title: Proteoglycans of reactive rat cortical astrocyte cultures: abundance of N-unsubstituted glucosamine-enriched heparan sulfate.
abstract: "Reactive" astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a >5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~20% and ~40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.
authors.affiliation: Department of Anatomy and Neurobiology, Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, United States; Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, United States. Electronic address: thomas.hering@case.edu.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Hering
authors.affiliation: Department of Anatomy and Neurobiology, Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, United States.
authors.forename: Justin A
authors.initials: JA
authors.lastname: Beller
authors.affiliation: Department of Anatomy and Neurobiology, Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, United States.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Calulot
authors.affiliation: Department of Anatomy and Neurobiology, Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, United States.
authors.forename: Adrian
authors.initials: A
authors.lastname: Centers
authors.affiliation: Department of Anatomy and Neurobiology, Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, United States.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Snow
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry*; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D011509:Proteoglycans / Q000737:chemistry*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D003470:Culture Media; D006497:Heparan Sulfate; D011509:Proteoglycans; D016212:Transforming Growth Factor beta
keywords: Astrocytes; Chondroitin sulfate; Glial scar; Heparan sulfate; N-unsubstituted glucosamine; Proteoglycans; Reactive astrogliosis