keratan-sulfate and Disease-Models--Animal

Mucopolysaccharidosis IVA (MPS IVA) is a rare disorder caused by mutations in the N-acetylgalactosamine-6-sulfate-sulfatase (

Topics: Animals; Chondrocytes; Chondroitin Sulfates; Chondroitinsulfatases; Disease Models, Animal; Humans; Keratan Sulfate; Mice; Mucopolysaccharidosis IV

Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage.. The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots.. Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively.. PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.

Topics: Animals; Anterior Cruciate Ligament; Cartilage, Articular; Cattle; Disease Models, Animal; Humans; Keratan Sulfate; Knee Joint; Matrix Metalloproteinase 3; Nitric Oxide; Osteoarthritis; Protein Kinase C; Rabbits; Stress, Mechanical; Superoxides; Tensile Strength; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-1

The serum level of a highly sulfated epitope present on long keratan sulfate chains provides a direct measure of the rate of catabolism of cartilage proteoglycans. Levels of the keratan sulfate epitope are elevated in patients with generalized osteoarthritis (OA), indicating these individuals have elevated rates of cartilage proteoglycan catabolism. In the Pond-Nuki model of canine OA, the serum level of the keratan sulfate epitope rises rapidly after the transection of the anterior cruciate ligament, long before OA lesions can be detected, and remains high for at least 13 weeks.

Topics: Animals; Biomarkers; Cartilage; Disease Models, Animal; Dogs; Keratan Sulfate; Osteoarthritis; Proteoglycans

Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.

Topics: Animals; Disaccharides; Disease Models, Animal; Interleukin-6; Keratan Sulfate; Lectins, C-Type; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

Topics: Alkalies; Animals; Biomarkers; Burns, Chemical; Corneal Diseases; Dermatan Sulfate; Disease Models, Animal; Extracellular Matrix; Eye Burns; Fluorescent Antibody Technique; Gene Expression; Glycosaminoglycans; Heparitin Sulfate; Keratan Sulfate; Mice; Proteoglycans

Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine-transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization.. A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively.. Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased.. Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

Topics: Animals; Corneal Stroma; Disease Models, Animal; Keratan Sulfate; Mice; Mice, Knockout; N-Acetylglucosaminyltransferases; Phenotype

Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here, we show that keratan sulfate (KS), one of the major glycosaminoglycans produced in the small airway, decreased in lungs of cigarette smoke-exposed mice. To confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO

Topics: Animals; Bronchoalveolar Lavage Fluid; Dexamethasone; Disaccharides; Disease Models, Animal; Disease Progression; Keratan Sulfate; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Models, Biological; Pancreatic Elastase; Pneumonia; Pulmonary Alveoli; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; RAW 264.7 Cells; Smoking; Sus scrofa

We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology [1]. To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in the liver and spleen, with detectable activity in the bone and brain. Second, newborn ERT was conducted after a tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th weeks were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in the liver and spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than that in adult mice treated with ERT; however, hyaline and fibrous cartilage cells in the femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mice.

Topics: Administration, Intravenous; Animals; Animals, Newborn; Bone Diseases; Cartilage; CHO Cells; Chondrocytes; Chondroitinsulfatases; Cricetulus; Disease Models, Animal; Enzyme Replacement Therapy; Growth Plate; Keratan Sulfate; Liver; Mice; Mice, Knockout; Mucopolysaccharidosis IV; Recombinant Proteins; Spleen; Tissue Distribution

Proteoglycans (PGs) are the components of extracellular matrices in the central nervous system (CNS). Keratan sulfate (KS) is a glycosaminoglycan that is included in the KSPG that acts as an inhibitory factor in nerve regeneration after CNS injury. To investigate the role of KS in immune diseases, we induced experimental autoimmune encephalomyelitis (EAE) in mice that were deficient in the N-acetylglucosamine (GlcNAc)-6-O-sulfotransferase 1 (GlcNAc6ST1) gene (KS-KO). KS-KO mice developed less severe EAE and showed repressed recall response in the induction phase. Furthermore, GlcNAc6ST1 might have roles in the passage of the pathogenic lymphocytes through the blood-brain barrier via adhesion molecules. Thus, modulation of KS may become a treatment for neuroimmunological diseases.

Topics: Adoptive Transfer; Animals; Carbohydrate Sulfotransferases; Cell Proliferation; Cytokines; Disease Models, Animal; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Keratan Sulfate; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Sulfotransferases; T-Lymphocytes

In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2β1, αMβ2, and αXβ2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.

Topics: Animals; Antibodies, Blocking; Bleomycin; Cell Differentiation; Cell Polarity; Chemical Fractionation; Chondroitin Sulfate Proteoglycans; Culture Media, Conditioned; Decorin; Disease Models, Animal; Fibroblasts; Humans; Integrin alpha2; Intercellular Signaling Peptides and Proteins; Keratan Sulfate; Lumican; Lung; Macrophages; Nerve Tissue Proteins; Peptides; Pulmonary Fibrosis; Trypsin; Tumor Necrosis Factor-alpha

The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1(G93A) ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3-positive activated or proliferating microglia of SOD1(G93A) ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1(G93A) mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1(G93A)/KSGal6ST(-/-) mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1(G93A)/GlcNAc6ST1(-/-) mice. These results indicate that both galactose-6- and N acteylglucosamine-6-sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation.

Topics: Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Animals; Brain; Carbohydrate Sulfotransferases; Disease Models, Animal; Epitopes; Female; Galectin 3; Humans; Keratan Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Middle Aged; Motor Neurons; Mutation, Missense; Spinal Cord; Sulfotransferases; Superoxide Dismutase

Pathological myopia (PM) is a hereditary ocular disease leading to severe loss of visual acuity and blindness. Lumican gene (LUM) is one of those candidate genes of PM. The purpose of this study was to establish a mutant Lumican transgenic mouse model, and to prepare for the further study of the pathogenesis of PM.. Experimental study. Mutation of LUM gene was created by site-directed mutagenesis. Recombinant DNA techniques were used for the construction of the pRP. EX3d-EF1A>LUM/flag>IRES/hrGFP transgene. The gene fragments were microinjected into the zygote male pronuclei of BDF1 mice, and then the zygote cells alive were transplanted into the oviduct of acceptor pregnant female ICR mice. The F0 generation transgenic mice obtained were named C57-TgN (LUM)CCMU. Genome DNA from mice tail was detected by PCR and Western blotting.. Six of 31 F0 generation mice were positive transgenic mice. The western blotting study showed that the flag-tag was expressed in the mouse tail tissue. Sixty-eight of 128 mice (F1 to F3 generation) were positive transgenic mice, the positive rate is 53.13%.. The mutant Lumican (cDNA 596T>C) transgenic mouse model has been established. This model will provide fundamental conditions for studies of the pathogenesis of PM. Also it will be the basis of further studies about the effect of Lumican mutation on the development of PM and structure and function of the extra cellular matrix.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Exons; Female; Keratan Sulfate; Lumican; Male; Mice; Mice, Inbred ICR; Mice, Transgenic; Mutagenesis, Site-Directed; Myopia; Plasmids

To evaluate the relevance between lumican expression patterns and the clinical course of patients with pancreatic ductal adenocarcinoma (PDAC), and to investigate the role of lumican in PDAC progression.. One hundred thirty-one patient tumors were chosen for tissue microarray staining, and Cox regression analysis was used to test the associations between lumican expression and clinical, pathologic, and oncologic outcomes in all patients. Primary PDAC cells and recombinant human lumican protein were used to establish a working model to mimic the in vivo interactions between stromal lumican and PDAC cells. Using this model, we tested the effects of lumican on EGFR signaling via Akt and hypoxia-inducible factor-1α (HIF1α) and its subsequent influence on glucose consumption, lactate production, intracellular ATP, and apoptotic cell death.. Lumican was present in the stroma surrounding PDAC cells in roughly one-half of primary tumors and the direct xenografts. Patients with stromal lumican were associated with a profound reduction in metastatic recurrence after surgery and 3-fold longer survival than patients without stromal lumican. In PDAC cells, extracellular lumican reduced EGFR expression and phosphorylation through enhanced dimerization and internalization of EGFR and the resultant inhibition of Akt kinase activity. Lumican also reduced HIF1α expression and activity via Akt. PDAC cells with enhanced HIF1α activity were resistant to lumican-induced inhibition of glucose consumption, lactate production, intracellular ATP, and apoptosis.. There is a positive association between stromal lumican in primary PDAC tumors and prolonged survival after tumor resection. Lumican plays a restrictive role in EGFR-expressing pancreatic cancer progression.

Topics: Animals; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; ErbB Receptors; Extracellular Space; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Keratan Sulfate; Lumican; Pancreatic Neoplasms; Patient Outcome Assessment; Prognosis; Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

On the basis of the role of small, leucine-rich proteoglycans (SLRPs) in fibrogenesis and inflammation, we hypothesized that they could be involved in cardiac remodeling and reverse remodeling as occurs during aortic stenosis and after aortic valve replacement. Thus, in a well-characterized aortic banding-debanding mouse model, we examined the SLRPs decorin and lumican and enzymes responsible for synthesis of their glycosaminoglycan (GAG) chains. Four weeks after banding of the ascending aorta, mice were subjected to a debanding operation (DB) and were subsequently followed for 3 or 14 days. Sham-operated mice served as controls. Western blotting revealed a 2.5-fold increase in the protein levels of glycosylated decorin in mice with left ventricular pressure overload after aortic banding (AB) with a gradual decrease after DB. Interestingly, protein levels of three key enzymes responsible for decorin GAG chain synthesis were also increased after AB, two of them gradually declining after DB. The inflammatory chemokine (C-X-C motif) ligand 16 (CXCL16) was increased after AB but was not significantly altered following DB. In cardiac fibroblasts CXCL16 increased the expression of the GAG-synthesizing enzyme chondroitin polymerizing factor (CHPF). The protein levels of lumican core protein with N-linked oligosaccharides increased by sevenfold after AB and decreased again 14 days after DB. Lumican with keratan sulfate chains was not regulated. In conclusion, this study shows alterations in glycosylated decorin and lumican core protein that might be implicated in myocardial remodeling and reverse remodeling, with a potential important role for CS/DS GAG chain-synthesizing enzymes.

Topics: Animals; Aortic Valve Stenosis; Blotting, Western; Cells, Cultured; Chemokine CXCL16; Chemokine CXCL6; Chondroitin Sulfate Proteoglycans; Decorin; Disease Models, Animal; Fibroblasts; Gene Expression Regulation, Enzymologic; Glucuronosyltransferase; Glycosylation; Glycosyltransferases; Inflammation Mediators; Keratan Sulfate; Lumican; Male; Mice; Mice, Inbred C57BL; Multifunctional Enzymes; Myocardial Contraction; Myocardium; N-Acetylgalactosaminyltransferases; Time Factors; Ultrasonography; Ventricular Pressure; Ventricular Remodeling

Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies.. Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM.. Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90-110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy.. Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling.

Topics: Animals; Biglycan; Chondroitin Sulfate Proteoglycans; Decidua; Diabetes Mellitus, Type 1; Disease Models, Animal; Disease Progression; Embryo Implantation, Delayed; Embryo Loss; Extracellular Matrix; Female; Fetal Diseases; Fibrillar Collagens; Gene Expression Regulation, Developmental; Interleukin-11; Keratan Sulfate; Lumican; Mice; Placentation; Pregnancy; Pregnancy in Diabetics; RNA, Messenger

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.

Topics: Animals; Biomarkers; Bone and Bones; Bone Density; Bone Diseases, Developmental; Chondrocytes; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Keratan Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidoses; Spinal Canal; X-Ray Microtomography

The aim of this study was to investigate the correlations of severity of osteoarthritis (OA) and serum biomarkers including keratan sulfate (KS), hyaluronic acid (HA) and chondroitin sulfate (CS) 846 epitope. We also investigated the effect of glucosamine and fish collagen peptide (FCP) on OA. OA was induced in 12 rabbits (12 weeks of age) by anterior cruciate ligament transection (ACLT). After the surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or FCP and glucosamine (FG group) for 4 weeks. The control group was provided water ad libitum (C group). Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT) for serum marker measurement. Biomarker levels were measured by using commercial kits. We evaluated OA severity both macroscopically and histologically. Macroscopic evaluation showed mildly eroded condylar surfaces in the C group. Histological findings were significantly different from the FG and other groups. There were no significant differences between each group at post-ACLT in terms of serum KS, HA and CS 846. Histological assessment and serum biomarker measurements performed at post-ACLT showed a significant correlation between HA concentration and OA severity. Variations in the CS 846 concentration at pre-ACLT and post-ACLT were significantly correlated with OA severity. Administration of glucosamine and FCP had chondroprotective effects in the ACLT model. Serum biomarker concentrations were significantly correlated with cartilage injury. Serum biomarker measurement would be useful for monitoring articular cartilage damage in the clinical setting.

Topics: Animals; Anterior Cruciate Ligament; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Collagen; Disease Models, Animal; Glucosamine; Histocytochemistry; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Rabbits; Statistics, Nonparametric

Mechanical ventilation used in patients with acute lung injury can damage pulmonary epithelial cells through production of inflammatory cytokines and excess deposition of the extracellular matrix protein lumican. Lumican participates in macrophage inflammatory protein (MIP)-2 and transforming growth factor-β₁ (TGF-β₁) signaling during the fibroproliferative phase of acute lung injury, which involves a process of epithelial-mesenchymal transition (EMT). The mechanisms regulating interactions between mechanical ventilation and lung injury are unclear. We hypothesized that lung damage and EMT by high tidal volume (Vt) mechanical stretch causes upregulation of lumican that modulates MIP-2 and TGF-β₁ through the extracellular signal-regulated kinase (ERK) 1/2 pathway.. Male C57BL/6 mice (either wild type or lumican null) aged 3 months and weighing between 25 and 30 g were exposed to low Vt (6 mL/kg) or high Vt (30 mL/kg) mechanical ventilation with room air for 2 to 8 h. Nonventilated mice were used as control subjects.. We found that high Vt mechanical ventilation increased microvascular permeability, neutrophil influx, production of free radicals, MIP-2 and TGF-β₁ proteins, positive staining of α-smooth muscle actin and S100A4/fibroblast-specific protein-1, Masson trichrome staining and extracellular collagen, and activation of lumican and ERK1/2 in wild-type mice. Decreased staining of the epithelial marker E-cadherin was also observed. Mechanical stretch-augmented EMT was attenuated with lumican-deficient mice and pharmacologic inhibition of ERK1/2 activity by PD98059.. The data suggest that lumican promotes high Vt mechanical ventilation-induced lung injury and EMT through the activation of the ERK1/2 pathway.

Topics: Animals; Cadherins; Chemokine CXCL2; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Epithelial-Mesenchymal Transition; Flavonoids; Keratan Sulfate; Lumican; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Kinase Inhibitors; Respiration, Artificial; Tidal Volume; Transforming Growth Factor beta1; Ventilator-Induced Lung Injury

Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen-associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by Toll-like receptor (TLR) 4, and neutrophil chemotaxis via integrins.. Here we investigated the role of lumican in the development of colitis mediated by intrarectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum(+/+) and Lum(-/-) mice.. The TNBS treated Lum(+/+) mouse colons showed marked increases in CXCL1, tumor necrosis factor alpha (TNF-α), and neutrophil infiltration, whereas these responses were significantly dampened in the Lum(-/-) mice. The nuclear factor kappa B (NF-κB) transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum(+/+) but not in Lum(-/-) colons. Also, nuclear translocation of NF-κB was delayed in LPS stimulated Lum(-/-) primary peritoneal macrophages.. The Lum(-/-) mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4(-/-) and MyD88(-/-) mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

Topics: Animals; Blotting, Western; Chondroitin Sulfate Proteoglycans; Colitis; Cytokines; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Female; Flow Cytometry; Haptens; Inflammation; Keratan Sulfate; Lipopolysaccharides; Lumican; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Toll-Like Receptor 4; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha

In a previous report, we found that the occurrence and amount of meningothelial cell nests in the subarachnoid space are significantly increased in glaucomatous optic nerves compared to normals. In order to allow research into the role of meningothelial cells during diseases of the optic nerve, an in vitro model is necessary. For this purpose, we developed a culture method for porcine meningothelial cells from the arachnoid layer covering the optic nerve.. Meningothelial cells were scraped from the arachnoid layer of porcine optic nerves and cultured for 2-3 weeks until the cells formed a monolayer. To eliminate contaminating fibroblasts from the culture, cells were negatively selected using magnetic anti-fibroblast beads after the first passage. Cells were detached using 0.05% Trypsin-EDTA, incubated with anti-fibroblast beads, separated using a magnetic column and the flow-through was collected. The purified primary meningothelial cells were characterized by electron microscopy and immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) and anti-keratan sulfate antibodies.. Primary cells grew out after dissection and formed a monolayer within 2-3 weeks, which was composed of two morphologically different cell types, flattened cells with round nuclei and fibroblast-like cells with long processes. The fibroblast-like cells in the culture could be labelled and selected using anti-fibroblast microbeads. The second cell type did not bind to the anti-fibroblast beads, and upon immunocytochemistry showed a marked expression of both GFAP and keratan sulphate. In addition, examination of these cells by electron microscopy revealed morphological characteristics of meningothelial cells, including hemidesmosomes and cytoplasmatic filaments.. The technique described in this paper for the primary culture of meningothelial cells from the subarachnoid space of the optic nerve and using magnetic beads for the removal of fibroblasts is effective in obtaining a highly enriched meningothelial cell culture.

Topics: Animals; Cell Culture Techniques; Disease Models, Animal; Fibroblasts; Glaucoma; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; Immunomagnetic Separation; Keratan Sulfate; Optic Nerve; Optic Nerve Diseases; Subarachnoid Space; Swine

Previous studies have demonstrated that the lack of lumican delayed corneal wound healing in lumican-null (Lum(-/-) ) mice. This defect is rescued by the addition of glycosylated lumican core protein to the injured corneas.. We examined the hypothesis that lumican is also required for the healing of cutaneous wounds using Lum(-/-) mice.. We demonstrated the basic thinner skin phenotypes in Lum(-/-) mice at different time points and the changes in arrangement of collagen fibres by transmission electron microscopy (TEM). A full skin thickness wound was generated by punch biopsy (6 mm diameter) in experimental Lum(-/-) and wild-type mice. The closure of injured skin was measured after various periods of time (3, 6, 12, 18 days). Specimens of injured and uninjured skin (serving as control) were then subjected to morphological examination with haematoxylin and eosin and Masson trichrome stains, and by TEM. Immunohistochemical staining with anti-CD68 antibody was used to assess the presence of macrophages in injured skin healing for various periods of time. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to elucidate the transforming growth factor (TGF)-β1-induced myofibroblast phenotypic genes.. Skin of adult Lum(-/-) mice (3 months and older) was much thinner (40% less) than that of age-matched wild-type mice. This phenomenon was aggravated in older mice. TEM revealed disoriented and irregular collagen fibrils in the dermis of Lum(-/-) mice. Delayed wound healing with an increase in inflammatory macrophages was compatible with the delayed response of the expression of TGF-β1, type I collagen α1 and fibronectin at the mRNA level by semiquantitative RT-PCR in the Lum(-/-) mice.. Our data demonstrate that lumican plays pivotal roles in skin collagen fibrillogenesis and wound healing.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; Fibronectins; Immunohistochemistry; Keratan Sulfate; Lumican; Mice; Mice, Knockout; Microscopy, Electron; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Transforming Growth Factor beta1; Wound Healing

Proteoglycans bearing keratan sulfate (KS), such as aggrecan, are components of the human cartilage extracellular matrix (ECM). However, the role of KS in influencing cartilage degradation associated with arthritis remains to be completely understood. KS side chains of the length found in human cartilage are not found in murine skeletal tissues. Using a murine model of inflammatory polyarthritis and cartilage explants exposed to interleukin-1α (IL-1α), we examined whether administering KS could influence intraarticular inflammation and cartilage degradation. Acute arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail, followed by an intraperitoneal injection of lipopolysaccharide. This treatment was followed by an intraperitoneal KS administration in half of the total mice to evaluate the therapeutic potential of KS for ameliorating arthritis. To investigate the therapeutic potential ex vivo, we examined cartilage fragility by measuring IL-1α-induced aggrecan release from cartilage explants treated with or without KS. Intraperitoneal KS administration ameliorated arthritis in DBA/1J mice. The aggrecan release induced by IL-1α was less in cartilage explants containing media with KS than in those without KS. Our data indicate that exogenous KS ameliorated arthritis in vivo and suppressed cartilage degradation ex vivo. KS may have important therapeutic potential in the treatment of inflammatory arthritis. The mechanism responsible for this requires further investigation, but KS may become a novel therapeutic agent for treating inflammatory diseases such as rheumatoid arthritis.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cartilage; Disease Models, Animal; Interleukin-17; Keratan Sulfate; Lymphotoxin-alpha; Mice; Mice, Inbred DBA; Tumor Necrosis Factor-alpha

Bioscaffolds have been successfully used to improve the healing of ligaments and tendons. In a rabbit model, the application of porcine small intestine submucosa (SIS) to the healing medial collateral ligament (MCL) resulted in improved mechanical properties with the formation of larger collagen fibrils. Thus, the objective of the study was to find out whether the SIS bioscaffold could improve the gene expressions of fibrillogenesis-related molecules, specifically, collagen types I, III, V, and small leucine-rich proteoglycans including decorin, biglycan, lumican, and fibromodulin, as well as collagen fibril morphology and organization, in the healing rabbit MCL at an early time point (6 weeks postinjury). Twenty skeletally mature rabbits were equally divided into two groups. In the SIS-treated group, a 6-mm gap was surgically created and a layer of SIS was sutured to cover the gap, whereas the gap was left open in the nontreated group. At 6 weeks postinjury, Masson's trichrome staining showed that the SIS-treated group had more regularly aligned collagen fibers and cells. Transmission electron microscopy revealed that the SIS-treated group had larger collagen fibrils with a diameter distribution from 24 to 120 nm, whereas the nontreated group had only small collagen fibrils (ranging from 26 to 87 nm, p < 0.05). Finally, the quantitative real-time PCR showed that the mRNAs of collagen type V, decorin, biglycan, and lumican in the SIS-treated group were 41, 58, 51, and 43% lower than those in the nontreated group, respectively (p < 0.05). Such significant reduction in the gene expressions are closely related to the improved morphological characteristics, which are known to be coupled with better mechanical properties, as previously reported in longer term studies.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Collagen; Collagen Type I; Collagen Type III; Collagen Type V; Decorin; Disease Models, Animal; Extracellular Matrix Proteins; Female; Fibromodulin; Intestinal Mucosa; Keratan Sulfate; Lumican; Medial Collateral Ligament, Knee; Microscopy, Electron, Transmission; Proteoglycans; Rabbits; RNA, Messenger; Swine; Tissue Engineering; Wound Healing

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.

Topics: Animals; Apoptosis; Autoimmune Diseases; Disaccharides; Disease Models, Animal; Female; Glomerulonephritis; Glycosaminoglycans; Hypergammaglobulinemia; In Situ Nick-End Labeling; Interferon-gamma; Interleukin-12; Keratan Sulfate; Lipopolysaccharides; Lymph Nodes; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Oligosaccharides; Phosphatidylinositol 3-Kinases; Protein Kinase C; T-Lymphocytes; Thy-1 Antigens

To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA).. Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4C(long mono), or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses.. Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4C(long mono) and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4C(long mono) was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4C(long mono) was elevated at 12 weeks after surgery.. Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4C(long mono) and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.

Topics: Aggrecans; Animals; Anterior Cruciate Ligament; Biomarkers; Calcium-Binding Proteins; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Collagen; Collagen Type II; Disease Models, Animal; Dogs; Epitopes; Extracellular Matrix Proteins; Female; Keratan Sulfate; Lectins, C-Type; Lumican; Osteoarthritis; Proteoglycans; Stifle; Synovial Fluid

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 microl of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and alpha-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFbeta1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

Topics: Actins; Adenoviridae; Animals; Cell Division; Chondroitin Sulfate Proteoglycans; Collagen Type VI; Disease Models, Animal; DNA-Binding Proteins; Epithelial Cells; Eye Injuries; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Keratan Sulfate; Lens Capsule, Crystalline; Lens, Crystalline; Lumican; Male; Mesoderm; Mice; Mice, Inbred C57BL; RNA, Messenger; Smad7 Protein; Trans-Activators; Transduction, Genetic; Transforming Growth Factor beta; Wound Healing

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-beta (TGF-beta) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-beta and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of alpha-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-beta signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Topics: Actins; Animals; Becaplermin; Biomarkers; Cell Differentiation; Cell Line; Cell Movement; Chondroitin Sulfate Proteoglycans; Collagen Type VI; Disease Models, Animal; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; Humans; Keratan Sulfate; Lumican; Mice; Mice, Inbred Strains; Mice, Knockout; Pigment Epithelium of Eye; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Retinal Detachment; Signal Transduction; Smad3 Protein; Swine; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitreoretinopathy, Proliferative

To minimize the neurotoxic injury by clot-derived substances after intracerebral hemorrhage (ICH) on the surrounding brain tissue, minimally invasive neurosurgical protocols have evolved evacuating the hematoma by stereotaxic injection of a fibrinolytic agent such as recombinant tissue plasminogen activator (rtPA), followed by aspiration of the lysed clot. However, the possible contribution of the presence of exogenous tPA itself to the toxic effects of hematoma-derived factors complicates the rationale and efficacy of this therapeutic approach. To clarify the role of exogenous rtPA on edema development, we examined the extent of edema formation in a murine model of collagenase-induced ICH, which included tPA-deficient (tPA-/-) and wild-type (wt) mice. In 16 (7 tPA-/- and 9 wt mice) out of 32 mice, 1 mg/kg rtPA was injected into the hematoma 5 h after ICH induction followed by aspiration of the liquefied clot 20 min later. In the control group (8 tPA-/- and 8 wt mice), only collagenase was injected. The edema volume was quantified using SPOT software on Luxol Fast Blue and Cresyl violet-stained cross-sections 24 h, 3, and 7 days post surgery. Twenty-four hours after ICH induction, tPA-/- mice had a significantly smaller edema volume (P< 0.01), even when rtPA was administered. Between days 3 and 7 after ICH, exogenous rtPA exerts its edema-promoting effect irrespective of the underlying genotype and exhibits an extensive microglial activation adjacent to the clot. In conclusion, the role of the endogenous tPA appears to be limited to the early phase of edema formation, whereas exogenous rtPA is edema-promoting between days 3 and 7 after ICH.

Topics: Animals; Blood Proteins; Brain; Brain Edema; Cerebral Hemorrhage; Collagenases; Disease Models, Animal; Fluorescent Antibody Technique; Indoles; Keratan Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Recombinant Proteins; Staining and Labeling; Time Factors; Tissue Plasminogen Activator

This study examined if viscosupplementation from intra-articular administration of a commercially available form of hyaluronan (HA) could promote the restoration of proteoglycan (PG) depletion induced by chymopapain and then if the repair could be maintained once HA treatment was discontinued.. Animals received cartilage injury with intra-articular chymopapain (2.0 mg) followed by weekly treatment with intra-articular HA. HA treated animals were compared to injured animals with no treatment, contralateral untreated joints and joints from normal controls. The effect of intra-articular HA alone on articular cartilage was also examined.. Serum keratan sulfate levels confirmed degradation of the cartilage PGs in the chymopapain-injected knees. Intra-articular chymopapain resulted in marked loss of PGs. There were no significant differences among the control groups (untreated control, HA/800 treatment only). HA treatment did not affect the loss of PGs caused by chymopapain after 42 days. However, in animals receiving chymopapain injury followed by weekly HA treatment for 42 days and then 42 days of free cage activity without HA, cartilage PG contents were significantly increased. Intra-articular HA alone had no effect on the articular cartilage.. The results in the present study suggest a potential protective effect of HA on chymopapain-induced acute articular cartilage injury in rabbits that, in time, permits damaged cartilage to resynthesize matrix PGs after the HA treatment is discontinued.

Topics: Animals; Cartilage, Articular; Chymopapain; Disease Models, Animal; Hindlimb; Hyaluronic Acid; Injections, Intra-Articular; Keratan Sulfate; Male; Protective Agents; Proteoglycans; Rabbits; Viscosity; Wound Healing

Lumican is a member of a small leucine-rich proteoglycan family. We previously found that lumican mRNA and its protein were ectopically and highly expressed in acinar cells in chronic pancreatitis (CP)-like lesions close to pancreatic cancer cells. CP-like lesions are characterized by acinar and ductal-ductular cell proliferation with expanding fibrosis. This finding suggests that lumican is ectopically synthesized by acinar cells under chronic inflammatory conditions and plays a role in fibrosis of the pancreas. However, the expression and role of lumican in acute inflammatory changes of the pancreas are not completely elucidated. In the present study, we aim to clarify whether lumican mRNA and its protein are expressed in exocrine or endocrine components in acute pancreatitis (AP). For experimental AP, Wistar rats received an intraperitoneal injection of L-arginine. Western blot analysis showed an intense 50-kDa band corresponding to the lumican protein in normal and L-arginine-treated rat pancreas. After L-arginine injection, three intense bands at 42, 57, and 92 kDa were detected on day 1. Immunohistochemically, the lumican protein was localized in ductal and a few centroacinar cells in the normal pancreas. After L-arginine injection, an immature fibrosis with fragmented and loose collagen fibers was observed in AP on day 4 and lumican immunoreactivity was detected in the collagen fibers. Lumican mRNA was faintly detected in islet cells in the normal pancreas, but it was strongly expressed in acinar and islet cells on day 1. Furthermore, lumican mRNA was expressed in many proliferating fibroblasts on day 4 by in situ hybridization. These findings indicate that lumican is transiently synthesized by acinar cells and fibroblasts in AP. Lumican proteins synthesized by acinar cells, islet cells, and fibroblasts may contribute to immature and transient fibrosis of AP.

Topics: Acute Disease; Amylases; Animals; Arginine; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; In Situ Hybridization; Keratan Sulfate; Lumican; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar

Keratan sulphate proteoglycan (KSPG) is a developmentally regulated barrier molecule, directing axonal growth during central nervous system (CNS) formation. The possible re-expression and functional significance of KSPG in preventing axon regeneration following spinal cord injury (SCI) is poorly understood. In the present investigation, the spatio-temporal expression of KSPG was studied following experimental SCI. There was no indication of sparing of axons at the lesion epicentre following severe compression injury. By 7 days post operation (p.o.) a diffuse increase of KSPG immunoreactivity (KSPG-IR) was observed in the parenchyma surrounding the lesion. This was followed by a delayed (21-28 days p.o.) and largely heterogeneous increase of KSPG-IR in the lesion epicentre, which revealed both cellular and extracellular matrix-like distribution patterns. Although no re-growth of anterogradely labelled corticospinal axons was observed, many 200-kDa neurofilament (NF)-positive axons could be detected growing into the connective tissue scar. This phase of spontaneous axonal re-growth was closely associated with a framework of glial cells (including Schwann cells from damaged local spinal nerve roots) that had migrated into the lesion site. The spontaneous nerve fibre re-growth could be detected in both KSPG-rich and KSPG-poor territories. The present data suggest that the lesion-induced up-regulation of KSPG-IR may have contributed to the lack of corticospinal axon re-growth. However, the lack of any direct spatio-temporal correlation between the distribution of raised KSPG-IR and spontaneous NF-positive axonal regeneration suggests that at least some populations of axons can resist the putative inhibitory effects of this extracellular matrix molecule.

Topics: Animals; Axons; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Extracellular Matrix Proteins; Keratan Sulfate; Lumican; Male; Nerve Regeneration; Rats; Rats, Wistar; Spinal Cord Injuries; Time Factors

Keratan sulfate proteoglycans (KSPGs) are extracellular matrix molecules that appear to establish boundaries for axonal growth in the developing brain and spinal cord. In vitro studies confirm that KSPGs define inhibitory boundaries to extending neurites. The aim of the current study was to investigate whether KSPGs are expressed after spinal cord injury (SCI) and thereby might act as potential inhibitors of axonal growth. Adult Fischer 344 rats were subjected to spinal cord lesions, and the temporal and spatial expression of KSPGs was examined using the 5D4 monoclonal anti-KSPG antibody. In the intact spinal cord, a subpopulation of microglia expressed 5D4-KSPG throughout the white and gray matter. Within 24 hr of injury, 5D4-KSPG immunoreactivity substantially increased and appeared on cellular profiles in close proximity to the spinal cord lesion site, peaking 3 d after injury. Double immunolabeling revealed that 5D4-KSPG expression arose from multiple cell types at the lesion site, including reactive microglia, macrophages, and oligodendrocyte progenitors. Astrocytes were not identified as a source of 5D4-KSPG. The robust and extensive production of 5D4-KSPG at sites of SCI precedes the expression of other putatively inhibitory proteoglycan molecules such as chondroitin sulfate proteoglycans. This is the first demonstration that KSPGs are expressed after SCI in a temporal and spatial relationship that could exert an early and important role in modulating axonal growth after SCI.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Disease Progression; Female; Immunohistochemistry; Keratan Sulfate; Lumican; Macrophages; Microglia; Oligodendroglia; Rats; Rats, Inbred F344; Spinal Cord; Spinal Cord Injuries; Stem Cells

Although none of these markers currently have any clinical applications, researchers have made strong advances in understanding molecular markers of OA and remain optimistic that the goal of identifying clinically useful markers of OA is attainable.(3-6,8,9) Much of this positive sentiment arises from the large array of molecules that have been identified.(8) Molecular markers of the greatest potential clinical use will be those that allow early detection of OA, permit disease progression to be monitored, or allow efficacy of various treatment regimens to be assessed. Earlier and more sensitive detection of OA changes may increase effective opportunities for treatment intervention during reversible phases of the OA disease process and allow more objective assessment of treatment efficacy and prognosis.(6)

Topics: Animals; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dog Diseases; Dogs; Femur; Keratan Sulfate; Matrix Metalloproteinase 3; Osteoarthritis; Synovial Fluid

Damage to the meniscus can lead to posttraumatic osteoarthritis. Early markers of joint injury and tissue disease may be useful in developing and administering clinical treatment. We investigated the effects of total medial meniscectomy on biomarkers measured serially in synovial lavage fluid each month for 3 months. Following meniscectomy in dogs, four biomarkers were evaluated: cartilage oligomeric matrix protein, keratan sulfate epitope (5D4), the 3B3(-) neoepitope of chondroitin-6-sulfate, and the 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate. Meniscectomy led to statistically significant elevations of all four biomarkers, with levels peaking at 4 weeks. By 12 weeks, the level of the 5D4 epitope returned to the preoperative baseline level whereas that of cartilage oligomeric matrix protein, 3B3(-), and 3B3(+) remained above the baseline. Concentrations of these biomarkers in the knees not operated on did not change significantly from the baseline. The levels of cartilage oligomeric matrix protein and 3B3(-) relative to 3B3(+) remained constant in all knees. In contrast, the level of 5D4 relative to 3B3(+) declined over time in the knee operated on but remained constant in the knee not operated on. These results demonstrate a quantitative change in the molecular components of synovial fluid after meniscectomy, as well as a qualitative change evinced by an alteration in the relative proportions of these epitopes. Extensive analyses showed a strong correlation between serum levels of 3B3(-) from the femoral and cephalic veins; however, serum 3B3(-) was not correlated with synovial fluid 3B3(-). These findings support the hypothesis that the concentrations of select cartilage biomarkers in synovial fluid are altered following meniscectomy and are promising tools for objectively monitoring the induction of osteoarthritis in this model system.

Topics: Animals; Bacterial Proteins; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Epitopes; Keratan Sulfate; Male; Membrane Proteins; Menisci, Tibial; Osteoarthritis; Synovial Fluid; Transferases

To relate the rate of bone resorption to serum levels of both hyaluronan (HA) and antigenic keratan sulfate (KS) in canine experimental osteoarthritis (OA) and to evaluate the effects of calcitonin on these parameters and the OA lesions of the unstable knee.. Twenty-two dogs underwent anterior cruciate ligament transection (ACLT) and 6 dogs underwent sham operation. Urinary pyridinium crosslinks were quantified by high-performance liquid chromatography. Immunoassays quantified hyaluronan (HA) and antigenic KS. Macroscopic and histologic OA lesions were scored. Calcitonin treatment was started on day 14 postsurgery and stopped on either day 49 or day 104 postsurgery. Control dogs and all treated dogs were killed on day 105.. All ACLT joints developed OA. In contrast to sham-operated animals, all operated dogs exhibited an early and sustained rise in the levels of their urinary and serum markers. Calcitonin markedly reduced the levels of these markers and the severity of OA lesions. Furthermore, the longer the period of calcitonin therapy, the lower the score of the OA lesions.. Bone, synovium, and articular cartilage all appear to be involved in the state of hypermetabolism that develops in unstable joints. Furthermore, the rate of bone resorption increases markedly in the early stages of this OA model and is likely to contribute to cartilage breakdown. Since calcitonin reduced the severity of OA changes, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury.

Topics: Amino Acids; Animals; Anterior Cruciate Ligament; Biomarkers; Bone and Bones; Bone Resorption; Calcitonin; Cartilage, Articular; Chromatography, High Pressure Liquid; Disease Models, Animal; Dogs; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Synovial Membrane

We studied the effects of intraarticular (ia) administration of hyaluronic acid (HA) (Mw approximately 9 x 10(5)) (Artz) on cartilage integrity and release into synovial fluid (SF) of keratan sulfate peptides (KS-pep) in an ovine model of early osteoarthritis (OA) induced by meniscectomy. Five consecutive weekly injections of HA (2 ml, 10 mg/ml) or saline (2 ml) were initiated 16 weeks after meniscectomy, and animals were sacrificed 5 weeks after the last injection. SF was sampled 8, 16, 23, and 26 weeks postoperation. In the saline injected animals KS-pep levels increased progressively in SF, relative to nonoperated controls (p < 0.05). KS-pep levels in SF of the HA treated group also increased, but were not statistically different from controls. Using a modified Mankin histological scoring system, cartilage at necropsy of HA injected joints showed less damage than similar regions of saline treated animals. A new mechanism for the protective effects of HA on cartilage is proposed.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Hyaluronic Acid; Injections, Intra-Articular; Keratan Sulfate; Knee Joint; Osteoarthritis; Sheep; Synovial Fluid

These studies seek to define the gene expression of proteoglycans and collagens in the developing hypertrophic phase of an experimental model of osteoarthritis (OA). Total RNA was extracted from articular cartilage of nonoperated and operated dog knees 10 weeks after induction of OA by transection of the anterior cruciate ligament. The relative amounts of mRNA for type II collagen and the core proteins for the aggrecan, biglycan, decorin, and fibromodulin were analyzed by Northern blotting. Total RNA in OA vs nonoperated knees was statistically significantly elevated 2.5x, the mRNA for type II collagen was elevated 8x, aggrecan 2x, biglycan 4x, fibromodulin 2x. The level for decorin was increased 1.6x, but this difference was not statistically significant. Chondrocytes respond actively to joint injury. Gene expression of proteoglycans and type II collagen is discoordinate in early experimental OA, and may contribute to the development of cartilage abnormalities.

Topics: Aggrecans; Animals; Biglycan; Carrier Proteins; Collagen; Decorin; Disease Models, Animal; Dogs; Extracellular Matrix Proteins; Fibromodulin; Gene Expression; Keratan Sulfate; Lectins, C-Type; Male; Osteoarthritis; Proteoglycans

The levels of proteoglycan aggregate components (link protein, keratan sulfate epitope, and total sulfated glycosaminoglycan) were determined in the synovial fluid lavages of dogs with experimental osteoarthritis or disuse atrophy. A model of experimental osteoarthritis was created by transection of the anterior cruciate ligament of the right knee; studies were carried out 6 and 12 weeks after surgery. Joint disuse was studied at 4 and 8 weeks after initiation of the disuse. Recovery after disuse also was studied in joints that had 3 weeks of remobilization after 4 or 8 weeks of disuse. Synovial fluid lavages from the right knee joints of untreated animals were used as controls. The concentrations of keratan sulfate epitope, sulfated glycosaminoglycan, and link protein in the synovial fluid lavages at 6 and 12 weeks after transection of the anterior cruciate were elevated compared with the control values. Similar analysis of the fluid after disuse showed that the levels of keratan sulfate epitope and sulfated glycosaminoglycan were increased compared with the control levels and the levels after transection. However, the concentration of link protein in the fluid after disuse was not significantly different from the control level. The levels of keratan sulfate epitope and sulfated glycosaminoglycan in the synovial fluid lavages after disuse with recovery were high, but the levels of link protein remained low. The results indicate that the catabolism of proteoglycan aggregates in articular cartilage during early osteoarthritis and disuse is different. The determination of keratan sulfate epitope in synovial fluid lavages appears to provide a relatively general indication of proteoglycan catabolism, whereas increased levels of link protein may be more indicative of cartilage degeneration.

Topics: Aggrecans; Analysis of Variance; Animals; Bone Matrix; Cartilage, Articular; Disease Models, Animal; Dogs; Enzyme-Linked Immunosorbent Assay; Epitopes; Extracellular Matrix Proteins; Glycosaminoglycans; Joint Diseases; Keratan Sulfate; Knee Joint; Lectins, C-Type; Osteoarthritis; Proteoglycans; Radioimmunoassay; Synovial Fluid

Arthritis refers to a heterogeneous class of diseases characterized by impairment of movement. Yet animal models of arthritis have traditionally been based on the utilization of animals housed without the capability of extended free movement and without adjunctive measurement of mobility. To define the determinants of mobility impairment, we have established a lipopolysaccharide (LPS)-induced arthritis model in the hamster that prominently features monitoring of mobility and compares mobility changes with histological and biochemical changes during arthritis. Intraarticular LPS induces a dose-dependent inhibition of the hamster's mobility as measured by decreased daily distance on a running wheel (normal distance 9 to 12 km/day). At low concentrations of LPS (0.1 and 1 microgram/knee), daily distances returned to normal after 4 and 6 days, respectively. At higher concentrations, the mobility was still markedly suppressed after 6 days, and, at 100 micrograms/knee, irreversible chondrocyte loss was observed on the femoral condylar margins. Further studies were therefore conducted using 1 microgram LPS/knee. Histological and biochemical changes were examined to determine which resolved at the time of restoration of mobility. At the time of restoration of mobility, the synovial capsule was still edematous and heavily infiltrated with leukocytes; proteoglycan loss from the medial femoral condyle was still increasing. Plasma keratan sulfate failed to correlate with either proteoglycan loss or mobility changes. Proteoglycan synthesis, which was maximally suppressed the second day after LPS, was enhanced over controls at the time of restoration of mobility, suggesting the onset of repair. These results suggest a possible association of mobility inhibition with local cytokine synthesis. This model provides an approach to define the causes of mobility impairment.

Topics: Animals; Arthritis; Cricetinae; Disease Models, Animal; Female; Keratan Sulfate; Lipopolysaccharides; Mesocricetus; Microscopy, Electron, Scanning; Motor Activity; Patella; Proteoglycans; Time Factors

The intra-articular injection of 0.02, 0.2, or 2.0 mg of chymopapain (CP) into the knee of adolescent rabbits caused the loss of more than 50% of the proteoglycans (PGs) in the cartilaginous tissues within the joint. Sequential measurements of cartilage-derived keratan sulfate epitope in serum and analyses of articular cartilage slices 2 days after the injection revealed that 0.02 mg of CP was nearly as effective as higher doses (0.2 or 2.0 mg of CP) in causing the depletion. The degradation and depletion of PGs in articular cartilage were shown to be localized to the joint and did not affect articular cartilage in the contralateral knee joint (no injection) or other cartilaginous tissues in the body. On day 9, partial replenishment of the articular cartilage PGs had occurred, irrespective of the dose used, and the articular surface within the joint remained intact. However, by day 21, articular cartilage in joints injected with 2.0 mg of CP had begun to show progressive degenerative changes, and these changes became more severe with time. In contrast, joints injected with 0.2 mg of CP continued to repair successfully by the reestablishment of a matrix that retained its integrity and appeared to remain functional for at least 6 months. These results suggest that the model may prove useful for the study of the repair processes that follow matrix injury and severe depletion of PGs from the articular cartilage matrix.

Topics: Animals; Cartilage Diseases; Cartilage, Articular; Chymopapain; Disease Models, Animal; Keratan Sulfate; Knee Joint; Male; Proteoglycans; Rabbits; Regeneration

Canine experimental models of osteoarthritis (OA) and disuse atrophy were used to study cartilage metabolism. The synovial fluids from the OA joints showed elevated levels of keratan sulfate (KS) epitope and link protein, indicating increased catabolism. Analysis of fluids from joints with disuse atrophy showed high levels of KS epitope, but no increase in link protein. Quantitation of a novel chondroitin sulfate (3B3) epitope showed it to be present only in the synovial fluids and articular cartilage of the OA joints. The results indicate that these may be important indicators, or markers, of degenerative joint disease.

Topics: Animals; Atrophy; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Keratan Sulfate; Osteoarthritis; Proteoglycans; Synovial Fluid

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

Topics: Animals; Cartilage, Articular; Chondroitin; Culture Techniques; Disease Models, Animal; Dogs; Extracellular Matrix Proteins; Female; Keratan Sulfate; Osteoarthritis; Proteoglycans; Synovial Fluid

Out of 30 adult rabbits, 20 had one knee immobilized with a plaster of Paris cast for 6 or 12 weeks, and 10 rabbits were used as untreated controls. Prior to immobilization, 10 knees were injected with high-molecular weight hyaluronic acid. The articular cartilage of the femoral condyles was studied by light microscopy, whereas that of the patella and tibia was analyzed biochemically. Degenerative changes of the articular cartilage similar to those seen in arthrosis were observed after 6 weeks. The intraarticular injection of hyaluronic acid did not prevent these changes; instead, the reparative processes seemed inhibited.

Topics: Animals; Arthritis; Cartilage, Articular; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Evaluation Studies as Topic; Hyaluronic Acid; Immobilization; Injections, Intra-Articular; Keratan Sulfate; Knee Joint; Rabbits; Uronic Acids

The effects of the chondroprotective agents (Arteparon, SP-54 and DH40J) on the release of proteoglycan degradation products (as keratan sulphate peptide fragments) from articular cartilage implanted into rat subcutaneous air pouches have been investigated by using an enzyme-linked immunosorbent-inhibition assay (ELISIA). The ELISIA technique was capable of quantitating the keratan sulphate peptides (KS peptides) in fluids within the range of 100-2,000 ng/ml by using the monoclonal antibody line 1/20/5-D-4 and human articular cartilage KS peptides as standard reagents. It was found that the levels of KS peptides present in the air-pouch fluid of rats treated with the chondroprotective drugs was significantly less than in fluid aspirated from the pouches of non-drug-treated control animals. On the basis of these findings we suggest that the assessment of KS peptide by ELISIAs may provide a useful means of monitoring proteoglycan breakdown products in biological fluids (e.g. synovial fluids or blood) and for evaluating the effects that antiarthritic drugs may have on this process.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Keratan Sulfate; Lumican; Male; Osteoarthritis; Pentosan Sulfuric Polyester; Peptide Fragments; Rats; Rats, Inbred Strains

Serum keratan sulfate (KS) levels were measured with an enzyme-linked immunosorbent assay using monoclonal antibody (MAb) 5-D-4 (anti-KS) in a rabbit model of osteoarthritis (OA) induced by partial meniscectomy. The partial medial meniscectomy produced pathologic changes of OA in the joints of the rabbits, which were seen when the animals were killed at 3, 6, 9, or 12 weeks postsurgery. Tibial or femoral osteophytes were seen in up to 90% of the operated joints; pitting and ulceration of medial femoral condyles were also frequently noted (77% of cases). Rabbits that underwent sham surgery, back-skin-operated rabbits, or nonoperated normal rabbits served as controls; the joints of these animals were normal at the time of killing. A rise in the level of serum KS was recorded in 50% of rabbits following partial meniscectomy, but this was matched by similar changes in the control groups. The mean serum KS level of the OA animals at serial intervals (3, 6, 9, or 12 weeks) following surgery was not significantly different from that in the control groups. When measured with a second MAb, 2-D-3, KS levels showed similar trends as with MAb 5-D-4, although lower assay values were obtained. These findings indicate that experimentally induced OA in rabbits is not associated with a significant rise in serum KS levels. KS levels did not differentiate OA from non-OA animals, nor did they parallel disease progression.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Keratan Sulfate; Osteoarthritis; Rabbits; Time Factors

Articular cartilage contains at least 2 proteoglycans (PGs) which aggregate with hyaluronate: one larger, richer in chondroitin sulphate (CSRPG); the other smaller, relatively richer in keratan sulphate (KSRPG). With maturation the ratio of CSRPG/KSRPG decreases. In order to test the hypothesis that osteoarthritic cartilage contains an increased amount of proteoglycans characteristic of immature cartilage, experimental osteoarthritis (OA) was induced in 11 dogs by transection of the anterior cruciate ligament. Proteoglycan populations were assessed by composite agarose polyacrylamide gel electrophoresis (CAPAGE). The OA cartilage had more proteoglycan and an increased proportion of the slower migrating band on CAPAGE, which corresponds to the CSRPG, supporting the hypothesis that OA cartilage contains an increased amount of proteoglycans characteristic of immature cartilage.

Topics: Animals; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Dogs; Keratan Sulfate; Lumican; Male; Osteoarthritis; Proteoglycans

We studied changes in subchondral bone and articular cartilage in an animal model of osteoarthrosis. In this model we applied repetitive impulsive loads to rabbits' knees. Their legs were held in short leg splints so the rabbits were unable to dampen the peak applied load with ankle flexion. After sacrifice, at 1 day to 6 weeks, we studied proximal tibial load-bearing cartilage histologically, biochemically, and with radioactive sulfate uptake. We also studied the subchondral bone under that cartilage histologically, histomorphometrically, with bone scan (99mTc pyrophosphate), and by tetracycline labeling. An increase in 99mTc labeling of the subchondral bone was the first reliable change observed. This was followed by an increase in tetracycline labeling, bone formation, and a decrease in porosity, which has been associated with relative stiffening of bone. Horizontal splitting and deep fibrillation of the overlying articular cartilage followed the early bone changes. All of these changes preceded changes in content and characterization of cartilage proteoglycans or increased chondrocyte activity as manifested by incorporation of radioactive sulfate. In this model the early bone changes preceded changes in the articular cartilage. The deep splitting of articular cartilage occurred prior to metabolic alteration of that tissue.

Topics: Animals; Calcium Pyrophosphate; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Female; Glycosaminoglycans; Keratan Sulfate; Knee Joint; Osteoarthritis; Proteoglycans; Rabbits; Synovial Membrane

An experimental model of osteoarthritis resulting from laxity of the joint was induced in eighteen mature dogs (at least two years old) by sectioning the anterior cruciate ligament of the right knee (stifle) with a stab incision, the left knee providing a control. A sham operation was also performed in three other dogs, in which a stab incision was made but the ligament left intact. The dogs were killed at various intervals from one to forty-eight weeks later. Morphological changes in bone, cartilage, synovial membrane and joint capsule were examined in all the joints and biochemical changes in the cartilage of three dogs killed after two, eight, and sixteen weeks. All the changes resulting from the operation progressed with time and became indistinguishable from those found in three dogs with natural osteoarthritis of the knee. There were no changes in the joints which had sham operations. As the time of onset is known, this experimental model in a larger species enables a study to be made of the biochemical as well as the morphological changes in the early stages of osteoarthritis.

Topics: Animals; Bone Development; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Female; Femur; Galactosamine; Glucosamine; Keratan Sulfate; Knee Joint; Ligaments, Articular; Male; Menisci, Tibial; Osteoarthritis; Proteoglycans; Stress, Physiological; Synovial Membrane; Tibia; Uronic Acids