Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind beta-amyloid protein (Abeta) 1-40 and 1-42, but are also potent enhancers of Abeta fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of Abeta fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on Abeta 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of Abeta fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of Abeta 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of Abeta amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) Abeta amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of Abeta amyloid deposition in Alzheimer's disease brain may also participate in the enhancement of Abeta amyloidosis.

Surgical options for cartilage resurfacing may be significantly improved by advances and application of biomaterials that direct tissue repair. A poly(ethylene glycol) diacrylate (PEGDA) hydrogel was designed to support cartilage matrix production, with easy surgical application. A model in vitro system demonstrated deposition of cartilage-specific extracellular matrix in the hydrogel biomaterial and stimulation of adjacent cartilage tissue development by mesenchymal stem cells. For translation to the joint environment, a chondroitin sulfate adhesive was applied to covalently bond and adhere the hydrogel to cartilage and bone tissue in articular defects. After preclinical testing in a caprine model, a pilot clinical study was initiated where the biomaterials system was combined with standard microfracture surgery in 15 patients with focal cartilage defects on the medial femoral condyle. Control patients were treated with microfracture alone. Magnetic resonance imaging showed that treated patients achieved significantly higher levels of tissue fill compared to controls. Magnetic resonance spin-spin relaxation times (T(2)) showed decreasing water content and increased tissue organization over time. Treated patients had less pain compared with controls, whereas knee function [International Knee Documentation Committee (IKDC)] scores increased to similar levels between the groups over the 6 months evaluated. No major adverse events were observed over the study period. With further clinical testing, this practical biomaterials strategy has the potential to improve the treatment of articular cartilage defects.

Cytotactin is an extracellular matrix protein that is involved in neuron-glia adhesion and is found in both neural and nonneural sites. It is synthesized by glia but not by neurons. In this study, we have examined the binding of cytotactin to a variety of extracellular matrix components using uniform microscopic beads (Covaspheres) that could be labeled and then linked to purified molecules. Cytotactin-coated beads bound well to neurons, and this binding was strongly inhibited by anti-cytotactin antibodies but not by anti-neural cell adhesion molecule (anti-N-CAM) antibodies. In contrast, the binding of N-CAM-coated beads to neurons was inhibited by anti-N-CAM antibodies and not by anti-cytotactin antibodies. To identify a neuronal ligand for cytotactin, we tested several molecules for their ability to block the binding of cytotactin-coated beads to cells. A proteoglycan-containing fraction that copurified with cytotactin from brain extracts strongly inhibited binding, whereas neither a heparan sulfate proteoglycan from Engelbreth-Holm-Swarm tumor cells nor soluble cytotactin itself had a significant inhibitory effect. The neural proteoglycan also inhibited the binding of cytotactin-coated beads to fibroblasts. Digestion with chondroitinase, heparitinase, and hyaluronidase as well as immunological analyses suggested that the predominant species in the active fraction was a chondroitin sulfate proteoglycan with a Mr280,000 core protein bearing HNK-1 antigenic determinants and also indicated that hyaluronic acid was present in this fraction. In experiments on in vitro synthesis, it was found that the proteoglycan was synthesized in culture by embryonic chicken brain tissue but not by embryonic chicken glial cells. A series of binding experiments was performed on appropriately derivatized beads to confirm that the proteoglycan is a ligand for cytotactin and to check for the possibility that other extracellular matrix proteins might interact with one or the other member of this binding couple. Proteoglycan-coated beads and cytotactin-coated beads coaggregated readily. The aggregation was inhibitable by anti-cytotactin antibodies, soluble cytotactin, or soluble proteoglycan. Addition of laminin inhibited the binding of cytotactin-coated beads to proteoglycan-coated beads or to cells; this is consistent with data indicating that laminin interacts with a component of the proteoglycan-containing fraction. In contrast, fibronectin bound to cytotactin, but it did not bind to proteoglycan or interfere with the binding of cytotactin to proteoglycan. The results of this study are in accord with the idea that the functions of extracellular matrix components during neural and nonneural development may be modulated both by competition for shared cell surface receptors and by a network of molecular interactions among the matrix components themselves.

The cell adhesion regulating extracellular matrix glycoprotein, thrombospondin (TSP), causes a loss of focal adhesion plaques from spread endothelial cells and fibroblasts. To localize the site on TSP that has focal adhesion-labilizing activity, we initially tested proteolytic fragments of TSP for activity. The heparin-binding fragment has significant focal adhesion-labilizing activity, whereas the nonheparin-binding 140-kDa fragment had no significant activity. These results were consistent with previous data that showed that both a monoclonal antibody to the heparin-binding domain of TSP (A2.5) and heparin neutralized TSP activity. Peptides from putative heparin binding sequences of the amino-terminal heparin-binding domain of TSP were synthesized and tested for their ability to cause loss of focal adhesions. The hep I peptide (amino acids 17-35) caused maximal loss of focal adhesions and was active at 0.1 microM, whereas peptide hep II (74-95) and peptide hep III (170-189) were inactive. The activity of the hep I peptide was neutralized by the addition of heparin and heparan sulfate but not by chondroitin sulfate. The basic amino acids in the hep I sequence appear to be required for focal adhesion-labilizing activity, because modification of the lysine residues at amino acids 24 and 32 rendered the peptide completely inactive. In addition, a peptide from the analogous sequence of mouse TSP 2, in which basic residues are conserved, was nearly as active as hep I from TSP1. These data show that the anti-adhesive activity of TSP is conserved in both TSP1 and TSP2 and that the active site is located in a 19-amino acid sequence in the heparin-binding domain of TSPs.

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine disaccharide units {[HexAbeta/alpha(1-3)GalNAcbeta(1-4)](n)()}. CS chains are polydisperse with respect to chain length, sulfate content, and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC-MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d(0)) or heavy (d(4)) form of 2-anthranilic acid (2-AA). Excess reagent was removed, and mixtures of the CS standard (d(0)) and unknown (d(4)) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometric detection in the negative ion mode. Tandem mass spectra were acquired, and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. The high accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization.

Various forms of heparan sulfate proteoglycan were solubilized from the mouse Engelbreth-Holm-Swarm (EHS) sarcoma by extraction with 0.5 M NaCl, collagenase digestion and extraction with 4 M guanidine. They could be separated into high (greater than or equal to 1.65 g/ml) and low (1.38 g/ml) buoyant density variants. The high-density form from the NaCl extract and collagenase digest had Mr = 130000 and So20,W = 4.5 S and contained 4-10% protein, indicating Mr = 5 000-12 000 for the protein core. This proteoglycan exhibited polydispersity as shown by rotary shadowing electron microscopy and ultracentrifugation. An average molecule consisted of four heparan sulfate chains (Mr = 29 000) each with a length of 32 +/- 10 nm. The low-density form (Mr about 400 000) could not be completely purified and contained about 50% protein. As shown by radioimmunoassay, the various proteoglycans shared similar protein cores. Labeling of the tumor in vivo or in vitro demonstrated preferential incorporation of radioactive sulfate in the high-density form. The high-density proteoglycan interacted in affinity chromatography by virtue of its heparan sulfate chains with laminin, fibronectin, the globular domain NC1 and the triple helix of collagen IV. These interactions were abolished at moderate concentrations of NaCl (0.1-0.2 M) and in the presence of heparin, chondroitin sulfate or dextran sulfate. Interactions with the globule NC1 could also be demonstrated by velocity band centrifugation in sucrose gradients and a binding constant of about 10(6) M-1 was derived.

Previous studies have suggested that heparin-like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell-specific, dose-dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 micrograms/ml; a half-maximal effect was observed at 1 microgram/ml. Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin-like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.

As a neurotropic virus, human immunodeficiency virus type 1 (HIV-1) invades the brain and causes severe neuronal, astrocyte, and myelin damage in AIDS patients. To gain access to the brain, HIV-1 must migrate through brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB). Given that BMECs lack the entry receptor CD4, HIV-1 must use receptors distinct from CD4 to enter these cells. We previously reported that cell surface proteoglycans serve as major HIV-1 receptors on primary human endothelial cells. In this study, we examined whether proteoglycans also impact cell-free HIV-1 invasion of the brain. Using an artificial BBB transmigration assay, we found that both heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are abundantly expressed on primary BMECs and promote HIV-1 attachment and entry. In contrast, the classical entry receptors, CXCR4 and CCR5, only moderately enhanced these processes. HSPGs and CSPGs captured HIV-1 in a gp120-dependent manner. However, no correlation between coreceptor usage and transmigration was identified. Furthermore, brain-derived viruses did not transmigrate more efficiently than lymphoid-derived viruses, suggesting that the ability of HIV-1 to replicate in the brain does not correlate with its capacity to migrate through the BBB as cell-free virus. Given that HIV-1-proteoglycan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups in proteoglycans, HIV-1 may exploit these interactions to rapidly enter and migrate through the BBB to invade the brain.

After injury to the mammalian central nervous system (CNS), neurons are not able to regenerate their axons and recovery is limited by restricted plasticity. Axon regeneration is inhibited by the presence of the various inhibitory molecules, including chondroitin sulfate proteoglycans (CSPGs) which are upregulated around the injury site. Plasticity after the end of critical periods is restricted by extracellular matrix changes, particularly the formation of CSPG-containing perineuronal nets. Enzymatic removal of chondroitin sulfate (CS) chains with chondroitinase ABC promotes axon regeneration and reactivates plasticity. This review details the structures and properties of the different CSPGs in the normal and damaged CNS, the use of the enzyme chondroitinase ABC to promote neural regeneration and plasticity, and discusses mechanisms of action and possible therapeutic uses of this enzyme.

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity-purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down-regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.

We report a carbohydrate microarray-based approach for the rapid, facile analysis of glycosaminoglycan-protein interactions. The key structural determinants responsible for protein binding, such as sulfate groups that participate in the interactions, were elucidated. Specificities were also readily compared across protein families or functional classes, and comparisons among glycosaminoglycan subclasses provided a more comprehensive understanding of protein specificity. To validate the approach, we showed that fibroblast growth factor family members have distinct sulfation preferences. We also demonstrated that heparan sulfate and chondroitin sulfate interact in a sulfation-dependent manner with various axon guidance proteins, including slit2, netrin1, ephrinA1, ephrinA5, and semaphorin5B. We anticipate that these microarrays will accelerate the discovery of glycosaminoglycan-binding proteins and provide a deeper understanding of their roles in regulating diverse biological processes.

Brevican is a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the central nervous system. The susceptibility of brevican to digestion by matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9, -10, and -13 and membrane type 1 and 3 MMPs) and aggrecanase-1 (ADAMTS4) was examined. MMP-1, -2, -3, -7, -8, -10, and -13 degraded brevican into a few fragments with similar molecular masses, whereas the degradation products of aggrecanase-1 had apparently different sizes. NH(2)-terminal sequence analyses of the digestion fragments revealed that cleavages of the brevican core protein by these metalloproteinases occurred commonly within the central non-homologous domain. MMP-1, -2, -3, -7, -8, -10, and -13 preferentially attacked the Ala(360)-Phe(361) bond, whereas aggrecanase-1 cleaved the Glu(395)-Ser(396) bond, which are similar to the cleavage sites observed with cartilage proteoglycan (aggrecan) for the MMPs and aggrecanase-1, respectively. These data demonstrate that MMP-1, -2, -3, -7, -8, -10, and -13 and aggrecanase-1 digest brevican in a similar pattern to aggrecan and suggest that they may be responsible for the physiological turnover and pathological degradation of brevican.

Chondroitin-sulfate containing proteoglycan (CSPG) of the extracellular matrix (ECM) was visualized in chick tissues and cell cultures with a monoclonal antibody, CS-56. Cultured cells of various origins contained dense punctate layers of CSPG on both the substrate and the cell surface, as determined by immunofluorescent and immunogold staining. Under culture conditions the CSPG-containing matrix was usually excluded from stable cell-to-substrate focal contacts. The substrate-attached CSPG exhibited remarkable chemical stability but could be successfully removed by pronase or chondroitinases ABC and AC. Incubation of living cells with CS-56 antibodies resulted in the clustering of surface CSPG into patches, indicating that the surface-bound CSPG is free to move laterally along the plasma membrane. The unique properties of the CSPG-containing ECM revealed by CS-56 antibodies and their relationships to specific types of cell contacts are discussed.

Infections with Plasmodium falciparum during pregnancy lead to the accumulation of parasitized red blood cells (infected erythrocytes, IEs) in the placenta. IEs of P. falciparum isolates that infect the human placenta were found to bind immunoglobulin G (IgG). A strain of P. falciparum cloned for IgG binding adhered massively to placental syncytiotrophoblasts in a pattern similar to that of natural infections. Adherence was inhibited by IgG-binding proteins, but not by glycosaminoglycans or enzymatic digestion of chondroitin sulfate A or hyaluronic acid. Normal, nonimmune IgG that is bound to a duffy binding-like domain beta of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) might at the IE surface act as a bridge to neonatal Fc receptors of the placenta.

To clone novel brain proteoglycans, we employed a strategy based on polyclonal antisera that recognize multiple proteoglycan core proteins. By using an antiserum raised against a fraction enriched for proteoglycans, we isolated three groups of cDNAs from a bovine brain lambda gt11 library. One of the cDNA groups has been fully sequenced and shown to encode a novel proteoglycan core protein of the aggrecan/versican family. This proteoglycan, named brevican, carries chondroitin sulfate chains, and, like other members of the family, contains a hyaluronic acid-binding domain in its N-terminal region, an epidermal growth factor-like repeat, a lectin-like and a complement regulatory protein-like domains in its C-terminal region. In contrast, the central region of brevican is much shorter than that of aggrecan, versican, or neurocan, and shows little homology with these proteoglycans. Brevican core protein exists as a 145 kDa full-length form and a 80 kDa N terminally truncated form. A significant amount of brevican devoid of any glycosaminoglycan chains is present in the brain, indicating that brevican is a "part-time" proteoglycan. Northern blot analysis reveals that a single 3.3-kilobase brevican transcript is present predominantly in the brain, and that it is expressed in primary cerebellar astrocytes but not in neurons.

Odontoblasts are responsible for formation of predentin, which is transformed to dentin when apatite crystals are formed and the fibrillar matrix becomes mineralized. Odontoblasts are specialized cells that synthesize and secrete a unique set of non-collagenous proteins (NCPs), as well as the collagenous matrix largely comprised of type I collagen. The NCPs consist of dentin specific and mineralized tissue specific proteins, as well as other proteins that are found in a variety of tissues. Three dentin specific proteins have been recognized to date: dentin phosphoprotein (DPP), also called phosphophoryn, AG1 (dentin matrix protein 1, Dmp1) and dentin sialoprotein (DSP). DPP appears to be made by odontoblasts and appears at the mineralization front within a short time. It may be secreted via odontoblastic processes. DPP binds to collagen and potentially initiates formation of apatite crystals. A second DPP function appears to be to bind to the 100 face of growing apatite crystals and to inhibit or slow their growth; thus, DPP may play a dual role by initiating mineralization and then affecting the crystal growth and perhaps the habit of the crystals. Although no function has been ascribed to AG1 or DSP, they should prove to be important markers for the odontoblast phenotype. A recent unique finding is that two separate genes appear to code for more than one DSP mRNA; other transcripts may result from differential splicing. Examples of mineralized tissue specific proteins expressed by osteoblasts as well as odontoblasts are bone sialoprotein (BSP) and osteocalcin. Some NCPs expressed by osteoblasts, odontoblasts and several other tissues include osteopontin (OPN) and the chondroitin sulfate containing proteoglycans, decorin and biglycan. We propose that characterization of odontoblasts in tissues and cultures should rely upon utilization of sets of markers for the above NCPs and their mRNAs. Similar approaches are commonly used in investigations on osteoblasts. Finally, dentin (like bone) contains other molecules such as growth factors, and serum derived proteins, found within the matrix; no functional significance has yet been placed upon this finding. Future experiments should focus upon the elucidation of the three dimensional structures of the collagenous fibrillar network and of the NCPs to determine the relationships to mineralization. The role played by odontoblasts in controlling extracellular events, such as by selective secretory routes, will require careful exploration.

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatment in vitro induced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.

Chondroitin sulfate proteoglycans (CSPGs) are upregulated in the CNS after injury and participate in the inhibition of axon regeneration mainly through their glycosaminoglycan (GAG) side chains. In the present study, we have identified a new way to alleviate the inhibition of axonal regeneration by CSPG GAGs. We have successfully decreased the amount of CSPG GAG produced by astrocytes by targeting chondroitin polymerizing factor (ChPF), a key enzyme in the CSPG biosynthetic pathway. Using short interfering RNA (siRNA), we reduced ChPF mRNA levels by 70% in both the Neu7 astrocyte cell line and primary rat astrocytes. This reduction leads to a decrease in ChPF protein levels and a reduced amount of CSPG GAG chains in the conditioned media (CM) of these cells. Secretion of neurocan by primary astrocytes and NG2 core protein by Neu7 cells transfected with ChPF siRNA is not decreased, suggesting that inhibiting GAG chain synthesis does not affect core protein trafficking from these cells. CM from siRNA-treated Neu7 cells is a less repulsive substrate for axons than CM from control cells. In addition, axonal outgrowth from cerebellar granule neurons is increased on or in CM from ChPF siRNA-treated Neu7 cells. These data indicate that targeting the biosynthesis of CSPG GAG is a potentially new therapeutic avenue for decreasing CSPG GAG produced by astrocytes after CNS injury.

The development of the vertebrate neural crest presents a particularly challenging problem in pattern formation. Several studies have revealed that a population of neural crest cells penetrates the sclerotomal mesenchyme of the somite only in its rostral half. In a search for molecular correlates of this pattern, we have observed that cytotactin and a chondroitin sulfate proteoglycan, two interactive extracellular matrix molecules, show a specialized distribution within the sclerotome. Cytotactin was localized in the rostral half of the sclerotome at about the time of neural crest cell invasion. The proteoglycan was initially diffuse throughout the sclerotome but became restricted to the caudal half after the appearance of cytotactin and invasion of neural crest cells in the rostral half. These distributions were crest cell-independent; they occurred on the same schedule even when all crest cells were removed by surgical extirpation of the neural tube. Furthermore, in tissue culture, somite cells synthesized high levels of both molecules. In vitro, crest cells rounded up in the presence of these molecules and cell migration assays revealed that neither cytotactin nor proteoglycan alone was as good a substratum for crest cell migration as fibronectin. In combination with fibronectin, however, cytotactin or proteoglycan only restricted cell movement but did not prevent it. Taken together, these observations support the hypothesis that cytotactin and the chondroitin sulfate proteoglycan may contribute to pattern formation during embryogenesis by means of their site-restricted distribution, their ability to alter migration on other substrates such as fibronectin, and their ability to induce cell-surface modulation.

To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other glycosaminoglycans. Enzymatic removal of cell surface glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.