Glycocalyx components affect platelet function, whole blood coagulation, and fibrinolysis: an in vitro study suggesting a link to trauma-induced coagulopathy.

BACKGROUND: The mechanisms of trauma induced coagulopathy (TIC) are considered multifactorial. Amongst others, however, shedding of the endothelial glycocalyx resulting in increased concentrations of glycocalyx fragments in plasma might also play a role. Thus, we hypothesized that shedded glycocalyx components affect coagulation and may act as humoral mediators of TIC. METHODS: To investigate effects of heparan sulfate, chondroitin sulfate, syndecan-1, versican, and thrombomodulin we added these fragments to in vitro assays of whole blood from healthy volunteers to yield concentrations observed in trauma patients. Platelet function, whole blood coagulation, and fibrinolysis were measured by standard coagulation tests, impedance aggregometry (IA), and viscoelastic tests (VET). To assess dose-response relationships, we performed IA with increasing concentrations of versican and VET with increasing concentrations of thrombomodulin. RESULTS: Intrinsically activated clotting times (i.e., activated partial thromboplastin time and intrinsically activated VET with and without heparinase) were unaffected by any glycocalyx fragment. Thrombomodulin, however, significantly and dose-dependently diminished fibrinolysis as assessed by VET with exogenously added rt-PA, and increased rt-PA-induced lysis Indices after 30 (up to 108% of control, p <  0,0001), 45 (up to 368% of control, p <  0,0001), and 60 min (up to 950% of control, p <  0,0001) in VET. Versican impaired platelet aggregation in response to arachidonic acid (up to - 37,6%, p <  0,0001), ADP (up to - 14,5%, p <  0,0001), and collagen (up to - 31,8%, p <  0,0001) in a dose-dependent manner, but did not affect TRAP-6 induced platelet aggregation. Clotting time in extrinsically activated VET was shortened by heparan sulfate (- 7,2%, p = 0,024), chondroitin sulfate (- 11,6%, p = 0,016), versican (- 13%, p = 0,012%), and when combined (- 7,2%, p = 0,007). CONCLUSIONS: Glycocalyx components exert distinct inhibitory effects on platelet function, coagulation, and fibrinolysis. These data do not support a 'heparin-like auto-anticoagulation' by shed glycosaminoglycans but suggest a possible role of versican in trauma-induced thrombocytopathy and of thrombomodulin in trauma-associated impairment of endogenous fibrinolysis.

Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

Under the ECM Dome: The Physiological Role of the Perinodal Extracellular Matrix as an Ion Diffusion Barrier.

Enriched Na+ channel clustering allows for rapid saltatory conduction at a specialized structure in myelinated axons, the node of Ranvier, where cations are exchanged across the axon membrane. In the extracellular matrix (ECM), highly negatively charged molecules accumulate and wrap around the nodal gaps creating an ECM dome, called the perinodal ECM. The perinodal ECM has different molecular compositions in the central nervous system (CNS) and peripheral nervous system (PNS). Chondroitin sulfate proteoglycans are abundant in the ECM at the CNS nodes, whereas heparan sulfate proteoglycans are abundant at the PNS nodes. The proteoglycans have glycosaminoglycan chains on their core proteins, which makes them electrostatically negative. They associate with other ECM molecules and form a huge stable ECM complex at the nodal gaps. The polyanionic molecular complexes have high affinity to cations and potentially contribute to preventing cation diffusion at the nodes.In this chapter, we describe the molecular composition of the perinodal ECM in the CNS and PNS, and discuss their physiological role at the node of Ranvier.

Chondroitin sulfate-D promotes neurite outgrowth by acting as an extracellular ligand for neuronal integrin αVß3.

BACKGROUND: Chondroitin sulfate (CS) chains are prominent extra/pericellular matrix components in the central nervous system (CNS) and can exert positive or negative regulatory effects on neurite outgrowth, depending on the CS structure and the amount. Despite the remarkable abilities of highly sulfated forms of CS chains to enhance neurite outgrowth, the neuronal recognition systems for such promotional CS chains, including CS-D polysaccharide, remain to be fully elucidated. METHODS: We explored the molecular basis of the CS-D-mediated neurite extension using primary hippocampal neurons cultured on substrate precoated with CS-D polysaccharides, and evaluated functional involvement of a distinct integrin heterodimer as a novel neuronal CS receptor for CS-D. RESULTS: We identified an extracellular matrix receptor, integrin αVß3, as a functional receptor for CS-D. CS-D, but not CS-C (a precursor form of CS-D) showed significant binding affinity toward recombinant integrin αVß3 heterodimer and activated intracellular signaling(s) involving focal adhesion kinase (FAK) and Src/Fyn kinase. Functional blockade of the respective players for integrin signaling abrogated the promotional effects of CS-D. We also found the existence of CS-D-induced integrin activation system in neuronal stem/progenitor cell population. CONCLUSIONS: The neuronal cell surface integrin αVß3 can function as a CS receptor for a highly sulfated CS subtype, CS-D. GENERAL SIGNIFICANCE: Our findings are the first to demonstrate that CS-dependent neurite outgrowth promotion is exerted via direct activation of specific integrin heterodimers on neuronal cell surfaces, providing new insights into understanding the CS-sensing machineries that regulate CNS development and regeneration.

[Influence of Synovial Fluid on Lubrication of Articular Cartilage in Vitro - A Review]. / Einfluss der Synovialflüssigkeit auf die Schmiereigenschaften von Gelenkknorpel in vitro.

Articular cartilage possesses unique tribological properties that are essential to reduce friction and wear. Especially under boundary lubrication conditions, synovial fluid as a whole, and its components ("biolubricants"), are important in assuring near frictionless/contactless lubrication of the joint surfaces. Therefore, several in vitro tribological models have been developed in recent years to investigate possible interdependencies. The aim of this article is to give a cursory overview of the influence of synovial fluid and its components on boundary lubrication of articular cartilage surfaces in vitro.

Positive and negative cues for modulating neurite dynamics and receptor expression.

Many current peripheral nerve repair strategies focus on delivering positive, growth promoting cues (e.g. extracellular matrix, ECM) while eliminating negative, growth inhibiting cues (e.g. chondroitin sulfate proteoglycans, CSPGs) at the injury site. We hypothesized that recapitulating the positive and negative cues of the peripheral nerve injury microenvironment would improve regeneration. First, we tested the effects of a characteristic CSPG, chondroitin sulfate A (CSA) on neurite dynamics of dissociated chick embryo dorsal root ganglion (DRG) neurons using time lapse video microscopy. DRG growth was recorded on different adhesive substrates, including a novel, porcine-derived spinal cord matrix (SCM). The SCM significantly increased frequency of neurite extension coordinated by a significant reduction in the neurites' time spent stalled. The SCM also mitigated inhibitory effects of CSA, producing longer neurites than the controls without CSA treatment. Next we aimed to elucidate receptors involved in mediating this behavior by testing the ability of CSA to upregulate cell-substrate binding receptors using flow cytometry. Our results showed a significant increase in syndecan-3 receptor expression in neurons treated with CSA. Furthermore, syndecans would most likely bind to the sulfated glycosaminoglycans measured in the SCM. Finally, we evaluated neurite growth on biomaterial scaffolds featuring CSA and SCM cues. Our results showed significantly increased neurite outgrowth on electrospun hyaluronic acid fibers with SCM and low levels of CSA. Higher incorporation of CSA maintained its inhibitory properties. Future work will evaluate coupling CSPGs with growth-permissive ECM to assess the combined effect on neurite outgrowth.

Germline ablation of dermatan-4O-sulfotransferase1 reduces regeneration after mouse spinal cord injury.

Chondroitin/dermatan sulfate proteoglycans (CSPGs/DSPGs) are major components of the extracellular matrix. Their expression is generally upregulated after injuries to the adult mammalian central nervous system, which is known for its low ability to restore function after injury. Several studies support the view that CSPGs inhibit regeneration after injury, whereas the functions of DSPGs in injury paradigms are less certain. To characterize the functions of DSPGs in the presence of CSPGs, we studied young adult dermatan-4O-sulfotransferase1-deficient (Chst14(-/-)) mice, which express chondroitin sulfates (CSs), but not dermatan sulfates (DSs), to characterize the functional outcome after severe compression injury of the spinal cord. In comparison to their wild-type (Chst14(+/+)) littermates, regeneration was reduced in Chst14(-/-) mice. No differences between genotypes were seen in the size of spinal cords, numbers of microglia and astrocytes neither in intact nor injured spinal cords after injury. Monoaminergic innervation and re-innervation of the spinal cord caudal to the lesion site as well as expression levels of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were similar in both genotypes, independent of whether they were injured and examined 6weeks after injury or not injured. These results suggest that, in contrast to CSPGs, DSPGs, being the products of Chst14 enzymatic activity, promote regeneration after injury of the adult mouse central nervous system.

Chondroitin-6-sulfate attenuates inflammatory responses in murine macrophages via suppression of NF-κB nuclear translocation.

Inflammation is a host protective response to noxious stimuli, and excessive production of pro-inflammatory mediators by macrophages (mφ) can lead to numerous pathological conditions. In this study, immunomodulatory effects of immobilized and soluble glycosaminoglycans (GAGs) on mouse-bone-marrow-derived mφ were compared by measuring nitric oxide (NO). We demonstrate here that all GAGs studied except for heparin were able to modulate interferon-γ/lipopolysaccharide (IFN-γ/LPS)-induced NO release by mφ to varying extents after 24h of incubation. In particular, the modulatory activities of soluble chondroitin-6-sulfate (C6S), hyaluronic acid and heparan sulfate altered markedly after covalent immobilization. Of these, soluble C6S exhibited the strongest NO inhibitory activity, and the inhibition was dose- and time-dependent. Moreover, C6S significantly reduced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α production by IFN-γ/LPS- or LPS-activated mφ. Specifically, the C6S-mediated suppression of mφ pro-inflammatory phenotype was accompanied by an increase in the IL-10 level, suggesting a possible switch towards anti-inflammatory/wound healing M2 state. In addition, the highest magnitude of inhibitory effects was obtained when cells were pre-treated with C6S prior to IFN-γ/LPS or LPS challenge, suggesting an additional role for C6S in protection against microbial infection. Further investigations reveal that the anti-inflammatory effects of C6S on activated mφ may be ascribed at least in part to suppression of NF-κB nuclear translocation.

Biosynthesis and function of chondroitin sulfate.

BACKGROUND: Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. SCOPE OF REVIEW: Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. MAJOR CONCLUSIONS: Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. GENERAL SIGNIFICANCE: Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.

The distribution and function of chondroitin sulfate and other sulfated glycosaminoglycans in the human bladder and their contribution to the protective bladder barrier.

PURPOSE: Glycosaminoglycan replenishment therapies are commonly applied to treat bladder inflammatory conditions such as bladder pain syndrome/interstitial cystitis. Although there is evidence that these therapies are clinically effective, much is still unknown about the location and function of different types of glycosaminoglycans in the bladder. We investigated the location of sulfated glycosaminoglycans in the bladder and evaluated their contribution to the urothelial barrier. MATERIALS AND METHODS: The location of different glycosaminoglycans (heparan sulfate, chondroitin sulfate and dermatan sulfate) in human and porcine bladders was investigated with immunofluorescence staining and isolating glycosaminoglycans using selective urothelial sampling techniques. Barrier function was evaluated with transepithelial electrical resistance measurements (Ω.cm(2)) on primary porcine urothelial cell cultures. The contribution of different glycosaminoglycans to the bladder barrier was investigated with specific glycosaminoglycan digesting enzymes and protamine. RESULTS: High glycosaminoglycan concentrations are located around the urothelial basal membrane and at the urothelial luminal surface. After removing the glycosaminoglycan layer, urothelial permeability increased. Natural recovery of the glycosaminoglycan layer takes less than 24 hours. Chondroitin sulfate was the only sulfated glycosaminoglycan that was located on the urothelial luminal surface and that contributed to urothelial barrier function. CONCLUSIONS: This study reveals an important role for chondroitin sulfate in bladder barrier function. Therapies aiming at restoring the luminal glycosaminoglycan layer in pathological conditions such as bladder pain syndrome/interstitial cystitis are based on a sound principle.

Chondroitin sulfate is a crucial determinant for skeletal muscle development/regeneration and improvement of muscular dystrophies.

Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.

Role of chondroitin sulfate C in the action of anthrax toxin.

Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax, and is responsible for the majority of disease symptoms. The toxin consists of 3 proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), which combine to form lethal and edema toxin. Glycosaminoglycans, which are present on the surface of cells, were investigated with regard to their role in toxicity resulting from anthrax toxin exposure. Lethal toxin-induced cytotoxicity of the RAW 264.7 cells was significantly inhibited by the addition of chondroitin sulfate C as determined by the MTT assay. By contrast, several other glycosaminoglycans, including heparin, heparan sulfate, and dermatan sulfate did not show significant levels of inhibition. Studies utilizing fluorescence-labeled PA demonstrated decreased PA binding to RAW 264.7 cells with the addition of chondroitin sulfate C. Formation of PA oligomers at the surface of cells after binding was also inhibited by chondroitin sulfate C. Interestingly, enzymatic degradation of endogenous chondroitin sulfate C from the cell surface with chondroitinase ABC was accompanied by increased sensitivity to the toxin. These findings were further confirmed by pretreating cells with sodium chlorate to reduce the degree of cell surface glycosaminoglycans sulfation. In addition, chondroitin sulfate C effectively inhibits edema toxin-induced cAMP accumulation in cells. Our results indicate that chondroitin sulfate C may play an important role in the toxicity of anthrax toxin.

Evaluation of host tissue integration, revascularization, and cellular infiltration within various dermal substrates.

Acellular dermal matrices are used in a variety of reconstructive and cosmetic procedures. There seems to be host tissue integration, revascularization, and recellularization into these products, but the exact timing and differences among these remain unknown. The purpose of this study is to determine and compare these properties of 4 different acellular dermal matrices (AlloDerm, DermACELL, DermaMatrix, and Integra) in an in vivo rat model. Tissue specimens were obtained at various time points. Histology and immunohistologic assays were used to quantify the extent of cellular infiltration and revascularization within the various matrices. A bimodal cellular response was observed in all products except for DermACELL. Cellular infiltration was highest in DermACELL and lowest in AlloDerm, and angiogenesis was evident by day 7. There were clear differences within the various products. It is undetermined whether these differences are advantageous or clinically significant. Future work is needed to define the specific roles for each.

Chondroitin sulfates in the developing rat hindbrain confine commissural projections of vestibular nuclear neurons.

BACKGROUND: Establishing correct neuronal circuitry is crucial to proper function of the vertebrate nervous system. The abundance of chondroitin sulfate (CS) proteoglycans in embryonic neural environments suggests that matrix proteoglycans regulate axonal projections when fiber tracts have not yet formed. Among the early-born neurons, the vestibular nucleus (VN) neurons initiate commissural projections soon after generation at E12.5 and reach the contralateral target by E15.5 in the rat hindbrain. We therefore exploited 24-hour cultures (1 day in vitro (DIV)) of the rat embryos and chondroitinase ABC treatment of the hindbrain matrix to reveal the role of CS moieties in axonal initiation and projection in the early hindbrain. RESULTS: DiI tracing from the VN at E12.5(+1 DIV) showed contralaterally projecting fibers assuming fascicles that hardly reached the midline in the controls. In the enzyme-treated embryos, the majority of fibers were unfasciculated as they crossed the midline at 90°. At E13.5(+1 DIV), the commissural projections formed fascicles and crossed the midline in the controls. Enzyme treatment apparently did not affect the pioneer axons that had advanced as thick fascicles normal to the midline and beyond, towards the contralateral VN. Later projections, however, traversed the enzyme-treated matrix as unfasciculated fibers, deviated from the normal course crossing the midline at various angles and extending beyond the contralateral VN. This suggests that CSs also limit the course of the later projections, which otherwise would be attracted to alternative targets. CONCLUSIONS: CS moieties in the early hindbrain therefore control the course and fasciculation of axonal projections and the timing of axonal arrival at the target.

Decorin and chondroitin-6 sulfate inhibit B16V melanoma cell migration and invasion by cellular acidification.

In vivo, cells are embedded in an environment generated and maintained by multiple cell-cell and cell-matrix interactions. While transiting the dermis metastasizing melanoma cells interact with the extracellular matrix (ECM) and fibroblasts. To study the roles of ECM components and fibroblasts in melanoma (B16V) cell migration and invasion, we established a co-culture system consisting of fibroblasts, their collagen-rich matrix and B16V cells. The crosstalk between B16V cells and fibroblasts was indicated by a clear increase in release and activity of matrix-metallo-protease-2. Time-lapse microscopy revealed that in B16V cells exposed to either decorin or chondroitin sulfates migration and invasion decreased by more than 50%. Decorin led to a reversible, chondroitin-6-sulfate to an irreversible, cytosolic acidification of B16V cells. Interestingly, decorin lowered NHE1 activity whereas chondroitin-6-sulfate did not. Furthermore, decorin and chondroitin-6-sulfate also acidified the pH at the cell surface which might prevent migration due to strong adhesion. In conclusion, the present co-culture system is an appropriate tool to analyze migration, invasion, and MMP release depending on cell-matrix interactions and the crosstalk between the invasive cells and those surrounded by their self-made matrix. We show a so far unknown function of decorin and chondroitin-6-sulfate: their ability to inhibit B16V cell migration by intracellular acidification.

Functions of chondroitin sulfate and heparan sulfate in the developing brain.

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.

Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages--the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses.

Effect of sulfated glycosaminoglycan digestion on the transverse permeability of medial collateral ligament.

Dermatan and chondroitin sulfate glycosaminoglycans (GAGs) comprise over 90% of the GAG content in ligament. Studies of their mechanical contribution to soft tissues have reported conflicting results. Measuring the transient compressive response and biphasic material parameters of the tissue may elucidate the contributions of GAGs to the viscoelastic response to deformation. The hypotheses of the current study were that digestion of sulfated GAGs would decrease compressive stress and aggregate modulus while increasing the permeability of porcine medial collateral ligament (MCL). Confined compression stress relaxation experiments were carried out on porcine MCL and tissue treated with chondroitinase ABC (ChABC). Results were fit to a biphasic constitutive model to derive permeability and aggregate modulus. Bovine articular cartilage was used as a benchmark tissue to verify that the apparatus provided reliable results. GAG digestion removed up to 88% of sulfated GAGs from the ligament. Removal of sulfated GAGs increased the permeability of porcine MCL nearly 6-fold versus control tissues. Peak stress decreased significantly. Bovine articular cartilage exhibited the typical reduction of GAG content and resultant decreases in stress and modulus and increases in permeability with ChABC digestion. Given the relatively small amount of GAG in ligament (<1% of tissue dry weight) and the significant change in peak stress and permeability upon removal of GAGs, sulfated GAGs may play a significant role in maintaining the apposition of collagen fibrils in the transverse direction, thus supporting dynamic compressive loads experienced by the ligament during complex joint motion.

Opposing functions of chondroitin sulfate and heparan sulfate during early neuronal polarization.

Axon-dendrite polarity of neurons is essential for information processing in the nervous system. Here we studied the functions of chondroitin sulfate (CS) and heparan sulfate (HS) in neuronal polarization using cultured dissociated hippocampal neurons. Immunohistochemical analyses of early cultured neurons indicated the distribution of these glycosaminoglycans to be quite different. While CS epitopes were accumulated in the focal contacts present in axons and cell bodies, those of HS were detected ubiquitously on the cell surface including on dendrites and axons. Treatment with chondroitinase (CHase) ABC, which degrades CS, and knockdown of a CS sulfotransferase, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST), which is involved in the biosynthesis of oversulfated structures, induced the formation of multiple axons in hippocampal neurons. Time-lapse recordings revealed the multiple axons of CHase ABC-treated neurons to be highly unstable, extending and retracting, repeatedly. CHase ABC-treatments suggested that CS is involved in the formation of phosphorylated focal adhesion kinase-positive focal contacts. Thus, CS may enhance integrin signaling in the nascent axons, supporting axon specification. On the other hand, when neurons were treated with heparitinases that specifically degrade HS, neurons with a single axon increased. The axons of HSase-treated neurons extended steadily and showed almost no retraction. These results suggest that CS stabilizes and HS destabilizes the growth of axons in an opposing manner, contributing to early neuronal polarization.