Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

Bacteroides thetaiotaomicron, a gram-negative anaerobe found in human colons, could utilize chondroitin sulfate, a tissue mucopolysaccharide, as its sole source of carbohydrate. The enzymes responsible for the breakdown of chondroitin sulfate by B. thetaiotaomicron were similar to those produced by Proteus vulgaris and Flavobacterium heparinum and included a lyase (EC 4.2.2.4), which degraded chondroitin sulfate into sulfated disaccharides, sulfatases (EC 3.1.6.4), which removed the sulfate residues, and a glucuronidase, which broke the unsulfated disaccharides into monosaccharide components. Chondroitin sulfate lyase, the first enzyme in the breakdown sequence, was not extracellular. It appeared to be located in the periplasmic space since lyase activity was released by treatment with ethylenediaminetetraacetate and lysozyme. Moreover, sodium polyanethole sulfonate, a high-molecular-weight inhibitor of chondroitin lyase, did not inhibit breakdown of chondroitin sulfate by intact bacteria. The sulfatase and glucuronidase appeared to be intracellular. None of these enzymes was strongly bound to membranes, and none of the steps in the breakdown of chondroitin sulfate was sensitive to oxygen.

Senile plaques of Alzheimer's disease brain contain, in addition to beta amyloid peptide (A beta), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, A beta at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar A beta(1-40) and A beta(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar A beta, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar A beta association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar A beta is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycan.A beta interaction. The ability of proteoglycans to bind fibrillar A beta and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimer's disease.

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.

Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.

Most proteoglycans bear either chondroitin sulfate or heparan sulfate chains linked to serine residues at Ser-Gly attachment sites on the core protein. However, only a fraction of proteins with Ser-Gly sites exhibit glycosaminoglycan chains. A variable proportion of these sites may be glycanated, and an unknown mechanism distinguishes whether these sites are for chondroitin sulfate or heparan sulfate. To evaluate the core protein features that determine whether and where chondroitin sulfate or heparan sulfate will be linked, we have studied mouse syndecan-1, a transmembrane proteoglycan that is invariably glycanated and can contain both chondroitin sulfate and heparan sulfate chains. The extracellular domain of the syndecan-1 core protein contains five Ser-Gly sites, three clustered near its N terminus and two adjacent to the transmembrane domain near its C terminus. We have established the distribution of glycosaminoglycans on these attachment clusters. In contrast to the C-terminal cluster, the N-terminal cluster was always glycanated, suggesting that this domain of the core protein contains sequences responsible for the invariable attachment of glycosaminoglycan chains. Solely chondroitin sulfate was found on the C-terminal cluster. This cluster contains the sequences EGSGE and ETSGE, both estimated to be on the protein surface in a hydrophilic environment. Heparan sulfate was found solely on the N-terminal cluster, which also bears some chondroitin sulfate. This cluster contains the sequences FSGSGTG and DGSGD, the former estimated to be in a hydrophobic pocket and the latter, similar to the sequence on the C-terminal cluster, in an exposed hydrophilic region. This glycosaminoglycan distribution was identical on mouse syndecan-1 produced by either mouse epithelial (NMuMG) or hamster mesenchymal (CHO) cells, suggesting that site-specific attachment of glycosaminoglycans is independent of cell type. These results implicate a cellular mechanism that distinguishes among the potential sites and attaches the correct glycosaminoglycan type unambiguously. Thus, structural elements of the core protein other than the Ser-Gly attachment sites determine if a site will be glycanated and, if so, whether with chondroitin sulfate or heparan sulfate.

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.

The absence of fiber regrowth in the injured mammalian CNS is influenced by several different factors and mechanisms. Besides the nonconducive properties of the glial scar tissue that forms around the lesion site, individual molecules present in CNS myelin and expressed by oligodendrocytes, such as NI-35/NI-250, bNI-220, and myelin-associated glycoprotein (MAG), have been isolated and shown to inhibit axonal growth. Here, we report an additional neurite growth-inhibitory activity purified from bovine spinal cord myelin that is not related to bNI-220 or MAG. This activity can be ascribed to the presence of two chondroitin sulfate proteoglycans (CSPGs), brevican and the brain-specific versican V2 splice variant. Neurite outgrowth of neonatal cerebellar granule cells and of dorsal root ganglion neurons in vitro was strongly inhibited by this myelin fraction enriched in CSPGs. Immunohistochemical staining revealed that brevican and versican V2 are present on the surfaces of differentiated oligodendrocytes. We provide evidence that treatment of oligodendrocytes with the proteoglycan synthesis inhibitors beta-xylosides can strongly influence the growth permissiveness of oligodendrocytes. beta-Xylosides abolished cell surface presentation of brevican and versican V2 and reversed growth cone collapse in encounters with oligodendrocytes as demonstrated by time-lapse video microscopy. Instead, growth cones were able to grow along or even into the processes of oligodendrocytes. Our results strongly suggest that brevican and versican V2 are additional components of CNS myelin that contribute to its nonpermissive substrate properties for axonal growth. Expression of these CSPGs on oligodendrocytes may indicate that they participate in the restriction of structural plasticity and regeneration in the adult CNS.

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.

The scientific and medical community remains skeptical regarding the efficacy of nutrition for osteoarthritis despite their broad acceptation by patients. In this context, this paper systematically reviews human clinical trials evaluating the effects of nutritional compounds on osteoarthritis. We searched the Medline, Embase, and Biosis databases from their inception to September 2005 using the terms random, double-blind method, trial, study, placebo, and osteoarthritis. We selected all peer-reviewed articles reporting the results of randomised human clinical trials (RCTs) in osteoarthritis that investigated the effects of oral interventions based on natural molecules. Studies on glucosamine and chondroitin sulfate were excluded. The quality of the RCTs was assessed with an osteoarthritic-specific standardised set of 12 criteria and a validated instrument. A best-evidence synthesis was used to categorise the scientific evidence behind each nutritional compound as good, moderate, or limited. A summary of the most relevant in vitro and animal studies is used to shed light on the potential mechanisms of action. Inclusion criteria were met by 53 RCTs out of the 2,026 identified studies. Good evidence was found for avocado soybean unsaponifiables. Moderate evidence was found for methylsulfonylmethane and SKI306X, a cocktail of plant extracts. Limited evidence was found for the Chinese plant extract duhuo jisheng Wan, cetyl myristoleate, lipids from green-lipped mussels, and plant extracts from Harpagophytum procumbens. Overall, scientific evidence exists for some specific nutritional interventions to provide symptom relief to osteoarthritic patients. It remains to be investigated whether nutritional compounds can have structure-modifying effects.

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K(d) for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 +/- 4.9 and 23.4 +/- 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized (125)I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of approximately 12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.

Exogenous transforming growth factor beta (TGF-beta 1) was shown in earlier studies to reversibly inhibit mouse mammary ductal growth. Using small plastic implants to treat regions of developing mammary glands in situ, we now report that TGF-beta 1 growth inhibition is associated with an ectopic accumulation of type I collagen messenger RNA and protein, as well as the glycosaminoglycan, chondroitin sulfate. Both macromolecules are normal components of the ductal extracellular matrix, which, under the influence of exogenous TGF-beta 1, became unusually concentrated immediately adjacent to the epithelial cells at the tip of the ductal growth points, the end buds. Stimulation of extracellular matrix was confined to aggregations of connective tissue cells around affected end buds and was not present around the TGF-beta 1 implants themselves, indicating that the matrix effect was epithelium dependent. Ectopic matrix synthesis was specific for TGF-beta 1 insofar as it was absent at ducts treated with other growth inhibitors, or at ducts undergoing normal involution in response to endogenous regulatory processes. These findings are consistent with the matrix-stimulating properties of TGF-beta 1 reported for other systems, but differ in their strict dependence upon epithelium. A possible role for endogenous TGF-beta 1 in modulating a mammary epithelium-stroma interaction is suggested.

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid beta were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples.

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.

In an initial report (Carbonetto, S. T., M. M. Gruver, and D. C. Turner (1982) Science 216: 897-899) we described the use of 2-hydroxyethylmethacrylate (HEMA) in preparing defined culture substrates for studying nerve fiber growth. In those studies fibronectin and collagen were conspicuous, among a variety of HEMA-embedded proteins, in supporting fiber growth of embryonic neurons from chick dorsal root ganglia. Here we further document and extend our preliminary studies, especially with regard to the interaction of growing nerve fibers with fibronectin. HEMA substrates were prepared with proteolytic fragments of fibronectin, each of which had one or more of the functional sites of the intact molecule. Only those fragments of fibronectin that retained the region of the molecule known to mediate myoblast attachment were active in supporting nerve fiber growth. When added in excess to the culture medium, the smallest of the active fragments inhibited fiber growth on substrates that contained intact fibronectin. In culture medium depleted of serum fibronectin, HEMA gels containing collagens purified from connective tissues (types I and III) or from basement membranes (type IV) were about as effective as HEMA substrates containing fibronectin in supporting fiber growth. Nerve fiber growth on collagen substrates proceeded in the absence of fibronectin in the culture medium. Several glycosaminoglycans (heparin, chondroitin sulfate, hyaluronic acid) were ineffective as substrates for fiber growth. Treatment of HEMA/ fibronectin gels with heparin or incorporation of heparin along with fibronectin in the gel profoundly diminished the efficacy of fibronectin in supporting nerve fiber growth. Our studies suggest that the growth of nerve fibers on fibronectin substrates results from direct interaction with a specific portion of the fibronectin molecule and that this interaction can be inhibited by heparin and possibly other glycosaminoglycans.

Monoclonal antibody Cat-301 recognizes a chondroitin sulfate proteoglycan (CSPG) expressed on the extracellular surface of cell bodies and proximal dendrites of specific subsets of neurons in many areas of the mammalian CNS, including the cat visual cortex. The Cat-301 CSPG is first detected at the close of the critical period in development, a period during which the pattern of neuronal activity determines the mature synaptic circuitry and neuronal phenotype. In the cat visual cortex, dark-rearing from birth prolongs the duration of the critical period and attenuates the expression of the Cat-301 antigen, implicating the Cat-301 CSPG in the cellular mechanisms that terminate the period of synaptic plasticity. Because the Cat-301 antigen is expressed on only a limited subset of neurons, we have further examined the molecular heterogeneity among neuronal cell-surface CSPGs and have asked (1) whether other neuronal subsets carry distinct CSPGs and (2) whether the activity-dependent expression of the Cat-301 CSPG is a property generalizable to related cell-surface CSPGs. Here, we report two new monoclonal antibodies, Cat-315 and Cat-316, which together with Cat-301 define a family of at least seven related yet distinct CSPGs. These three antibodies define nonidentical subsets of neurons in the cat visual cortex. The expression of normal levels of these CSPGs is reduced by dark-rearing. Together, these data show that the family of cell-surface CSPGs is molecularly diverse, that different sets of neurons express distinct complements of cell-surface antigens, and that the regulation of CSPG expression by activity may be a general feature of neuronal cell-surface CSPGs.

Protocols for analyzing the fine structure of hyaluronan and chondroitin sulfate using fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone-derivatized hyaluronidase/chondroitinase digestion products were adapted for direct analysis of previously characterized cartilage-derived samples. The chondroitin sulfate disaccharide compositions for fetal and 68 year human aggrecan from FACE analyses were DeltaDi4S (50%), DeltaDi6S (43%), and DeltaDi0S (7%); and DeltaDi4S (3%), DeltaDi6S (96%), and DeltaDi0S (1%), respectively. The nonreducing terminal structures included predominantly 4S-galNAc with minor amounts of 6S-galNAc and Di6S for the fetal aggrecan sample and, in addition, included 4,6S-galNAc in the 68 year aggrecan sample. FACE analysis of a proteinase K digest of rat chondrosarcoma tissue gave an internal disaccharide composition for its chondroitin sulfate chains of DeltaDi0S (7%) and DeltaDi4S (93%) with no DeltaDi6S and DeltaDi4, 6S detected, while DeltaDiHA from hyaluronan was 5% of the total. Analysis of nonreducing terminal structures indicated the presence of 4S-galNAc (51%), galNAc (27%), and Di4S (22%) with no 4,6S-galNAc or Di6S detected. Unexpectedly, FACE analysis detected putative linkage oligosaccharide structures from the chondroitin sulfate chains including both unsulfated (85%) and 4-sulfated (15%) linkage oligosaccharides. Finally, the number averaged chain length estimated from the ratio of the molar fluorescence of the Deltadisaccharides to that of the nonreducing termini or the linkage oligosaccharide structures was calculated as approximately 16 kDa. A tissue glucose concentration of 0.72 g/l was also measured. These results for both samples as determined by FACE analysis were similar to results previously reported, using more labor and time intensive procedures, validating the FACE protocols.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.

The monoclonal antibody O4 has been used to define a biologically distinct stage of the oligodendroglial lineage in vitro. Furthermore, O4+ oligodendroglial progenitors have been found in cell cultures derived from mature tissue, leading to speculation about the presence of oligodendroglial progenitors in the adult central nervous system (CNS). However, the existence of adult oligodendroglial progenitors has yet to be conclusively demonstrated in the intact animal. We have investigated the expression of O4 immunoreactivity in the developing and mature rat forebrain and the relationship of these cells to cells expressing the early oligodendroglial progenitor markers GD3 ganglioside and NG2 chondroitin sulfate proteoglycan, and to differentiated galactocerebroside expressing oligodendroglia. By the day of birth O4+ cells were already widely distributed throughout the formative corpus callosum and increased in number in the white matter and cortical gray matter over the first 2 postnatal weeks. In contrast to cell culture observations, most O4+ cells seen over this period failed to express GD3, although the majority did express NG2. Beginning at postnatal day 4, NG2+/O4-progenitors in the corpus callosum and cerebral cortical gray matter underwent a wave of differentiation into NG2+/O4+ cells and then into galactocerebroside-positive oligodendroglia. Interestingly, not all cells underwent this progression: a population remained as O4+/NG2+ progenitors. Furthermore, this O4+/NG2+ population persisted into adulthood and failed to express their GD3, galactocerebroside, RIP, or myelin basic protein (MBP). They were also distinguishable from glial fibrillary acidic protein+ and glutamine synthetase+ astrocytes and OX-42+ microglia. We therefore propose that these O4+/NG2+ cells represent adult oligodendroglial progenitors hitherto only described in cell culture.

The cellular properties of semilunar cardiac valve leaflets may be more complex than previously assumed. In particular, the cells of the leaflet matrix which are likely critical to proper cusp function are a poorly described population to date. We hypothesized that, similar to the matrix cells of atrioventricular valves, aortic valve leaflet interstitial cells (AoLIC's) possess characteristics of both fibroblasts (matrix secretion) and smooth muscle cells (contraction). Porcine AoLIC's were structurally examined for contractile and stress fiber protein assemblies using transmission electron microscopy and immunocytochemistry. Contractile function in response to vasoactive stimuli was directly assessed using AoLIC's cultured on flame-polymerized silicone, with cell contraction identified by the appearance of wrinkles in the substratum after challenge with each agent. The structural analyses showed cellular microfilaments were often organized into various contractile arrangements including polygonal networks, and that AoLIC's are rich in smooth muscle-specific alpha-actin. Incomplete basal laminae often associated with myofibroblasts were observed. Contraction experiments indicated a responsivity of similar latency, but variable peak and duration to 10(-7) M L-epinephrine, 3.2 x 10(-7) M angiotensin II, 110 microM carbachol, 50 mM KCl, 3.2 x 10(-7) M bradykinin, 110 microM isoproterenol, and 5 x 10(-7) M endothelin I. Soluble and insoluble matrix secretion was confirmed with FITC-conjugated monoclonal antibodies to chondroitin sulfate, fibronectin, and prolyl-4-hydroxylase. These data show that the AoLIC's are best designated as myofibroblasts. The unusual features of the myofibroblast may be central to lifelong aortic leaflet durability.

Observations were made on the behavior of chondrocytes grown under various conditions in vitro. The chondrocytes in 10-day embryonic chick vertebrae were grown as cultures of intact vertebrae, as pellets of chondrocytes liberated from their matrix, and as monodispersed cells plated out on plasma clots. Cartilage matrix was stained metachromatically with toluidine blue. Radioautographs were made of incorporated H(3)-thymidine, H(3)-proline, and S(35)-sulfate to determine the extent of DNA synthesis, collagen synthesis, and chondroitin sulfate synthesis, respectively. Chondrocytes in intact vertebrae or in pellets are rounded and actively synthesizing chondroitin sulfate and collagen. There is little DNA synthesis by cells in either vertebrae or pellets. Chondrocytes grown as monodisperse cells rapidly cease synthesizing cytologically detectable chondroitin sulfate and are induced to synthesize DNA and divide. There is a change in the shape of these chondrocytes from a rounded to a more stellate condition which accompanies the shift in metabolic activity. Conversely, when the cells attain a certain cell density, they reacquire a rounded shape, cease dividing, and again synthesize chondroitin sulfate. Clusters of chondrocytes synthesize more chondroitin sulfate than isolated chondrocytes. It is concluded that most chondrocytes synthesizing chondroitin sulfate do not concurrently synthesize DNA. Interaction between associated chondrocytes is important in inducing and maintaining chondroitin sulfate synthesis in genetically determined chondrocytes. Failure of interaction between chondrocytes leads to DNA synthesis and cell multiplication.

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.