We previously observed that the cortical neuronal cell adhesion mediated by midkine (MK), a heparin (Hep)-binding growth factor, is specifically inhibited by oversulfated chondroitin sulfate-E (CS-E) (Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413) and that CS-E exhibits neurite outgrowth promoting activities toward embryonic rat hippocampal neurons. We have also shown oversulfated CS chains in embryonic chick and rat brains and demonstrated that the CS disaccharide composition changes during brain development. In view of these findings, here we tested the possibility of CS-E interacting with Hep-binding growth factors during development, using squid cartilage CS-E. The binding ability of Hep-binding growth factors (MK, pleiotrophin (PTN), fibroblast growth factor-1 (FGF-1), FGF-2, Hep-binding epidermal growth factor-like growth factor (HB-EGF), FGF-10, FGF-16, and FGF-18) toward [(3)H]CS-E was first tested by a filter binding assay, which demonstrated direct binding of all growth factors, except FGF-1, to CS-E. The bindings were characterized further in an Interaction Analysis system, where all of the growth factors, except FGF-1, gave concentration-dependent and specific bindings. The kinetic constants k(a), k(d), and K(d) suggested that MK, PTN, FGF-16, FGF-18, and HB-EGF bound strongly to CS-E, in comparable degrees to the binding to Hep, whereas the intensity of binding of FGF-2 and FGF-10 toward CS-E was lower than that for Hep. These findings suggest the possibility of CS-E being a binding partner, a coreceptor, or a genuine receptor for various Hep-binding growth factors in the brain and possibly also in other tissues.

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.

Cardiac valve formation is a complex process that involves cell signaling events between the myocardial and endocardial layers of the heart across an elaborate extracellular matrix. These signals lead to marked morphogenetic movements and transdifferentiation of the endocardial cells at chamber boundaries. Here we identify the genetic defect in zebrafish jekyll mutants, which are deficient in the initiation of heart valve formation. The jekyll mutation disrupts a homolog of Drosophila Sugarless, a uridine 5'-diphosphate (UDP)-glucose dehydrogenase required for heparan sulfate, chondroitin sulfate, and hyaluronic acid production. The atrioventricular border cells do not differentiate from their neighbors in jekyll mutants, suggesting that Jekyll is required in a cell signaling event that establishes a boundary between the atrium and ventricle.

The basic concept, that specialized extracellular matrices rich in hyaluronan, chondroitin sulfate proteoglycans (aggrecan, versican, neurocan, brevican, phosphacan), link proteins and tenascins (Tn-R, Tn-C) can regulate cellular migration and axonal growth and thus, actively participate in the development and maturation of the nervous system, has in recent years gained rapidly expanding experimental support. The swift assembly and remodeling of these matrices have been associated with axonal guidance functions in the periphery and with the structural stabilization of myelinated fiber tracts and synaptic contacts in the maturating central nervous system. Particular interest has been focused on the putative role of chondroitin sulfate proteoglycans in suppressing central nervous system regeneration after lesions. The axon growth inhibitory properties of several of these chondroitin sulfate proteoglycans in vitro, and the partial recovery of structural plasticity in lesioned animals treated with chondroitin sulfate degrading enzymes in vivo have significantly contributed to the increased awareness of this long time neglected structure.

The NG2 chondroitin sulfate proteoglycan is a valuable marker for several types of incompletely-differentiated precursor cells, including oligodendrocyte progenitors in the central nervous system, developing mesenchymal cells in cartilage, muscle, and bone, and pericytes/smooth muscle cells in developing vasculature. In addition to extending our knowledge about the developmental roles of these cell types, current studies on NG2 are also providing information about the molecular mechanisms through which the proteoglycan itself influences progenitor development. This research suggests that interaction of NG2 with extracellular and intracellular ligands regulates signaling events that are important for both cell proliferation and cell migration.

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B beta-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4 degrees C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.

OBJECTIVE

To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes.

METHODS

Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes.

RESULTS

Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum.

CONCLUSIONS

A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.

The extracellular matrix component hyaluronan (HA) exists physiologically as a high m.w. polymer but is cleaved at sites of inflammation, where it will be contacted by dendritic cells (DC). To determine the effects of HA on DC, HA fragments of different size were established. Only small HA fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (m.w. 80, 000-200,000) or high m.w. HA (m.w. 1,000,000-600,000) induced immunophenotypic maturation of human monocyte-derived DC (up-regulation of HLA-DR, B7-1/2, CD83, down-regulation of CD115). Likewise, only sHA increased DC production of the cytokines IL-1beta, TNF-alpha, and IL-12 as well as their allostimulatory capacity. These effects were highly specific for sHA, because they were not induced by other glycosaminoglycans such as chondroitin sulfate or heparan sulfate or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA receptors CD44 or the receptor for hyaluronan-mediated motility, because DC from CD44-deficient mice and wild-type mice both responded similarly to sHA stimulation, whereas the receptor for hyaluronan-mediated motility is not detectable in DC. However, TNF-alpha is an essential mediator of sHA-induced DC maturation as shown by blocking studies with a soluble TNFR1. These findings suggest that during inflammation, interaction of DC with small HA fragments induce DC maturation.

We investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF164 (rrSCF164) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of "connective tissue-type mast cells" (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF164 induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC. BMCMC maintained in rrSCF164 not only proliferated but also matured. Prior to exposure to rrSCF164, the BMCMC were alcian blue positive, safranin negative, and berberine sulfate negative; had a histamine content of 0.08 +/- 0.02 pg per cell; and incorporated [35S]sulfate into chondroitin sulfates. After 4 wk in rrSCF164, the BMCMC were predominantly safranin positive and berberine sulfate positive, had a histamine content of 2.23 +/- 0.39 pg per cell, and synthesized 35S-labeled proteoglycans that included substantial amounts (41-70%) of [35S]heparin. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.

We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor III (ECMR III), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HT1080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonoverlapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O- and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.

OBJECTIVE

To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA).

METHODS

Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3.

RESULTS

Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immunoreactive products were also seen, with an apparent size of>> 250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein.

CONCLUSIONS

The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373-alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.

Urine contains compounds that modulate the nucleation, growth and aggregation of crystals as well as their attachment to renal epithelial cells. These compounds may function to protect the kidneys against: 1, the possibility of crystallization in tubular fluid and urine, which are generally metastable with respect to calcium salts, 2, crystal retention within the kidneys thereby preventing stone formation and 3, possibly against plaque formation at the nephron basement membrane. Since oxalate is the most common stone type, the effect of various modulators on calcium oxalate (CaOx) crystallization has been examined in greater details. Most of the inhibitory activity resides in macromolecules such as glycoproteins and glycosaminoglycans while nucleation promotion activity is most likely sustained by membrane lipids. Nephrocalcin, Tamm-Horsfall protein, osteopontin, urinary prothrombin fragment 1, and bikunin are the most studied inhibitory proteins while chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) are the best studied glycosaminoglycans. Crystallization modulating macromolecules discussed here are also prominent in cell injury, inflammation and recovery. Renal epithelial cells on exposure to oxalate and CaOx crystals produce some of the inflammatory molecules such as monocyte chemoattractant protein-1 (MCP-1) with no apparent role in crystal formation. In addition, macrophages surround the CaOx crystals present in the renal interstitium. These observations indicate a close relationship between inflammation and nephrolithiasis.

Recent cDNA cloning of the glycosyltransferases involved in the synthesis of the sulfated glycosaminoglycan sidechains of proteoglycans has provided important clues to answering long-standing questions concerning the mechanisms of both chain polymerization and the biosynthetic sorting of glucosaminoglycans (heparin/heparan sulfate) and galactosaminoglycans (chondroitin/dermatan sulfate). These biosynthetic mechanisms are crucial to the expression and regulation of the biological functions of glycosaminoglycans in development and pathophysiology.

Synthetic hydrogel mimics of the extracellular matrix (ECM) were created by crosslinking a thiol-modified analog of heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol) diacrylate (PEGDA). The covalently bound heparin provided a crosslinkable analog of a heparan sulfate proteoglycan, thus providing a multivalent biomaterial capable of controlled release of basic fibroblast growth factor (bFGF). Hydrogels contained >97% water and formed rapidly in <10min. With as little as 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content), the rate of release of bFGF in vitro was substantially reduced. Total bFGF released increased with lower percentages of heparin; essentially quantitative release of bFGF was observed from heparin-free hydrogels. Moreover, the hydrogel-released bFGF retained 55% of its biological activity for up to 28 days as determined by a cell proliferation assay. Finally, when these hydrogels were implanted into subcutaneous pockets in Balb/c mice, neovascularization increased dramatically with HA and CS hydrogels that contained both bFGF and crosslinked heparin. In contrast, hydrogels lacking bFGF or crosslinked heparin showed little increase in neovascularization. Thus, covalently linked, heparin-containing glycosaminoglycan hydrogels that can be injected and crosslinked in situ constitute highly promising new materials for controlled release of heparin-binding growth factors in vivo.

Extracellular modulation of phenotype is an emerging paradigm in this current postgenomics age of molecular and cell biology. Glycosaminoglycans (GAGs) are primary components of the cell surface and the cell-extracellular matrix (ECM) interface. Advances in the technology to analyze GAGs and in whole-organism genetics have led to a dramatic increase in the known important biological role of these complex polysaccharides. Owing to their ubiquitous distribution at the cell-ECM interface, GAGs interact with numerous proteins and modulate their activity, thus impinging on fundamental biological processes such as cell growth and development. Many recent reviews have captured important aspects of GAG structure and biosynthesis, GAG-protein interactions, and GAG biology. GAG research is currently at a stage where there is a need for an integrated systems or glycomics approach, which involves an integration of all of the above concepts to define their structure-function relationships. Focusing on heparin/heparan (HSGAGs) and chondroitin/dermatan sulfate (CSGAGs), this review highlights the important aspects of GAGs and summarizes these aspects in the context of taking a glycomics approach that integrates the different technologies to define structure-function relationships of GAGs.

Axonal regeneration is minimal after CNS injuries in adult mammals and medical treatments to recover neurological deficits caused by axon disconnection are extremely limited. The failure of axonal elongation is principally attributed to the nonpermissive environment and reduced intrinsic growth capacity. In this report, we studied the role of glycogen synthase kinase-3 (GSK-3) inactivation on neurite and axon growth from adult neurons via combined in vitro and in vivo approaches. We found that the major CNS inhibiting substrates including chondroitin sulfate proteoglycans could inactivate protein kinase B (Akt) and activate GSK-3beta signals in neurons. GSK-3 inactivation with pharmacologic inhibitors enhances neurite outgrowth of dorsal root ganglion neurons derived from adult mice or cerebellar granule neurons from postnatal rodents cultured on CNS inhibitors. Application of GSK-3 inhibitors stimulates axon formation and elongation of mature neurons whether in presence or absence of inhibitory substrates. Systemic application of the GSK-3 inhibitor lithium to spinal cord-lesioned rats suppresses the activity of this kinase around lesion. Treatments with GSK-3 inhibitors including a clinical dose of lithium to rats with thoracic spinal cord transection or contusion injuries induce significant descending corticospinal and serotonergic axon sprouting in caudal spinal cord and promote locomotor functional recovery. Our studies suggest that GSK-3 signal is an important therapeutic target for promoting functional recovery of adult CNS injuries and that administration of GSK-3 inhibitors may facilitate the development of an effective treatment to white matter injuries including spinal cord trauma given the wide use of lithium in humans.

Cortical plasticity is most evident during a critical period in early life, but the mechanisms that restrict plasticity after the critical period are poorly understood. We found that a developmental increase in the 4-sulfation/6-sulfation (4S/6S) ratio of chondroitin sulfate proteoglycans (CSPGs), which are components of the brain extracellular matrix, leads to the termination of the critical period for ocular dominance plasticity in the mouse visual cortex. Condensation of CSPGs into perineuronal nets that enwrapped synaptic contacts on parvalbumin-expressing interneurons was prevented by cell-autonomous overexpression of chondroitin 6-sulfation, which maintains a low 4S/6S ratio. Furthermore, the increase in the 4S/6S ratio was required for the accumulation of Otx2, a homeoprotein that activates the development of parvalbumin-expressing interneurons, and for functional maturation of the electrophysiological properties of these cells. Our results indicate that the critical period for cortical plasticity is regulated by the 4S/6S ratio of CSPGs, which determines the maturation of parvalbumin-expressing interneurons.

Axotomized neurons within the damaged CNS are thought to be prevented from functional regeneration by inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors. Here, we provide a transgenic test of the role of CSPGs in limiting regeneration, using the gfap promotor to express a CSPG-degrading enzyme chondroitinase ABC (ChABC) in astrocytes. Corticospinal axons extend within the lesion site, but not caudal to it, after dorsal hemisection in the transgenic mice. The presence of the gfap-ChABC transgene yields no significant improvement in motor function recovery in this model. In contrast, functionally significant sensory axon regeneration is observed after dorsal rhizotomy in transgenic mice. These transgenic studies confirm a local efficacy for reduced CSPG to enhance CNS axon growth after traumatic injury. CSPGs appear to function in a spatially distinct role from myelin inhibitors, implying that combination-based therapy will be especially advantageous for CNS injuries.

BACKGROUND

Pericytes are integral members of the neurovascular unit. Using mouse models lacking endothelial-secreted platelet derived growth factor-B (PDGF-B) or platelet derived growth factor receptor beta (PDGFRβ) on pericytes, it has been demonstrated that PDGF-B/PDGFRβ interactions mediate pericyte recruitment to the vessel wall in the embryonic brain regulating the development of the cerebral microcirculation and the blood-brain barrier (BBB). Relatively little is known, however, about the roles of PDGF-B/PDGFRβ interactions and pericytes in the adult brain in part due to a lack of adequate and/or properly characterized experimental models. To address whether genetic disruption of PDGFRβ signaling would result in a pericyte-specific insult in adult mice, we studied the pattern and cellular distribution of PDGFRβ expression in the brain in adult control mice and F7 mice that express two hypomorphic Pdgfrβ alleles containing seven point mutations in the cytoplasmic domain of PDGFRβ that impair downstream PDGFRβ receptor signaling.

RESULTS

Using dual fluorescent in situ hybridization, immunofluorescent staining for different cell types in the neurovascular unit, and a fluorescent in situ proximity ligation assay to visualize molecular PDGF-B/PDGFRβ interactions on brain tissue sections, we show for the first time that PDGFRβ is exclusively expressed in pericytes, and not in neurons, astrocytes or endothelial cells, in the adult brain of control 129S1/SvlmJ mice. PDGFRβ co-localized only with well-established pericyte markers such as Chondroitin Sulfate Proteoglycan NG2 and the xLacZ4 transgenic reporter. We next confirm pericyte-specific PDGFRβ expression in the brains of F7 mutants and show that these mice are viable in spite of substantial 40-60% reductions in regional pericyte coverage of brain capillaries.

CONCLUSIONS

Our data show that PDGFRβ is exclusively expressed in pericytes in the adult 129S1/Sv1mJ and F7 mouse brain. Moreover, our findings suggest that genetic disruption of PDGFRβ signaling results in a pericyte-specific insult in adult F7 mutants and will not exert a primary effect on neurons because PDGFRβ is not expressed in neurons of the adult 129S1/SvlmJ and F7 mouse brain. Therefore, mouse models with normal and deficient PDGFRβ signaling on a 129S1/SvlmJ background may effectively be used to deduce the specific roles of pericytes in maintaining the cerebral microcirculation and BBB integrity in the adult and aging brain as well as during neurodegenerative and brain vascular disorders.