Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.     

The mode of action of heparan and dermatan sulfates in the regulation of hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHO pgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met forms in vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

The interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides

Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.     

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or on the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 μg/liver per day, more than 90% of which was secreted into the medium within 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate and hyaluronic acid.     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

Dermatan sulfate binds and potentiates activity of keratinocyte growth factor (FGF-7)

FGF-7 is induced after injury and induces the proliferation of keratinocytes. Like most members of the FGF family, the activity of FGF-7 is strongly influenced by binding to heparin, but this glycosaminoglycan is absent on keratinocyte cell surfaces and minimally present in the wound environment. In this investigation we compared the relative activity of heparan sulfate and chondroitin sulfate B (dermatan sulfate), glycosaminoglycans that are present in wounds. A lymphoid cell line (BaF/KGFR) containing the FGF-7 receptor (FGFR2 IIIb) was treated with FGF-7 and with various glycosaminoglycans. FGF-7 did not support cell proliferation in the absence of glycosaminoglycan or with addition of heparan sulfate or chondroitin sulfate A/C but did stimulate BaF/KGFR division in the presence of dermatan sulfate or highly sulfated low molecular weight fractions of dermatan. Dermatan sulfate also enabled FGF-7-dependent phosphorylation of mitogen-activated protein kinase and promoted binding of radiolabeled FGF-7 to FGFR2 IIIb. In addition, dermatan sulfate and FGF-7 stimulated growth of normal keratinocytes in culture. Thus, dermatan sulfate, the predominant glycosaminoglycan in skin, is the principle cofactor for FGF-7.     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Characterization of glycosaminoglycans stored in mucopolysaccharidosis III A: evidence for a generally occuring degradation of heparan sulfate by endoglycosidases.

The characterization of intracellularly stored glycosaminoglycans from organs of a patient suffering from mucopolysaccharidosis III A (Sanfilippo A disease) is described. Both heparan sulfate and galactosamine-containing glycosaminoglycans (chondroitin sulfate, dermatan sulfate) are accumulated in the liver, whereas in the other organs (spleen, kidney, heart, cerebrum, cerebellum) heparan sulfate is almost the only glycosaminoglycan stored. It is shown by [3H]NaBH4 reduction and subsequent identification of the 3H-labelled sugar alcohols that heparan sulfate is degraded in all organs by at least two endoglycosidases, an endoglucuronidase and an endoglucosaminidase, to fragments of low molecular weight (Mr approximately 2 000-6 600).     

Urinary excretion of glycosaminoglycans and albumin in experimental diabetes mellitus

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.     

Age-related changes in the content and composition of glycosaminoglycans isolated from the mouse skeletal muscle: normal and dystrophic conditions

Glycosaminoglycans were isolated from the skeletal muscle of either normal or dystrophic mice aged from 3 to 18 weeks. The glycosaminoglycan content of the normal muscle, based on the tissue weight, decreased slightly during the period from 3 to 10 weeks, and remained almost unchanged after 10 weeks. The major glycosaminoglycan in normal muscle was hyaluronate, the relative amount of which increased slightly (from 70% to 80%) with age. Both dermatan sulfate and heparan sulfate were also obtained. The relative amounts of these sulfated glycosaminoglycans tended to decrease with age. On the other hand, the glycosaminoglycan content of the dystrophic muscle was higher than that of normal muscle even at 3 weeks. The proportion of hyaluronate was almost constant (about 65%) throughout the age range examined. The relative amount of dermatan sulfate increased from 20% to 30% with a compensatory decrease in the amount of heparan sulfate. Further, the incorporation of [35S]sulfate into glycosaminoglycans by the dystrophic muscle was reduced to about 60% of the normal. These differences in glycosaminoglycan composition and [35S]sulfate incorporation between the normal and the dystrophic muscles may be related to the progressive muscular dysfunction seen in this disease.     

Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.     

Binding and internalization of exogenous glycosaminoglycans in weakly and highly metastatic rhabdomyosarcoma cells

The fate of exogenous glycosaminoglycans in cultures of strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cells was studied. The time course and concentration dependence of binding and internalization of the radiolabeled sulfated glycosaminoglycans were determined. Weakly metastatic cells took up heparin, heparan and dermatan sulfates into their pericellular compartment at a higher rate than the strongly metastatic RMS 0 cells. The RMS 8 cells exhibited about two times more binding sites for these iduronic acid containing glycosaminoglycans, and internalized higher amounts of them than the RMS 0 cells. The uptake of the chondroitin sulfate into the peri- and intracellular compartments of both cell types was about 5-15% of that of the other glycosaminoglycans studied. The specificity of displacement of the pericellular heparin and dermatan sulfate by the unlabeled glycosaminoglycans indicates the involvement of specific structural features of the polysaccharide chains in the interactions of glycosaminoglycans with the surface of rhabdomyosarcoma cells, beside ionic forces due to the polyanionic character of the glycosaminoglycans. Heparin and heparan sulfate degradation products, mainly large oligosaccharides, were recovered from the surface of RMS 0 cells but were absent on the surface of the RMS 8 cells. About 30% of the internalized heparin and heparan sulfate was present in the partially degraded form in both cell types. Oligosaccharides derived from glycosaminoglycans were not released into the medium. The decrease in the amount of iduronic acid containing glycosaminoglycans internalized by the highly invasive cells seems to be correlated with an increased cell-associated degradation and with an apparent loss of glycosaminoglycan binding sites on the cell surface.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.