Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs(∗)163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals' fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.

We have revealed that in Caenorhabditis elegans, non-sulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, whereas heparan sulfate is essential for embryonic morphogenesis in the later stages of development. To clarify the roles of chondroitin sulfate and heparan sulfate in early embryogenesis in mammals, we generated glucuronyltransferase-I (GlcAT-I) knock-out mice by gene targeting. GlcAT-I is an enzyme required for the synthesis of both chondroitin sulfate and heparan sulfate. Here we report that mice with a deletion of GlcAT-I showed remarkable reduction of the synthesis of chondroitin sulfate and heparan sulfate and embryonic lethality before the 8-cell stage because of failed cytokinesis. In addition, treatment of wild-type 2-cell embryos with chondroitinase ABC had marked effects on cell division, although many heparitinase-treated embryos normally developed to blastocysts. Taken together, these results suggest that chondroitin sulfate in mammals, as with non-sulfated chondroitin in C. elegans, is indispensable for embryonic cell division.

A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.

Chondroitin sulfate (CS) dermatan sulfate (DS), and CS/DS hybrid chains are biologically active like heparan sulfate, and structurally the most complex species of the glycosaminoglycan family along with heparan sulfate. They exist at the cell surface and extracellular matrix in the form of proteoglycans. They function as regulators of functional proteins such as growth factors, cytokines, chemokines, adhesion molecules, and lipoproteins through interactions with the ligands of these proteins via specific saccharide domains. Structural alterations have been often implicated in pathological conditions, such as cancer and atherosclerosis. Recent microsequencing of CS/DS oligosaccharides that bind growth factors, such as pleiotrophin, and various monoclonal antibodies against CS/DS, have revealed a considerable number of unique oligosaccharide sequences. This review focuses on recent advances in the study of the structure-function relation of CS, DS and their hybrid chains in physiological and pathological conditions.

Chondroitin sulfate proteoglycans (CSPGs) play a pivotal role in many neuronal growth mechanisms including axon guidance and the modulation of repair processes following injury to the spinal cord or brain. Many actions of CSPGs in the central nervous system (CNS) are governed by the specific sulfation pattern on the glycosaminoglycan (GAG) chains attached to CSPG core proteins. To elucidate the role of CSPGs and sulfated GAG chains following traumatic brain injury (TBI), controlled cortical impact injury of mild to moderate severity was performed over the left sensory motor cortex in mice. Using immunoblotting and immunostaining, we found that TBI resulted in an increase in the CSPGs neurocan and NG2 expression in a tight band surrounding the injury core, which overlapped with the presence of 4-sulfated CS GAGs but not with 6-sulfated GAGs. This increase was observed as early as 7 days post injury (dpi), and persisted for up to 28 dpi. Labeling with markers against microglia/macrophages, NG2+ cells, fibroblasts, and astrocytes showed that these cells were all localized in the area, suggesting multiple origins of chondroitin-4-sulfate increase. TBI also caused a decrease in the expression of aggrecan and phosphacan in the pericontusional cortex with a concomitant reduction in the number of perineuronal nets. In summary, we describe a dual response in CSPGs whereby they may be actively involved in complex repair processes following TBI.

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re-growth across the gradient. The use of selective enzyme inhibitors revealed that MMP-2 enables immature astrocytes to cross the proteoglycan rim. The bridge-building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC-induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced.

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G ->> A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.

A proteoglycan was isolated from ascites fluid produced by a rat yolk sac tumor. The glycosaminoglycan chains of the proteoglycan are all sensitive to digestion with chondroitinase ABC and about 90% are sensitive to chondroitinase AC. The proteoglycan contains 5% protein. Amino acid analysis revealed a high content of serine and glycine which together constitute 37% of the amino acids. Glutamic acid (glutamine) and aspartic acid (asparagine) are also abundant. Galactosamine accounts for 97% of the hexosamine and the remainder is glucosamine. These characteristics indicate that the glycosaminoglycan side chains of this proteoglycan are predominantly chondroitin sulfate with a smaller amount of dermatan sulfate. Antibodies to the proteoglycan were prepared by immunization of a rabbit with purified alkali-treated proteoglycan. Affinity-purified antibodies from the antiserum immunoprecipitated (35S)sulfate-labeled radioactivity from culture media of the yolk sac tumor cells known to contain chondroitin sulfate proteoglycan. This binding was inhibited by the intact purified proteoglycan but not by proteoglycan treated with papain, suggesting dependence of the reactivity of the antibodies on integrity of the protein part of the proteoglycan. Immunofluorescence of the cultured yolk sac tumor cells revealed localization of immune reactive proteoglycans at the cell surface.

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 A. The overall structure reveals an unusual "beads-on-a-string"-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K(d), of about 10 nM was determined for the interaction between cathepsin K and C4-S.

Versican, a large extracellular matrix proteoglycan accumulates in tumor stroma and plays a key role in both malignant transformation and tumor progression. Increased versican expression has been observed in a wide range of malignant tumors, and has been associated with both cancer relapse and poor patient outcomes in breast, prostate, and many other cancer types. Through negatively-charged chondroitin and dermatan sulfate side chains or interactions of the G1 and G3 domains, versican is able to regulate many cellular processes including cell adhesion, proliferation, apoptosis, migration, angiogenesis, invasion and metastasis. In this review, the biological roles that versican plays in cancer development are presented. Therapeutic targeting of versican in malignant tumors is also discussed.

A mechanical insult to the brain drastically alters the microenvironment as specific cell types become reactive in an effort to sequester severely damaged tissue. Although injury-induced astrogliosis has been investigated, the relationship between well-defined biomechanical inputs and acute astrogliotic alterations is not well understood. We evaluated the effects of strain rate on cell death and astrogliosis using a three-dimensional (3-D) in vitro model of neurons and astrocytes within a bioactive matrix. At 21 days post-plating, co-cultures were deformed to 0.50 shear strain at strain rates of 1, 10, or 30 s(-1). We found that cell death and astrogliotic profiles varied differentially based on strain rate at 2 days post-insult. Significant cell death was observed after moderate (10 s(-1)) and high (30 s(-1)) rate deformation, but not after quasi-static (1 s(-1)) loading. The vast majority of cell death occurred in neurons, suggesting that these cells are more susceptible to high rate shear strains than astrocytes for the insult parameters used here. Injury-induced astrogliosis was compared to co-cultures treated with transforming growth factor beta, which induced robust astrocyte hypertrophy and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Quasi-static loading resulted in increased cell density and CSPG secretion. Moderate rate deformation increased cell density, GFAP reactivity, and hypertrophic process density. High rate deformation resulted in increased GFAP reactivity; however, other astrogliotic alterations were not observed at this time-point. These results demonstrate that the mode and degree of astrogliosis depend on rate of deformation, demonstrating astrogliotic augmentation at sub-lethal injury levels as well as levels inducing cell death.

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.

Although brain tumors are classified as if their lineage were well understood, the relationship between the molecular events that specify neural cell lineage and brain tumors remains enigmatic. Traditionally, cell surface membrane antigens have served as biomarkers that distinguish brain tumor origin and malignancy. In this study, membrane proteins were identified from a terminally differentiated mouse astrocyte (AC) and CT-2A astrocytoma (CT-2A) cell line using liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). A total of 321 and 297 protein groups with at least one unique peptide were identified in the AC and CT-2A cells. Using a label-free quantitative MS approach, 25 plasma membrane proteins in CT-2A were found significantly up- or down-regulated compared with those in AC. Three of the up-regulated proteins, chondroitin sulfate proteoglycan-4 (Cspg4), interferon-induced transmembrane protein-2 (IFITM2) and -3 (IFITM3) were further validated by semi-quantitative RT-PCR analysis. In addition, a third member of the IFITM family, interferon-induced transmembrane protein-1 (IFITM1) was also analyzed. Expression of Cspg4, IFITM1 and IFITM3 was significantly greater in the CT-2A cells than that in the AC cells. Interestingly, Cspg4, also known as neuronal/glial 2 (NG2) proteoglycan in human, is an oligodendrocyte progenitor marker. Therefore, our data suggest that the CT-2A tumor may be derived from NG2 glia rather than from fully differentiated astrocytes. Moreover, the CT-2A cells also express a series of interferon-induced signature proteins that may be specific to this tumor. These data highlight the utility of LC-MS/MS for the identification of brain tumor membrane biomarkers.

We have previously shown that all perineuronal nets (PNNs) bearing neurons express a hyaluronan synthase (HAS), a link protein (usually cartilage link protein-1; Crtl1) and a chondroitin sulfate proteoglycan (usually aggrecan). Animal lacking Crtl1 in the CNS lacks normal PNNs. PNNs are implicated in the control of neuronal plasticity, and interventions to modulate PNN formation will be useful for manipulating plasticity. We have developed an in vitro model which demonstrates how the structural components of PNNs trigger their formation, using human embryonic kidney cells, which do not normally produce a pericellular matrix. Expression of HAS3 leads to the production of a diffuse matrix. It was converted into a compact PNN-like structure when the cells also expressed Crtl1 and aggrecan. This matrix was stained by Wisteria floribunda, contained Crtl1 and aggrecan, and like PNNs, could only be solubilized in 6 M urea. In the absence of hyaluronan produced by HAS3, aggrecan and Crtl1 dissipated into the medium, but when the cells were transfected to produce a hyaluronan matrix, Crtl1 and aggrecan were incorporated into it. Cells lacking any one of these molecules showed impaired integrity of the PNNs. Cells expressing HAS3 and Crtl1 were able to incorporate exogenous aggrecan into their pericellular matrix.

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.

The distinct axonal tracts of the mature nervous system are defined during development by sets of substrate-bound and diffusible molecular signals that promote or restrict axonal elongation. In the adult cerebral cortex, efferent and afferent axons are segregated within the white matter. To define the relationship of growing efferent and afferent axons in the developing murine cortex to chondroitin sulfate proteoglycans (CSPGs) in the pericellular and extracellular matrix, we used the fluorescent tracer Dil to determine axonal trajectories and immunolabeling to disclose the distribution of CSPGs. Axons of neurons in the preplate are the first to leave the cortex; they arise in the CSPG-rich preplate and extend obliquely across it to enter the CSPG-poor intermediate zone. Slightly later, axons of cortical plate neurons extend directly across the CSPG-rich subplate, and then turn abruptly to run in the upper intermediate zone. In contrast, once afferent axons from the thalamus reach the developing cortical wall, their intracortical trajectory is centered on the CSPG-rich subplate, above the path taken by efferent axons. Our findings demonstrate a molecular difference between the adjacent but distinct efferent and afferent pathways in developing neocortex. Early efferents cross the subplate and follow a pathway that contains very little CSPG, while afferents preferentially travel more superficially within the CSPG-rich subplate. Thus, CSPGs and associated extracellular matrix (ECM) components in the preplate/subplate do not form a barrier to axonal initiation or outgrowth in the neocortex as they may in other locations. Instead, their distribution suggests a role in defining discrete axonal pathways during early cortical development.

Spinal cord injuries (SCIs) result in devastating lifelong disability for patients and their families. The initial mechanical trauma is followed by a damaging secondary injury cascade involving proapoptotic signaling, ischemia, and inflammatory cell infiltration. Ongoing cellular necrosis releases ATP, DNA, glutamate, and free radicals to create a cytotoxic postinjury milieu. Long-term regeneration of lost or injured networks is further impeded by cystic cavitation and the formation of an inhibitory glial-chondroitin sulfate proteoglycan scar. In this article, we discuss important neuroprotective interventions currently applied in clinical practice, including surgical decompression, blood pressure augmentation, and i.v. methylprednisolone. We then explore exciting translational therapies on the horizon, such as riluzole, minocycline, fibroblast growth factor, magnesium, and hypothermia. Finally, we summarize the key neuroregenerative strategies of the next decade, including glial scar degradation, Rho-ROCK inhibition, cell-based therapies, and novel bioengineered adjuncts. Throughout, we emphasize the need for combinatorial approaches to this multifactorial problem and discuss relevant studies at the forefront of translation. We conclude by providing our perspectives on the future direction of SCI research.

CONCLUSIONS

Spinal cord injuries (SCIs) result in devastating, lifelong disability for patients and their families. This article discusses important neuroprotective interventions currently applied in clinical practice, including surgical decompression, blood pressure augmentation, and i.v. methylprednisolone. Translational therapies on the horizon are discussed, such as riluzole, minocycline, fibroblast growth factor, magnesium, and hypothermia. The key neuroregenerative strategies of the next decade are summarized, including glial scar degradation, Rho-ROCK inhibition, cell-based therapies, and novel bioengineered adjuncts. The need for combinatorial approaches to this multifactorial problem is emphasized, relevant studies at the forefront of translation are discussed, and perspectives on the future direction of SCI research are presented.

Spinal cord injury (SCI) causes permanent debilitation due to the inability of axons to grow through established scars. Both the sugar chains and core proteins of chondroitin sulfate proteoglycans (CSPGs) are inhibitory for neurite regrowth. Chondroitinase ABC (ChABC) degrades the sugar chains and allows for synaptic plasticity, suggesting that after the sugar chain cleavage additional steps occur promoting a permissive microenvironment in the glial scar region. We report that the clearance of the core protein by the tissue plasminogen activator (tPA)/plasmin proteolytic system partially contributes to ChABC-promoted plasticity. tPA and plasmin are upregulated after SCI and degrade the deglycosylated CSPG proteins. Mice lacking tPA (tPA(-/-)) exhibit attenuated neurite outgrowth and blunted sensory and motor recovery despite ChABC treatment. Coadministration of ChABC and plasmin enhanced the tPA(-/-) phenotype and supported recovery in WT SCI mice. Collectively, these findings show that the tPA/plasmin cascade may act downstream of ChABC to allow for synergistic sensory and motor improvement compared with each treatment alone and suggest a potential new approach to enhance functional recovery after SCI.

The formation of the distinct layers of the cerebral cortex begins when cortical plate neurons take up positions within the extracellular matrix (ECM)-rich preplate, dividing it into the marginal zone above and the subplate below. We have analyzed this process in the reeler mutant mouse, in which cortical lamination is severely disrupted. The recent observation that the product of the reeler gene is an ECM-like protein that is expressed by cells of the marginal zone indicates a critical role for ECM in cortical lamination. We have found that preplate cells in normal cortex that are tagged during their terminal division with bromodeoxyuridine (BrdU) are closely associated with chondroitin sulfate proteoglycans (CSPGs), which were identified by immunolabeling; this association is maintained in the marginal zone and subplate after the preplate is divided by cortical plate formation. Cortical plate cells do not aggregate within the preplate in reeler; instead, preplate cells remain as an undivided superficial layer containing abundant CSPGs, and cortical plate neurons accumulate below them. These findings indicate that preplate cells are responsible for the formation of a localized ECM, because the association of CSPGs with preplate cells is maintained even when these cells are in abnormal positions. The failure of cortical plate neurons to aggregate within the framework of the preplate and its associated ECM and to divide it is one of the earliest structural abnormalities detectable in reeler cortex, suggesting that this step is important for the subsequent formation of cortical layers.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.