Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA)

Summary Recent immunofluorescent and histochemical data [1] did not detect changes in the concentration of proteoglycans between noncalcified and calcified cartilage in fetal bovine growth plate or metaphyseal bone. These fidings were constant, regardless of prior fixation before demineralization with disodium ethylenediaminetetraacetate (EDTA) or prior demineralization before fixation. Previous experience has shown that EDTA can extract proteoglycans from calcified cartilage. With this in mind, we determined the amount of proteoglycan extracted from calcified cartilage in metaphyseal bone and uncalcified growth plate cartilages during decalcification of unfixed fresh tissues with EDTA. To this end, fresh growth plate cartilages and metaphyses were decalcified at 5°C for 48 hours in a buffered solution of EDTA to which several protease inhibitors were added. Under these conditions 20–25% of the total proteoglycan (measured as uronic acid and hexosamine) was extracted from mineralized cartilage but only about 1% from the uncalcified (growth plate) cartilages. Thus, histochemical and immunohistochemical studies appear to be insensitive measures of proteoglycan concentrations in histological sections of mineralized tissue and may not give quantitative information.     

The response of the canine intervertebral disc to immobilization produced by spinal arthrodesis is dependent on constitutional factors

Posterior lumbar spinal fusion was performed on five mature greyhounds. Two months prior to death, all of the surgical and five age-matched control greyhounds were given Na2(35)SO4 (1.0 mCi/kg) intravenously. All fusion animals were killed 6 months postoperation, and discs beneath the fusion mass as well as those adjacent to it (parafusion discs) were sampled separately and dissected into the nuclei pulposi and annuli fibrosi (AF). Proteoglycans (PGs) were extracted with 4.0 M GuHCl and then purified by CsCl density gradient ultracentrifugation. These PG monomers were subjected to Sepharose CL-2B chromatography, and their hydrodynamic size and ability to aggregate were determined. The level, extractability, and hydrodynamic size of PGs in the AF of fusion discs were found to be greater than those in control discs, as were the keratan sulfate core protein complexes prepared by chondroitin ABC lyase digestion. The ability of the 60-day-old PG subunit populations, isolated from fusion discs, to aggregate was also higher than controls. There was, however, no difference between the galactosamine/glucosamine, galactosamine/protein, glucosamine/protein, or hexuronate/protein ratios of PGs in fusion and control discs.     

Structural changes in reassembled growth plate aggregates

To investigate possible structural changes in reassembled proteoglycan aggregates during cartilage mineralization, we examined the molecular architecture and dimensions of growth plate proteoglycan aggregates by electron microscopy. The ends of fetal bovine femurs and tibias were separated into three regions: the epiphysis; the cartilage growth plate, consisting of the proliferative zone and the unmineralized portion of the hypertrophic zone; and the calcified portion of the hypertrophic zone along with part of the metaphysis. Aggregates from all three regions had the same molecular architecture. They consisted of central hyaluronic filaments with multiple attached monomers. Monomers consisted of two segments: a peripheral thick segment, which represents primarily the chondroitin sulfate-rich region, and a thin segment attached directly to the hyaluronic acid filament. The length of aggregated monomers did not differ between the growth plate cartilage and the metaphysis, nor did the lengths of the thin and thick segments, indicating that the chondroitin sulfate-rich region of aggregated monomers is not degraded during cartilage mineralization. Between the growth plate cartilage and the metaphysis, aggregates became shorter and had fewer monomers and wider spacing between monomers. These structural alterations in proteoglycan aggregates may be one of the events that prepares the matrix for mineralization.     

Newly synthesized proteoglycans in pseudoachondroplasia

In an attempt to elucidate the biochemical defect in pseudoachondroplasia, proteoglycan metabolism was investigated in cartilage from a patient with the dominant form of this condition. Iliac-crest cartilage was radioactively labeled with ³⁵S-sulfate and the newly synthesized proteoglycans examined for their hydrodynamic size and glycosaminoglycan composition. The banding pattern of the purified proteoglycans was analyzed by gel-electrophoresis using large pore polyacrylamide-agarose. We found a normal chain-length of glycosaminoglycans and a normal ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. The proteoglycans were not enriched in keratan sulfate. Gel electrophoretic analysis of the proteoglycans disclosed a banding pattern comparable to that of two normal controls. In contrast to the findings of other authors no differences between the proteoglycans of pseudoachondroplastic and normal cartilage were detected.     

Effects of chondroitinase-ABC on proteoglycans and swelling properties of fibrocartilage in bovine flexor tendon

Fibrocartilaginous regions of bovine deep flexor tendon were treated with chondroitinase-ABC and trypsin in order to extract proteoglycans from the extracellular matrix and thereby investigate the contribution of proteoglycan and collagen organization to tissue material properties. Chondroitinase-ABC digestion of tendon specimens for 24 h resulted in extraction of 60% of tissue glycosaminoglycan and leaching of the degraded large proteoglycan from the tissue residue. The totally degraded core protein of the small dermatan sulfate proteoglycan remained with the tissue residue, indicating that it is specifically associated with the tissue residue and that this association is not dependent on the glycosaminoglycan chains. Treatment of residues with trypsin after chondroitinase-ABC digestion depleted the specimens of proteoglycan. Bulk swelling tests on enzyme-extracted specimens showed that the distinct swelling properties of the fibrocartilaginous regions of the distal flexor tendon could be partially accounted for by elevated levels of proteoglycan. Swelling tests also showed that the distinct collagen organization of this region contributes significantly to the tissue's material properties. These results suggest that the fibrocartilaginous organization and composition of the articulating layer of distal tendon are adapted for mechanical requirements unique to this site, which receives compressive and frictional loads in addition to tensile loads.     

Effect of nitric oxide synthase inhibitor on proteoglycan metabolism in repaired articular cartilage in rabbits

Nitricoxide (NO )isanimportantregulatoryfactorandasignal transductionmoleculeintheprocessofcellgrowthanddifferentiation .Recentreportshaveindicatedthatnitricoxideiscloselyrelatedtothedisintegrationofarticularcartilage,becausehighexpressionofinducednitricoxidesynthase (iNOS)inhibitormRNAisdetectedinthearticularcartilageandiNOScanamelioratethecartilagemetabolisminpatientswithosteoarthritisorrheumaticarthritis .1,2 OurpreviousstudyshowedthatiNOScanamelioratethe qualityofrepairedcartilages ,t…     

Mechanisms of proteoglycan inhibition of hydroxyapatite growth

Summary Purified bovine nasal cartilage proteoglycans (aggregate and subunit containing fractions) and to a lesser degree, chondroitin 4-sulfate of physiological size, retard seeded hydroxyapatite (HA) growthin vitro. The large hydrodynamic size and high charge density of these macromolecules are believed to be associated with the ability of proteoglycans to inhibit HA formation and growth. We now demonstrate the involvement of the negative charges of proteoglycans in this inhibition (a) by comparing the inhibitory ability of chondroitin 4-sulfate and its desulfated analog, and (b) by comparing the growth of HA seed crystals coated either with proteoglycan aggregates or chondroitin 4-sulfate to that of uncoated crystals. In the desulfation experiments, desulfated chondroitin sulfate was a less efficient HA growth inhibitor than untreated, undesulfated chondroitin sulfate of similar molecular size. Dextran sulfate showed higher inhibitory effectiveness than unchanged neutral dextran. Both experiments suggest that sulfate groups play an important role in the regulation of mineral deposition by proteoglycans. In the coating experiment, precoating of HA seed crystals with proteoglycan aggregates decreased the amount of HA precipitated as a function of time, suggesting proteoglcans may block the active nucleating sites on HA surface and slow down the growth process. Chondroitin 4-sulfate has a similar but weaker coating effect. Neural dextran, having little affinity for HA, had no effect.     

The ultrastructural localization of sulfated proteoglycans is identical in the amyloids of Alzheimer's disease and AA,AL, senile cardiac and medullary carcinoma-associated amyloidosis

Summary The cationic dyes cuprolinic blue and ruthenium red were used to ultrastructurally localize proteoglycans (PGs) within the neuritic plaque and neurofibrillary tangle of Alzheimer's disease. Highly sulfated PGs were specifically localized to the amyloid fibril of the neuritic plaque and the paired filaments of the neurofibrillary tangle. This demonstrates that highly sulfated PGs either comprise part of the Alzheimer's amyloid fibril and paired filament or are intimately associated with them. Four unrelated types of amyloid — AA (inflammation-associated), AL (immunoglobulin light chain), senile cardiac (prealbumin) and medullary carcinoma-associated amyloid (procalcitonin) — showed an identical pattern of localization of highly sulfated PG to the different amyloid fibrils. This constant close spatial relationship between PGs and diverse amyloid proteins suggests that PGs may play a role in amyloidogenesis.Supported by Grant MT 3153 from the Medical Research Council of Canada and by a Dean's MRC award from Queen's University     

Interleukin-1 and nerve growth factor induce hypersecretion and hypersulfation of neuroblastoma proteoglycans which bind β-amyloid

Inflammation and the response to injury may play an important role in the process of amyloidosis in Alzheimer's disease. We investigated the effect of interleukin-1 (IL-1) and nerve growth factor (NGF) on the metabolism of neuroblastoma proteoglycans. IL-1 and NGF increased the net charge and the net secretion of neuroblastoma proteoglycans. NGF also specifically increased the relative amount of cell-associated and secreted heparan sulfate proteoglycans in these cells. We previously demonstrated that neuroblastoma heparan sulfate proteoglycan binds specifically to the amyloid β-amyloid peptide involved in Alzheimer's disease. Heparan sulfate glycosaminoglycans synthesized by IL-1-stimulated cells demonstrated an increased relative binding affinity for the β-amyloid peptide. Thus, IL-1 and NGF induce the hypersecretion and hypersulfation of neuroblastoma heparan sulfate proteoglycans which bind β-amyloid. These studies link the process of inflammation and repair with alterations in the metabolism of heparan sulfate proteoglycans and amyloid formation in Alzheimer's disease and other disorders.