Surface charge of endothelial cells estimated from electrophoretic mobility

The surface charge density of endothelial cells was estimated from cell electrophoresis. Cultured endothelial cells from the bovine pulmonary artery were suspended in saline and placed in the lumen of a glass capillary. A voltage was applied across the capillary ends and the velocity imparted to the cells was measured with a microscope. Erythrocyte mobility was also measured. The mobility in (micron/s)/(V/cm) was 0.74 +/- 0.08 for endothelial cells and 1.03 +/- 0.15 for erythrocytes. Charge density in esu/cm2 was calculated as 2.62 x 10(4) and 0.91 x 10(4) for endothelial and red cells, respectively. Removal of sialic acid did not affect the mobility of endothelial cells, but it reduced that of red cells to near zero. Endothelial cell mobility decreased either with ionic strength or calcium concentration. Our results strongly suggest that the surface charge of endothelial cells is dependent on sulfated glycosaminoglycans.     

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.     

Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans

Heparin is known to bind to cultured endothelial cells. This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern. Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium. The increase is apparent as early as 8 h after heparin administration. Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form. Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion. Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change. These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.     

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.     

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells

Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.     

The role of vascular-derived perlecan in modulating cell adhesion,proliferation and growth factor signaling

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α₂β₁ integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.     

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.     

Pregnancy-specific glycoprotein 1 induces endothelial tubulogenesis through interaction with cell surface proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.     

Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells

The synthesis of sulfated glycosaminoglycans was analysed in mouse fibroblasts during the transition from exponential growth to quiescent monolayers. 'Normal' Swiss 3T3 fibroblasts were compared with SV40 transformed 3T3, C6, ST1 and HeLa cells. p-Nitrophenyl-beta-D-xyloside, an artificial acceptor for glycosaminoglycans synthesis, was used as a probe. Exponentially growing 'normal' 3T3 cells synthesized both dermatan sulfate and chondroitin 4-sulfate, retaining the latter and releasing the former to the medium. Upon reaching quiescence these cells switched to retention of dermatan sulfate and release of chondroitin 4-sulfate. SV3T3 cells synthesized several fold less sulfated glycosaminoglycans than 'normal' 3T3. Even though SV3T3 cells are able to synthesize dermatan sulfate, they only retained chondroitin 4-sulfate, never switching to retention of dermatan sulfate. These results indicated that the transition from rapidly proliferating to resting G0 state in normal cells is accompanied by a switch from chondroitin-sulfate rich to dermatan-sulfate-rich cells. This switching was not observed with transformed cells, which are unable to enter the G0 state. Phenylxyloside caused a several fold increase in glycosaminoglycans released to the medium in both cell types, but it did not interfere with either growth rate or cell morphology. Particularly the phenylxyloside treatment led to an increase of more than 10-fold in production of dermatan and chondroitin sulfate by SV3T3, C6, ST1 and HeLa cells. This demonstrated that transformed cells have a high capacity for glycosaminoglycan synthesis. Analysis of enzymatic degradation products of glycosaminoglycans, synthesized in the presence of phenylxyloside, by normal and transformed cells, led to the finding of 4- and 6-sulfated iduronic and glucuronic acid-containing disaccharides. This result indicated that the xyloside causes the synthesis of a peculiar chondroitin sulfate/dermatan sulfate, in both normal and transformed cells.     

Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells

Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and is specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.     

Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (K_i = 2.5 ± 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.     

Heparan sulfate proteoglycans as trastuzumab targets in anoikis-resistant endothelial cells

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.     

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn⁺², HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn²⁺. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.     

The morphogenic properties of oligomeric endostatin are dependent on cell surface heparan sulfate

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells is low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70–80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.     

Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol beta-D-xylose and cis/trans-decahydro-2-naphtol beta-D-xylose. Besides stimulating the synthesis, O-nitrophenyl-beta-D-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.