Biomedical subjects

D R Eyre

Publications and source records attributed to D R Eyre.

At least 19 recordsLinked to original sources

PubMed2006

Articular cartilage collagen: an irreplaceable framework?

Adult articular cartilage by dry weight is two-thirds collagen. The collagen has a unique molecular phenotype. The nascent type II collagen fibril is a heteropolymer, with collagen IX molecules covalently linked to the surface and collagen XI forming the filamentous template of the fibril as a whole. The functions of collagens IX and XI in the heteropolymer are far from clear but, evidently, they are critically important since mutations in COLIX and COLXI genes can result in chondrodysplasia syndromes. Here we review what is known of the collagen assembly and present new evidence that collagen type III becomes covalently added to the polymeric fabric of adult human articular cartilage, perhaps as part of a matrix repair or remodelling process.

Amino Acid Sequence↗

PubMed2005

Urinary levels of type II collagen C-telopeptide crosslink are unrelated to joint space narrowing in patients with knee osteoarthritis.

OBJECTIVE: To determine whether urinary concentrations of the cross linked C-telopeptide of type II collagen (CTx-II) distinguish subjects with progressive radiographic or symptomatic knee osteoarthritis from those with stable disease. METHODS SUBJECTS: were 120 obese women with unilateral knee osteoarthritis who participated in a 30 month, randomised, placebo controlled trial of structure modification by doxycycline, in which a standardised semiflexed anteroposterior view of the knee was obtained at baseline and 30 months. Subjects were selected from a larger sample to permit comparisons of urinary CTx-II levels between 60 progressors and 60 non-progressors with respect to medial joint space narrowing. Each group contained 30 subjects who, across five semi-annual assessments, reported on at least two occasions an increase of > or =20% in 50 ft walk pain (minimum = 1 cm on a 10 cm visual analogue scale), relative to the previous visit. The remainder reported no increases in knee pain. Urine samples were obtained semi-annually for determination of the CTx-II and creatinine concentrations. RESULTS: In an analysis of the placebo group only, the frequency of radiographic progressors in the upper and middle tertiles (48% and 60%, respectively) of the baseline CTx-II distribution was not significantly different than that in the lower tertile (64%). These results were unchanged after inclusion of data from subjects in the doxycycline group. Furthermore, serial CTx-II levels did not distinguish subjects with progressive radiographic or symptomatic knee osteoarthritis from those with stable disease. CONCLUSIONS: In this pilot study, urinary CTx-II concentration was not a useful biomarker of osteoarthritis progression.

Biomarkers↗

PubMed2005

Sport-related differences in biomarkers of bone resorption and cartilage degradation in endurance athletes.

OBJECTIVE: By measuring urinary cross-linked N-telopeptide (NTx) as a bone resorption marker and urinary C-telopeptide of type II collagen (CTx-II) as a cartilage degradation marker, we asked whether differences in skeletal stresses in college athletes undergoing high-intensity training for diverse types of aerobic sports affect their skeletal metabolism and, if so, differentially or in unison. METHODS: The study was cross-sectional at a Division 1 college campus with 60 student athletes representing crew, cross-country running and swimming. Controls were 16 non-athlete undergraduates. Urine samples were collected for NTx and CTx-II analysis by enzyme-linked immunosorbent assay, normalizing results to creatinine. Two-way analysis of variance models and pair-wise comparisons were used to test whether biomarker levels differed by sport and the significance when adjusted for body mass index (BMI). RESULTS: NTx and CTx-II showed significant differences between groups before and after adjusting for BMI. NTx was highest in the rowers, and higher in rowers and runners than in swimmers or controls. CTx-II was significantly higher in runners than in crew, swimmers or controls, when unadjusted for BMI. After adjusting for BMI, these group differences remained significant except for runners over crew. CONCLUSION: Athletes in-training in the three sports show significant differences in these markers of bone resorption and cartilage collagen degradation. The results suggest that crew undergo the highest bone remodeling and runners the highest cartilage degradation. The results also show how these markers can vary physiologically between individuals, at extremes of skeletal exercise.

Adolescent↗

PubMed2005

Acetabular augmentation at six- to 30-year follow-up. A biochemical and histological analysis.

In developmental dysplasia of the hip, a deficient acetabulum may be augmented by placing local autogenous iliac osseous graft, or the ilium itself, over the head of the femur with the expectation that the added bone will function as a bearing surface. We analysed this bone obtained en bloc during subsequent surgery which was performed for degenerative osteoarthritis in three patients at 6, 25 and 30 years after the initial augmentation procedure. In each patient, the augmentation comprised of red cancellous bone covered on its articulating surface by a distinct layer of white tissue. Microscopy of this tissue showed parallel rows of spindle-shaped cells lying between linearly arranged collagen bundles typical of joint capsule. Biochemical analysis showed type I collagen, the principal collagen of joint capsule and bone, with no significant quantity of type II collagen, the principal collagen of cartilage. While the added bone produced by acetabular augmentation was durable, histological and biochemical analyses suggested that it had not undergone cartilage metaplasia. The augmented acetabulum articulates with the head of the femur by means of an interposed hip joint capsule.

Acetabulum↗

PubMed2004

The association of lumbar spondylolisthesis with collagen IX tryptophan alleles.

Two collagen type IX gene polymorphisms that introduce a tryptophan residue into the protein's triple-helical domain have been linked to an increased risk of lumbar disc disease. To determine whether a particular subset of symptomatic lumbar disease is specifically associated with these polymorphisms, we performed a prospective case-control study of 107 patients who underwent surgery of the lumbar spine. Patients were assigned to one of five clinical categories (fracture, disc degeneration, disc herniation, spinal stenosis without spondylolisthesis and spinal stenosis with spondylolisthesis) based on history, imaging results, and findings during surgery. Of the 11 tryptophan-positive patients, eight had spinal stenosis with spondylolisthesis and three had disc herniation. The presence of the tryptophan allele was significantly associated with African-American or Asian designation for race (odds ratio 4.61, 95% CI 0.63 to 25.35) and with the diagnosis of spinal stenosis with spondylolisthesis (odds ratio 6.81, 95% CI 1.47 to 41.95). Our findings indicate that tryptophan polymorphisms predispose carriers to the development of symptomatic spinal stenosis associated with spondylolisthesis which requires surgery.

Adolescent↗

PubMed2003

Short-term calcium supplementation has no effect on biochemical markers of bone remodeling in early postmenopausal women.

INTRODUCTION: This study was designed to assess changes in biochemical markers of bone remodeling in early postmenopausal women receiving calcium supplementation. MATERIALS AND METHODS: In a randomized cross-over study of eighteen weeks duration, the effect of a 6-week calcium supplementation (1000 mg calcium carbonate) on biochemical markers of bone resorption (collagen type I cross-linked C- and N-telopeptides) and bone formation (osteocalcin, total and bone-specific alkaline phosphatase), and total serum calcium was assessed in 27 early postmenopausal women. RESULTS: While total serum calcium levels increased significantly due to calcium supplementation (p<0.05), biochemical markers of both bone resorption and formation remained virtually unchanged. CONCLUSION: In contrast to other investigations, there was no significant short-term effect of calcium supplementation on biochemical markers of either bone resorption or formation.

Biomarkers↗

PubMed2003

Bone remodeling and bone mineral density during pregnancy.

INTRODUCTION: The effect of pregnancy upon the maternal skeleton is not fully understood. The information that has been gathered by recent studies is conflicting with regard to overall loss or gain of bone during pregnancy. The aim of the present longitudinal, controlled study, therefore, was to investigate the effect of pregnancy on lumbar spine, wrist, and hip bone mineral density, and to describe bone remodeling during pregnancy as indicated by biochemical markers of both bone resorption and formation. MATERIALS AND METHODS: Thirty healthy women (15 subjects seeking pregnancy and 15 non-pregnant controls) were studied. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry before conception and within 2 weeks after parturition. Markers of bone resorption (urinary cross-linked type I collagen N-telopeptides, serum type I collagen C-telopeptides) and bone formation (total and bone specific alkaline phosphatase, osteocalcin), and total serum calcium were analyzed before, during (once in each trimester), and after pregnancy. RESULTS: During pregnancy, BMD decreased significantly by 3.4+/-4.1% at the lumbar spine and 4.3+/-3.9% at the trochanter, while there was a slight but significant increase in BMD at the proximal 1/3 of the forearm (1.3+/-1.9%). Total hip and femoral neck BMD did not change significantly, nor did total and ultradistal forearm BMD. Bone resorption increased during pregnancy with peak levels in the third trimester (N-telopeptides) or post partum (C-telopeptides), respectively. The increase in bone resorption was accompanied by a significant decrease in serum calcium in the third trimester. Markers of bone formation showed a biphasic pattern with decreases from baseline to the first (total and bone specific alkaline phosphatase) or second trimester (osteocalcin), respectively, followed by a significant increase in the third trimester and post partum. There was no change in any parameter in the control group throughout the study. CONCLUSION: In conclusion, pregnancy is characterized by high bone turnover with resorption preceding formation. During the first and second trimester bone remodeling is uncoupled. Serum calcium decreases as bone resorption peaks in late pregnancy. There are significant decreases in bone mineral density at sites rich in trabecular bone, such as the lumbar spine and the trochanter.

Adult↗

PubMed2002

Collagen polymorphisms of the intervertebral disc.

The mechanical function and the collagen phenotype of the disc are complex, each a hybrid of elements of ligament and cartilage. In detail, the collagen properties are unique. Collagens I and II provide the bulk of the tissue fabric interwoven in opposing radial concentration gradients. From analysis of isolated cross-linked peptides, some degree of commingling of these major fibrillar collagens occurs down to the molecular level. Collagens V, VI, IX, XI, XII and XIV all contribute to the matrix. Collagen IX is the short molecular form that lacks a non-collagenous (NC)4 domain, not the long form found in most hyaline cartilages. Protein sequence and reverse transcriptase-PCR analysis confirmed this was the result of expression from the alternative transcription start site, not proteolysis of the long form. In view of recent reports that common single nucleotide polymorphisms in COL9A2 and COL9A3 are linked to chronic sciatica associated with disc pathology, the specific interactions and role of collagen IX in disc tissue are important to define.

Animals↗

PubMed2002

Recent developments in cartilage research: matrix biology of the collagen II/IX/XI heterofibril network.

Research on cartilage is intensifying as efforts expand to discover disease-modifying drugs to treat or prevent osteoarthritis. Proteolytic damage to the collagen fabric of cartilage is a critical, and probably early, component of the pathogenesis of degenerative joint disease. Here we summarize recent findings on the unique heteromeric structure of cartilage collagen fibrils, including the key role of collagen IX, a covalently bonded fibril-adapter molecule. A highly specific pattern of cross-linking sites that involves all three component gene products strongly suggests that collagen IX has evolved to function as an interfibrillar network-bonding agent. This is supported from the genetic evidence that mutations in all three collagen IX genes can produce a phenotype in which cartilage matrix integrity and early-onset osteoarthritis are a feature. From the structure of the cartilage collagen heteropolymer we also predict a pivotal role for telopeptide (non-triple-helical) proteolytic cleavages in the remodelling and degradation of collagen fibrils.

Animals↗

PubMed2002

Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors.

OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro. In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined. METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes. In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage. RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number. Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen. Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization. The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2. Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix. To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used. Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18. Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation. Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization. CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.

Adult↗

PubMed2001

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

ADAM Proteins↗

PubMed2001

Molecular properties of matrilin-3 isolated from human growth cartilage.

Matrilin-3 is a recently identified matrix protein of cartilage that shows sequence homology to matrilin-1 (cartilage matrix protein or CMP). Here we identify and characterize the molecular properties of matrilin-3 from human growth cartilage by immunochemical and mass spectrometry methods. Extracts of fetal skeletal cartilage were resolved by SDS-PAGE and candidate matrilin subunits were identified by electrospray mass spectrometry of tryptic peptides. Matrilin-3 and matrilin-1 were both present in disulfide-bonded tetrameric components. Polyclonal antisera to synthetic peptides specific to each subunit confirmed the identities by Western blotting and further demonstrated the existence of several forms of tetramer. A homotetramer (matrilin-3)4 and more than one species of heterotetramer containing matrilin-3 and matrilin-1 chains were resolved. Immunohistochemistry of tissue sections confirmed that both matrilin-1 and matrilin-3 are widely codistributed throughout human skeletal growth cartilage.

Amino Acid Sequence↗

PubMed2000

Two-dimensional peptide mapping of cross-linked type IX collagen in human cartilage.

Type IX collagen is a quantitatively minor component of hyaline cartilage that is essential for the normal structural integrity of the tissue. Purification and analysis are difficult because the mature protein is insoluble as a cross-linked integral component of the fibrillar matrix. In order to view a peptide map of the total pool of type IX collagen in a cartilage sample, a selective method based on Western blot analysis was developed for displaying collagen IX peptides in a cyanogen bromide digest of tissue. Digests were partially resolved by reverse-phase HPLC, individual fractions were run on SDS-PAGE and then transblotted to membrane, and the collagen IX fragments were revealed using an anti-collagen IX rabbit antiserum. All major CB-peptides from alpha1(IX), alpha2(IX), and alpha3(IX) chains in the resulting two-dimensional display were identified by amino-terminal sequence analysis. Cross-linked peptides originating from sites of covalent interaction between collagen types IX and II and between IX and IX were also defined. By comparison with an analysis of soluble type IX collagen from chondrocyte culture medium, the results showed that the pool of type IX collagen molecules in fetal and adult human cartilage is extensively cross-linked intermolecularly at sites previously revealed by other methods using purified protein. This sensitive, direct method has the potential to screen for abnormalities in the content and properties of type IX collagen in tissue samples, for example, in the study of heritable chondrodysplasia syndromes and the pathogenesis of cartilage destruction in osteoarthritis.

Adult↗

PubMed2000

A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy.

Multiple epiphyseal dysplasia (MED) is a degenerative cartilage condition shown in some cases to be caused by mutations in genes encoding cartilage oligomeric matrix protein or type IX collagen. We studied a family with autosomal dominant MED affecting predominantly the knee joints and a mild proximal myopathy. Genetic linkage to the COL9A3 locus on chromosome 20q13.3 was established with a peak log(10) odds ratio for linkage score of 3.87 for markers D20S93 and D20S164. Reverse transcription-PCR performed on the muscle biopsy revealed aberrant mRNA lacking exon 3, which predicted a protein lacking 12 amino acids from the COL3 domain of alpha3(IX) collagen. Direct sequencing of genomic DNA confirmed the presence of a splice acceptor mutation in intron 2 of the COL9A3 gene (intervening sequence 2, G-A, -1) only in affected family members. By electron microscopy, chondrocytes from epiphyseal cartilage exhibited dilated rough endoplasmic reticulum containing linear lamellae of alternating electron-dense and electron-lucent material, reflecting abnormal processing of mutant protein. Type IX collagen chains appeared normal in size and quantity but showed defective cross-linking by Western blotting. The novel phenotype of MED and mild myopathy is likely caused by a dominant-negative effect of the exon 3-skipping mutation in the COL9A3 gene. Patients with MED and a waddling gait but minimal radiographic hip involvement should be evaluated for a primary myopathy and a mutation in type IX collagen.

Cartilage↗

PubMed2000

Autoreactivity against matrilin-1 in a patient with relapsing polychondritis.

Relapsing polychondritis (RP) is a rare inflammatory disease of cartilage. Chondritis of the auricular, nasal, and tracheal cartilages predominates in this disease, suggesting a response to a tissue-specific antigen. One potential antigen is matrilin-1, a cartilage matrix protein found uniquely in the tracheal, auricular, and nasal cartilage of adults. We describe herein a patient with RP who had both a humoral and a cellular immune response directed toward the cartilage matrix protein matrilin-1.

Aged↗

PubMed2000

Hypercalcemia during the osteogenic phase after rat marrow ablation coincides with increased bone resorption assessed by the NTx marker.

Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.

Animals↗

PubMed2000

Proteolysis of human bone collagen by cathepsin K: characterization of the cleavage sites generating by cross-linked N-telopeptide neoepitope.

An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.

Adult↗

PubMed2000

Report of five novel and one recurrent COL2A1 mutations with analysis of genotype-phenotype correlation in patients with a lethal type II collagen disorder.

Achondrogenesis II-hypochondrogenesis and severe spondyloepiphyseal dysplasia congenita (SEDC) are lethal forms of dwarfism caused by dominant mutations in the type II collagen gene (COL2A1). To identify the underlying defect in seven cases with this group of conditions, we used the combined strategy of cartilage protein analysis and COL2A1 mutation analysis. Overmodified type II collagen and the presence of type I collagen was found in the cartilage matrix of all seven cases. Five patients were heterozygous for a nucleotide change that predicted a glycine substitution in the triple helical domain (G313S, G517V, G571A, G910C, G943S). In all five cases, analysis of cartilage type II collagen suggested incorporation of the abnormal alpha1(II) chain in the extracellular collagen trimers. The G943S mutation has been reported previously in another unrelated patient with a strikingly similar phenotype, illustrating the possible specific effect of the mutation. The radiographically less severely affected patient was heterozygous for a 4 bp deletion in the splice donor site of intron 35, likely to result in aberrant splicing. One case was shown to be heterozygous for a single nucleotide change predicted to result in a T1191N substitution in the carboxy-propeptide of the proalpha1(II) collagen chain. Study of the clinical, radiographic, and morphological features of the seven cases supports evidence for a phenotypic continuum between achondrogenesis II-hypochondrogenesis and lethal SEDC and suggests a relationship between the amount of type I collagen in the cartilage and the severity of the phenotype.

Achondroplasia↗