We have used a slot-blot radioimmunoassay to quantitate the levels of hyaluronan-binding chondroitin sulfate proteoglycans in developing rat brain from embryonic day 14 (E 14) to eight months postnatal. Recombinant nonhomologous regions of the core proteins were used for immunization to obtain polyclonal antibodies specific for aggrecan, the alpha and beta domains of versican mRNA splice variants, and N- and C-terminal portions of neurocan, while brevican was quantitated using a specific monoclonal antibody. The concentration of aggrecan increased steadily during brain development up to 5 months of age, when it reached a level that was 18-fold higher than at E14. Alternatively spliced versican isoforms containing the alpha domain of the glycosaminoglycan attachment region were present at a relatively low level during the late embryonic and early postnatal period, decreased by approximately 50% between 1 and 2 weeks postnatal, and then increased steadily in concentration to reach a maximum at 100 days that was 7-fold that present at 10 days postnatal. In contrast to these results, versican isoforms containing the beta domain more than doubled in concentration between E14 and birth, after which they decreased by greater than 90% to reach a low "mature" level that remained unchanged between 2 and 8 months. The N- and C-terminal portions of neurocan (produced by a developmentally-regulated proteolytic cleavage in the middle of its chondroitin sulfate attachment region) both increased in embryonic brain during development, reached a peak in the early postnatal period, and then declined thereafter. As in the case of aggrecan, only traces of brevican were detected in embryonic brain and its concentration increased steadily after birth to reach an adult level that was approximately 14-fold higher than that present in neonatal brain. These striking and distinctive changes in the concentrations of the different members of this family of structurally related proteoglycans in developing brain, including changes in opposite directions for versican mRNA splice variants, indicate that the individual proteoglycans and their isoforms probably serve unique functions during nervous tissue histogenesis.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

Plasmodium falciparum malaria is a major cause of mortality and severe morbidity. Its virulence is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is central to both. Here, we present evidence of a P. falciparum evasion mechanism not previously documented: the masking of PfEMP1-specific IgG epitopes by nonspecific IgM. Nonspecific IgM binding to erythrocytes infected by parasites expressing the PfEMP1 protein VAR2CSA (involved in placental malaria pathogenesis and protective immunity) blocked subsequent specific binding of human monoclonal IgG to the Duffy binding-like (DBL) domains DBL3X and DBL5ε of this PfEMP1 variant. Strikingly, a VAR2CSA-specific monoclonal antibody that binds outside these domains and can inhibit IE adhesion to the specific VAR2CSA receptor chondroitin sulfate A was unaffected. Nonspecific IgM binding protected the parasites from FcγR-dependent phagocytosis of VAR2CSA(+) IEs, but it did not affect IE adhesion to chondroitin sulfate A or lead to C1q deposition on IEs. Taken together, our results indicate that the VAR2CSA affinity for nonspecific IgM has evolved to allow placenta-sequestering P. falciparum to evade acquired protective immunity without compromising VAR2CSA function or increasing IE susceptibility to complement-mediated lysis. Furthermore, functionally important PfEMP1 epitopes not prone to IgM masking are likely to be particularly important targets of acquired protective immunity to P. falciparum malaria.

The functional role of reactive astrocytes after stroke is controversial. To elucidate whether reactive astrocytes contribute to neurological recovery, we compared behavioral outcome, axonal remodeling of the corticospinal tract (CST), and the spatio-temporal change of chondroitin sulfate proteoglycan (CSPG) expression between wild-type (WT) and glial fibrillary acidic protein/vimentin double knockout (GFAP(-/-) Vim(-/-) ) mice subjected to Rose Bengal induced cerebral cortical photothrombotic stroke in the right forelimb motor area. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the CST axons. Compared with WT mice, the motor functional recovery and BDA-positive CST axonal length in the denervated side of the cervical gray matter were significantly reduced in GFAP(-/-) Vim(-/-) mice (n = 10/group, P < 0.01). Immunohistological data showed that in GFAP(-/-) Vim(-/-) mice, in which astrocytic reactivity is attenuated, CSPG expression was significantly increased in the lesion remote areas in both hemispheres, but decreased in the ischemic lesion boundary zone, compared with WT mice (n = 12/group, P < 0.001). Our data suggest that attenuated astrocytic reactivity impairs or delays neurological recovery by reducing CST axonal remodeling in the denervated spinal cord. Thus, manipulation of astrocytic reactivity post stroke may represent a therapeutic target for neurorestorative strategies.

The large and medium-sized arteries in elderly people show varying degrees of intimal and medial change. The medial change is known as age-related medial degeneration and sclerosis (ARMDS). The ARMDS results in systolic hypertension and left ventricular hypertrophy of the heart as a result of loss of arterial elasticity. It also causes aortic dilatation, or even aortic aneurysm. The ARMDS and atherosclerosis are distinct entities, but are often overlapped and confused with each other. The present review mainly focuses on ARMDS and briefly addresses atherosclerosis, and aging of arterioles, capillaries and veins. The smooth muscle cells in the inner half of the aortic media of elderly people degenerate and undergo apoptosis. This causes degradation of elastin fibers and the accumulation of collagen fibers in the media, but the inflammatory infiltrates are scarce. Biochemical studies showed an age-related decrease of elastin and its crosslinks, and an increase of collagen and its crosslink. Because the turnover of elastin is very long, it likely suffers from glycation (Maillard reaction) and glyco-oxidative reaction. The advanced glycation end-products accumulate in the aortic media with increasing age. Alcian-blue positive mucin accumulates in aortic media in elderly people. The major component of the increase of aortic mucin is chondroitin-6-sulfate. Microcalcification is frequent in the inner acellular portion of the aortic media in elderly people. Calcium contents increase with age. In conclusion, the ARMDS is a distinct pathological entity with clinical significance. The pathogenesis of ARMDS is unclear; the mechanical stress of elastin, endothelial dysfunction, and glycation of elastin are proposed.

Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens.

The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of approximately 3-4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201-36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd approximately 10-20 nM) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.

Chondroitin sulfate proteoglycans are major constituents of the extracellular matrix and form perineuronal nets. Information regarding the growth-inhibitory activity of these molecules after injury is rapidly expanding. However, less is known about their physiological role in the adult undamaged CNS. Here, we investigated the function of chondroitin sulfate proteoglycans in maintaining the proper structure of Purkinje axons in the cerebellum of adult rats. To this end, we examined the morphology and distribution of intracortical Purkinje neurites after intraparenchymal injection of chondroitinase ABC. Staining with the lectin Wisteria floribunda agglutinin or 2B6 antibodies showed that this treatment efficiently removed chondroitin sulfate proteoglycans from wide areas of the cerebellar cortex. In the same sites, there was a profuse outgrowth of terminal branches from the Purkinje infraganglionic plexus, which invaded the deeper regions of the granular layer. In contrast, myelinated axon segments were not affected and maintained their normal relationship with oligodendroglial sheaths. Purkinje axon sprouting was first evident at 4 d and increased further at 7 d after enzyme application. Within 42 d, the expression pattern of chondroitin sulfate proteoglycans gradually recovered, whereas axonal modifications progressively regressed. Our results show that, in the absence of injury or novel external stimuli, degradation of chondroitin sulfate proteoglycans is sufficient to induce Purkinje axon sprouting but not the formation of long-lasting synaptic contacts. Together with other growth-inhibitory molecules, such as myelin-associated proteins, chondroitin sulfate proteoglycans restrict structural plasticity of intact Purkinje axons to maintain normal wiring patterns in the adult cerebellar cortex.

Undifferentiated human melanoma cell lines produce a large chondroitin sulfate proteoglycan, different from the well-known melanoma-specific proteoglycan mel-PG (Heredia and colleagues, Arch Biochem Biophys, 333: 198-206, 1996). We have identified this proteoglycan as versican and analyzed the expression of versican in several human melanoma cell lines. Versican isoforms are expressed in undifferentiated cell lines but not in differentiated cells, and the isoform expression pattern depends on the degree of cell differentiation. The V0 and V1 isoforms are found on cells with an early degree of differentiation, whereas the V1 isoform is present in cells with an intermediate degree of differentiation. We have also characterized some functional properties of versican on human melanoma cells: the purified proteoglycan stimulates cell growth and inhibits cell adhesion when cells are grown on fibronectin or collagen type I as substrates, and thus may facilitate tumor cell detachment and proliferation. Furthermore, we have analyzed the expression of versican in human melanocytic nevi and melanoma: 10 benign melanocytic nevi, 10 dysplastic nevi, 11 primary malignant melanomas, and 8 metastatic melanomas were tested. Immunoreactivity for versican was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas. Our results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis.

Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(DeltaL)). Confocal microscopy demonstrated that both TMG and TMG(DeltaL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(DeltaL)-expressed cells grew singly. Overexpression of TMG, but not TMG(DeltaL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function.

Proteoglycan synthesis by slices of adult bovine articular cartilage is stimulated two-to threefold when tissue is cultured in the presence of fetal calf serum for 5-6 days. After this, essentially steady-state conditions are achieved for up to 14 days in which the high synthetic rates are maintained and the amount of proteoglycan in the tissue remains nearly constant. In the absence of fetal calf serum, synthesis declines to a lower level and there is a gradual, net loss of proteoglycan from the tissue. Tissue maintained without serum for several days rapidly increases synthetic rates to the higher levels over 2-3 days after transferring into medium with serum, and vice versa, indicating that the response of the chondrocytes to serum factors is reversible. The structures of the proteoglycans synthesized under all medium conditions were typical for cartilage. Only small differences in glycosaminoglycan chain sizes and a consistent decrease in the relative amount of keratan sulfate to chondroitin sulfate during the first days in the culture were observed. The net capacity of the cells for chondroitin sulfate synthesis, as estimated by incubation in the presence of exogenous beta-xyloside acceptor, increased (or decreased) in parallel with the changes in endogenous proteoglycan synthesis when cultures were transferred from medium without to medium with serum (or vice versa), suggesting that changes in the net amounts of the enzymes for chondroitin sulfate synthesis are closely coordinated with changes in the amount of core protein being processed to proteoglycans. The responses of calf articular cartilage in the same system were somewhat different. Serum in the medium was required to maintain initial high levels of synthesis. The proteoglycans synthesized contained a lower proportion of keratan sulfate than those initially synthesized in the adult tissue, and there was no change in this proportion with time in culture. The maintenance of steady-state conditions for proteoglycan metabolism by either adult or calf tissue in the presence of serum in these cultures should provide a useful model for studying the regulation of synthesis and catabolism of proteoglycans by chondrocytes residing in a nearly normal extracellular matrix for long periods of time.

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.

Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has been shown to bind to extracellular matrix (ECM) with relatively high affinity, but the ECM components that mediate this interaction have not been identified. These studies show that radiolabeled IGFBP-5 specifically coprecipitates with two ECM proteins, thrombospondin-1 (TSP-1) and osteopontin (OPN). As TSP-1 binds avidly to heparin, as does IGFBP-5, the effect of glycosaminoglycans on the TSP-1/IGFBP-5 interaction was analyzed. Heparan and dermatan sulfate inhibited binding, whereas heparin increased binding. Chondroitin sulfate A and B had no effect. In contrast, both heparin and heparan sulfate significantly inhibited the OPN-IGFBP-5 interaction and chondroitin sulfate A, B, and C had no effect. To determine the region of IGFBP-5 that was involved in each interaction, synthetic peptides that spanned several regions of IGFBP-5 were tested for their capacity to competitively inhibit coprecipitation. A peptide that contained the amino acids between positions 201 and 218 resulted in 76% and 86% inhibition of binding to TSP-1 and OPN, respectively. Three other synthetic peptides that spanned regions ofIGFBP-5 with several charged residues had no effect. IGFBP-5 mutants that contained substitutions for basic residues in the 201-218 region were tested for their ability to bind to TSP-1 or OPN. A mutant with substitutions for amino acids at positions R201 and K202 and a mutant with substitutions for K211, R214, K217, and R218 had the greatest reduction in binding to TSP-1. Mutants containing substitutions for R214 alone and the combined K217A, R218A mutant had the greatest reductions in OPN binding. When the smooth muscle cell growth response to these components was assessed, IGF-I plus IGFBP-5 or the combination of TSP-1 or OPN with IGF-I potentiated the IGF-I effect. The addition of IGFBP-5 to these combinations resulted in further significant growth stimulation. Both OPN and TSP-1 specifically bind to IGFBP-5 with high affinity. These interactions may be important for concentrating intact IGFBP-5 in extracellular matrix and for modulating the cooperative interaction between the IGF-I receptor and integrin receptor signaling pathways.

The ultrastructural organization and the composition of newly synthesized glycosaminoglycan (GAG) in the epithelial basal lamina of mouse embryo submandibular glands were assessed. The labeled GAG accumulating in the lamina is distinct from that in its tissue of origin, the epithelium, or from that in the surrounding mesenchyme. In the lamina, hyaluronic acid accounts for approximately 50% of the labeled GAG, chondroitin-4-sulfate is twice the chondroitin-6-sulfate, and there is a low proportion of chondroitin. This composition is constant regardless of whether the lamina is labeled by whole glands or, in the absence of mesenchyme, by isolated epithelia retaining a lamina and by isolated epithelia generating a lamina de novo. The results andicate that the labeled GAG are bona fide components of the lamina, and suggest that laminar GAG is deposited in units of constant composition. Ultrastructural observations following ruthenium red staining or tannic acid fixation extablish that the lamina is a highly ordered specialization of the basal cell surface. Discrete structures in macroperiodic arrays apparently attached to the plasmalemma are visualized. This organization is seen in intact glands and in the laminae produced by epithelia in the absence of mesenchyme or biological substrate. The data are interpreted as indicating that the basal lamina contains supramolecular complexes of hyaluronic acid and proteoglycan which are organized into an extracellular scaffolding which imposes structural form on the epithelium.

Adult axons in the mammalian central nervous system do not elicit spontaneous regeneration after injury, although many affected neurons have survived the neurotrauma. However, axonal regeneration does occur under certain conditions. These conditions include: (a) modification of regrowth environment, such as supply of peripheral nerve bridges and transplantation of Schwann cells or olfactory ensheathing glia to the injury site; (b) application of neurotrophic factors at the cell soma and axon tips; (c) blockade of growth-inhibitory molecules such as Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein; (d) prevention of chondroitin-sulfate-proteoglycans-related scar tissue formation at the injury site using chondroitinase ABC; and (e) elevation of intrinsic growth potential of injured neurons via increasing intracellular cyclic adenosine monophosphate level. A large body of evidence suggests that these conditions achieve enhanced neuronal survival and axonal regeneration through sometimes overlapping and sometimes distinct signal transduction mechanisms, depending on the targeted neuronal populations and intervention circumstances. This article reviews the available information on signal transduction pathways underlying neurotrophic-factor-mediated neuronal survival and neurite outgrowth/axonal regeneration. Better understanding of signaling transduction is important in helping us develop practical therapeutic approaches for encouraging neuronal survival and axonal regeneration after traumatic injury in clinical context.

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)

The formation of the glial scar following a spinal cord injury presents a significant barrier to the regenerative process. It is primarily composed of chondroitin sulfate proteoglycans (CSPGs) that can inhibit axonal sprouting and regeneration. Although the inhibitory effects on neurons are well documented, little is known about their effects on oligodendrocyte progenitor cells (OPCs). In this study, we examined the effects of CSPGs on OPC process outgrowth and differentiation in vitro. The results show that specific CSPGs, in particularly those highly up-regulated following spinal cord injury, inhibit OPC process outgrowth and differentiation, and that treatment with chondroitinase ABC can completely reverse this inhibition. Additionally, treatment with the Rho kinase inhibitor Y-27632 also reverses the observed inhibition, implicating the activation of Rho kinase in the CSPG inhibition of OPC growth. Taken together, these findings demonstrate that the CSPGs found within the glial scar are not only inhibitory to neurons, but also to OPCs. Moreover, this study shows that chondroitinase ABC treatment, having shown promise in promoting axonal regeneration, may also enhance remyelination.

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.

The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, beta1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair.

In a previous study it was found that with age there is an increase in the frequency of paranodal profiles of myelinated nerve fibers in the cerebral cortex of monkeys. This indicates that there is an increase in the number of internodal myelin segments, and raises the question of whether additional oligodendrocytes are necessary to generate the increased numbers of internodal myelin segments. The present study shows that in layer 4C beta of monkey primary visual cortex there is an age-related increase in the number of oligodendrocytes. When young (4-10 years of age) and old (25-35 years of age) monkeys are compared, the increase is found to be approximately 50%, and it begins in middle age (12-19 years old). It is also shown that although there is no increase in the population of astrocytes in layer 4C beta with age, there appears to be a slight increase in the frequency of microglial cells. As their numbers increase, oligodendrocytes in pairs, rows and groups become more common, which suggests that additional oligodendrocytes are being generated by cell division. Since there is little evidence that mature oligodendrocytes can divide, it is probable that the new oligodendrocytes are generated from progenitor cells which, as many studies have shown, can be labeled by antibodies to NG2, a chondroitin sulfate proteoglycan. By comparing the appearance of these NG2-labeled cells with cells encountered in thin sections of normally prepared tissue, it is shown that the NG2-positive cells have the features of neuroglial cells that were previously described as beta astrocytes.

The use of stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of cartilaginous defects. The multilineage potential of a population of putative mesodermal stem cells obtained from human lipoaspirates, termed processed lipoaspirate cells, was previously characterized. The chondrogenic potential of those cells was confirmed with a combination of histological and molecular approaches. Processed lipoaspirate cells under high-density micromass culture conditions, supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid, formed well-defined nodules within 48 hours of induction and expressed the cartilaginous markers collagen type II, chondroitin-4-sulfate, and keratan sulfate. Reverse transcription polymerase chain reaction analysis confirmed the expression of collagen type II and the cartilage-specific proteoglycan aggrecan. In summary, human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis in vitro.

A scar tissue interface forms rather than a normal ligament insertion site following attachment of a tendon graft to bone. The specific cell types that initiate the process of tendon-to-bone healing are unknown. We hypothesized that inflammatory cell accumulation following tendon-to-bone repair results in this scar interface. We used a rodent model to examine the temporal and spatial pattern of accumulation of hematopoietic lineage cells in the early phase of tendon-to-bone healing. Thirty-six Lewis rats underwent anterior cruciate ligament (ACL) reconstruction in the left knee using a flexor digitorum longus tendon graft. Six animals were sacrificed at 4, 7, 11, 14, 21, and 28 days after surgery. Serial sections were analyzed for proliferating cells (PCNA), recruited macrophages (ED1), resident macrophages (ED2), neutrophils, T-lymphocytes (CD3), mast cells, immature progenitor cells/pericytes (expressing the NG2 cell-surface chondroitin sulfate proteoglycan), and newly-formed blood vessels (Factor VIII). Neutrophils, ED1(+) and ED2(+) macrophages accumulated sequentially in the healing tendon graft, with progressive cell ingrowth from the interface towards the inner tendon. Neutrophils and ED1(+) cells were seen in the tendon-bone interface at 4 days after surgery, while ED2(+) macrophages were not identified until 11 days. These cells progressively repopulated the tendon graft. NG2-positive progenitor cells were found along the edge of the bone tunnel in the interface, but these cells did not invade the tendon. Occasional T-lymphocytes and mast cells were seen in the tendon-bone interface. There was no proliferation of intrinsic tendon cells, indicating that the tendon does not directly contribute to healing. We hypothesize that cytokines produced by infiltrating macrophages are likely to contribute to the formation of a fibrous scar tissue interface rather than a normal insertion site.