while treatment from day 1 proved effective only in the chronic period. The effects were confirmed by improved total antioxidant status and γ-glutamyltransferase activity. CS administered under a pre-treatment regimen was also able to reduce the production of pro-inflammatory cytokines, C-reactive protein in plasma, phagocytic activity and the intracellular oxidative burst of neutrophils. CONCLUSIONS: CS proved to be effective in slowing down AAR development and in reducing disease markers, thus supporting its beneficial activity as a drug in humans.

Lymphatic vessels in human eyelids: an immunohistological study in dermatochalasis and chalazion.

We investigated lymphatic morphology and expression of endothelin (ET-1) axis molecules in human eyelids affected by an inflammatory state (chalazion) and an age-related degenerative condition (dermatochalasis). Lymphatics were immunohistologically detected by D2-40/LYVE-1 staining. Absorbing lymphatic vessels were localized in papillary dermis and around skin appendages with distinctive morphology. In chalazion, D2-40 reactive flattened lymphatic profiles were compressed by inflammatory infiltrate; in dermatochalasis, large fully opened lymphatics were observed, with a significantly wider total area (lymphatic lumina/200x field; p < 0.05). The lymphatic density (number/200x field) in the two groups was within the same range. Lymphatic dilation is possibly dependent on reduction and fragmentation of the dermal elastic network as well as of oxytalanic fibers in the papillary dermis of dermatochalasis, as shown by Weigert's reaction. Multifunctional peptide ET-1, involved in vasomotion, inflammation and connective proliferation, was faintly and discontinuously localized on lymphatics, as was its type A receptor. In contrast, the consistent expression of type B receptor indicates that lymphatic endothelium is a physiological target for ET-1, whose effects are modulated by multiple pathophysiological conditions. Thus, vasoactive factors play a role in the physiology of richly vascularized eyelids, and therefore, morphofunctional characterization of lymphatic vessels may be useful in suggesting treatment options.

Methylsulfonylmethane and mobilee prevent negative effect of IL-1ß in human chondrocyte cultures via NF-κB signaling pathway.

Nutraceuticals are compounds that serve as nutrition with an easy accessibility and favourable safety profile. Recent studies showed their potential activity on osteoarthritis (OA) inflammation and cartilage metabolism. We investigated the effect of methylsulfonylmethane (MSM) and mobilee in human OA chondrocyte cultures exposed to interleukin (IL)-1ß. OA cartilage was obtained from femoral heads of five patients undergoing total replacement surgery. Chondrocytes were incubated with mobilee (200 and 500 µM) and MSM (2000 and 6000 µM) in presence of IL-1ß (10 ng/mL) and nuclear factor (NF)-κB inhibitor (BAY 11-7082, 1 µM), for 24 and 48 h. Viability and apoptosis were performed by MMT and flow cytometry. The metalloproteinase (MMP)-1,-3,-13 and type II collagen (Col2a1) were analyzed by qRT-PCR and ELISA, and NF-κB activation by immunofluorescence. IL-1ß stimulus determined a significant regulation of survival, apoptotic ratio, as well as of gene expression and serum levels of MMP-1,-3,-13 and Col2a1 in OA chondrocytes compared to baseline. Mobilee and MSM incubation significantly reversed the effect of IL-1ß. IL-1ß significantly induced NF-κB p50 nuclear translocation, which was significantly counteracted by the pre-treatment of OA chodrocytes with the tested compounds. BAY11-7082 significantly modulated MMPs and Col2a1 expression respectively to basal state. Co-treatment of IL-1ß with mobilee, MSM and BAY11-7082 didn't cause changes of MMPs or Col2a1 beyond that caused by each single treatment. We demonstrated that MSM and mobilee have a beneficial effect on OA chondrocytes metabolism, probably due to the modulation of NF-κB pathway, providing a powerful rationale for the use of these substances in OA treatment.

Degradation of high-molar-mass hyaluronan by an oxidative system comprising ascorbate, Cu(II), and hydrogen peroxide: inhibitory action of antiinflammatory drugs--naproxen and acetylsalicylic acid.

Changes in dynamic viscosity of the solutions of a high-molar-mass hyaluronan (HA) were monitored using a rotational viscometer. The degradative conditions generated in the HA solutions by a system comprising ascorbate plus Cu(II) plus H(2)O(2) were studied either in the presence or absence of a drug--naproxen or acetylsalicylic acid. Continual decrease of the dynamic viscosity of HA solution was indicative of the polymer degradation. Addition of the drug retarded/inhibited the HA degradation in a concentration-dependent manner. The characteristics of the fragmented polymers were investigated by FT-IR spectroscopy and by two different liquid chromatographic techniques, namely by size-exclusion chromatography equipped with a multi-angle light scattering photometric detector and by high-performance liquid chromatography connected on-line to a spectrofluorometer.

Lymphatic vessels in human sural nerve: immunohistochemical detection by D2-40.

Lymphatics were detected in the epineurium of the human sural nerve by D-240 immunostaining and confirmed by ultrastructural examination.

Structural and functional modifications of bovine trypsin by heparins. Influence of heparin molecular mass and structure.

Heparins with different structures, charge density and molecular mass were evaluated for their capacity to induce structural and functional alterations of bovine trypsin in a low ionic strength buffer (20 mM Tris-HCl pH 7.4). Unfractionated heparin, and slow and fast moving heparin species increased the fluorescence peak emission of trypsin to the same extent (about +40.0%), whilst partially desulfated and re-N-acetylated heparin with a charge density of 1.47 modified the fluorescence at 330 nm by about +27% and natural heparan sulfate with a sulfate-to-carboxyl ratio < 1 by about +13%. Heparin fractions with narrow polydispersity and the same charge density (produced by chemical depolymerization in the presence of free radicals and further gel-permeation chromatography) having molecular mass lower than about 6000 interact with trypsin to a less extent, even though fractions with molecular mass of about 4500 and 3600 partially retain this property. No modification of fluorescence peak emission of trypsin with heparin was appreciable when the ionic strength of the buffer was increased to 0.3 mM NaCl. An altered ability to reduce cytochrome c was observed for heparins of different charge density; fragments with molecular mass lower than approximately 4000 were also unable to produce superoxide. Trypsin was degraded into fragments by heparin and derivatives after 3 h incubation at 37 degrees C. After electrophoresis in polyacrylamide-gels the trypsin bands disappeared and fragments with lower molecular mass were more evident. This effect depended on the molecular mass of heparin, and was more evident for unfractionated heparin and for a heparin fraction with a molecular mass of 7820. The esterolytic activity of trypsin was inhibited to the same extent by heparin derivatives of various structure and charge density while activity undermet minor changes in the presence of heparin fractions of Mr lower than 4000.

Inhibition of human leukocyte elastase activity by heparins: influence of charge density.

Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography. Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a M(r) greater than about 2000-3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a M(r) of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity.

Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma.

Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.

Characterization of a small chondroitin sulfate proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater (Mollusca Gastropoda).

A small chondroitin sulfate proteoglycan was isolated and partially characterized for core protein and glycosaminoglycan structures from the mucus surrounding embryos in the developmental pouch of Viviparus ater (Mollusca Gastropoda). The protein bearing polysaccharide nature was confirmed by gel-permeation chromatography separation of fractions positive to the uronic acid dosage, 7.5% SDS-PAGE under reducing conditions, sequential staining with alcian blue and ammoniacal silver. Its molecular mass was calculated at about 228,800. After degradation of the galactosaminoglycan components by chondroitinase ABC in the presence of proteinase inhibitors, the molecular mass of the core protein was determined at about 72,200. Treatment of the proteoglycan with keratanase did not modify its electrophoretic migration. Isoelectric focusing of the core protein demonstrated a micro-heterogeneity with the presence of two isoforms with different isoelectric point, pI=8.2 and 6.6, in a ratio of about 1:2.2. The glycosaminoglycan component of the proteoglycan was characterized as chondroitin sulfate with a molecular mass of about 30,750 composed of 5% non-sulfated unsaturated disaccharide, 94% monosulfated disaccharides (4-monosulfated to 6-monosulfated disaccharide ratio of 1.36) and 1. 5% disulfated disaccharides (in particular 1.3% 2,6-disulfated disaccharide) with a sulfate to carboxyl ratio of 0.96. Degradation of the chondroitin sulfate with chondroitinase ABC and ACII permitted to determine a percentage of glucuronic acid of about 78.4. The proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater is formed by a small core protein bearing about five chondroitin sulfate chains (80% chondroitin sulfate/20% dermatan sulfate) with potential function in the developmental processes of molluscs embryos.

The protective effect on Cu2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure.

The effect of various glycosaminoglycans on Cu(2+)- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t(lag)) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t(lag) and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu(2+)- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics.

Physico-chemical properties and the structure of dermatan sulfate fractions purified from plasma after oral administration in healthy human volunteers.

Dermatan sulfate (DS) was administered by oral route in healthy human volunteers. The structure, physico-chemical properties and biological activity of DS purified from human plasma after oral administration were studied and compared with those of native DS. DS extracted and purified from pig mucosa has a relative molecular mass (Mr) of about 23,100 and is composed of about 10% nonsulfated disaccharide, 80% monosulfated disaccharides and about 10% disulfated disaccharides, with a sulfate to carboxyl ratio of 1.00 and a heparin cofactor II (HCII) activity of about 160 units/mg. This native polysaccharide is composed of about 94% iduronic acid. One gram of native DS was orally administered to five healthy human volunteers, and 50 ml of blood were collected after 4 h. DS possibly present in plasma after oral administration was extracted and purified. About 130 +/- 42 micrograms of DS per 50 ml of blood were detected by agarose-gel electrophoresis and DMB assay. This DS shows a broad Mr range. After oral absorption, substantial amounts of species with a Mr of about 7,500 are detected in blood but chains with Mr ranging from 7,500 to 20,000 are also found. Moreover, some very low-Mr species are detected, with a prevalence of disaccharides. After oral absorption, DS is sulfated above all in position 4 of the N-acetyl-galactosamine (60%), with a sulfate to carboxyl ratio of about 0.64, demonstrating that DS is desulfated during or after oral absorption by about 30-40%, A small amount of disulfated disaccharide (in particular 2,4-disulfated, 1.4%) is preserved from catabolic processes, as DS extracted from human plasma is able to inhibit thrombin activity mediated by HCII (about 16 U/mg).

Effects of chondroitin sulfates with different structures on leukemia cells: U-937 cell proliferation and differentiation.

Chondroitin sulfates extracted and purified by different manufacturers were tested to evaluate their effects on proliferation and differentiation processes of U-937 cells. The different chondroitin sulfates were evaluated for purity, structure and physicochemical properties. The three chondroitin sulfates utilized did not present other contaminant glycosaminoglycans and proteins and had about the same relative molecular mass but different disaccharide patterns and charge density. Chondroitin sulfates with small amounts of disulfated disaccharides and low charge density, at 5 micrograms/ml concentration, doubled (about + 133%) cell proliferation in comparison to controls. In contrast, chondroitin sulfates with large amounts of disulfated disaccharides and high sulfate to carboxyl ratio were less effective (about + 15%) in stimulating cell proliferation at low concentration. A decrease of U-937 cell proliferation was observed in proportion to the increased amounts of chondroitin sulfate with low sulfate to carboxyl ratio. On the contrary, chondroitin sulfate with large amounts of disulfated disaccharides produced increased cell proliferation depending on concentration. Small amounts (5-10 micrograms/ml) of chondroitin sulfates with low charge density reduced the differentiative process of U-937 cells. Chondroitin sulfate with large amounts of disulfated disaccharides and high charge density seemed to be able to produce a significant decrease of differentiative processes only at very high concentrations (1000 micrograms/ml). These contrasting effects of chondroitin sulfates with different disaccharide patterns (and structure) and charge density on a leukemia cell line could help to explain the regulation of proliferative and/or differentiative processes of hemopoietic cells. This is underlined by the changes of types, physicochemical properties and structure of glycosaminoglycans induced by different extracellular factors and agents.

Differential activity of glycosaminoglycans on colony-forming cells from cord blood. Preliminary results.

Heparin, heparan sulfate and chondroitin sulfate were evaluated for their possible role on proliferation and differentiation of hematological precursor cells from cord blood. For these purposes, different concentrations of glycosaminoglycans were added to methyl-cellulose in colony assay performed with human cord blood derived cells. A volume of 10 microg/ml heparin induces a significant increase of both granulocyte-monocyte and granulocyte colonies, and a decrease of erythroid-colonies, more evident in the presence of 100 microg/ml. Heparan sulfate-treatment induces a significant increase of all granulocyte-monocyte colonies derived from CFU-granulocyte-monocyte, CFU-granulocyte and CFU-monocyte precursors. A significant decrease of multipotent cells was also observed. On the other hand, chondroitin sulfate induces an increase of granulocyte-colonies and a decrease of erythroid-colonies. Glycosaminoglycans with different structure may be useful to increase the number of specific colonies. The selective and differential binding of glycosaminoglycans with several growth factors and the regulation of their activities is discussed.

Adsorption of glycosaminoglycans onto coral--a new possible implant biomaterials for regeneration therapy.

The adsorption of glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, highly sulfated chondroitin sulfate, chondroitin sulfate, and hyaluronan) onto coral has been investigated. Granules of natural coral of specific diameter, between 100 and 500 microm, having high content of calcium (> 98%) and a homogeneous surface adsorb glycosaminoglycans with different capacity. Heparin (maximum adsorption 1.29 +/- 0.10 mg/20 mg of coral, 6.45% w/w) is adsorbed more than highly sulfated chondroitin sulfate species (maximum adsorption of 0.90 +/- 0.06 mg/20 mg of coral, 4.50% w/w), chondroitin sulfate (maximum adsorption of 0.72 +/- 0.06 mg/20 mg of coral, 3.60% w/w), dermatan sulfate (maximum adsorption of 0.70 +/- 0.06 mg/20 mg of coral, 3.50% w/w) and heparan sulfate (maximum adsorption of 0.72 +/- 0.07 mg/20 mg of coral, 3.60% w/w). Hyaluronan is not adsorbed onto granules of coral. The percentage adsorption of polyanions onto coral depends mainly on their charge density, with sulfate groups being more important than carboxyl groups. This study found no evidence that iduronic acid is more important than glucuronic acid and no role of molecular mass on the adsorption of polysaccharides onto coral was found. The adsorption of glycosaminoglycans is driven by electrostatic interactions with calcium sites of coral that are dependent on pH and blocked in the presence of large amounts of salt. Due to these peculiar properties, the combination of granules of natural coral with glycosaminoglycans makes this material potentially useful in osseointegration in bone metabolism or periodontal therapy.