Decorin is a Zn2+ metalloprotein.

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by leucine-rich repeat motifs. We show here that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells has a high affinity for Zn2+ as demonstrated by equilibrium dialyses. The Zn2+-binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in a spacing reminiscent of a zinc finger. A recombinant 41-amino acid long peptide representing the N-terminal domain of decorin has full Zn2+ binding activity and binds two Zn2+ ions with an average KD of 3 x 10(-7) M. Binding of Zn2+ to this peptide results in a change in secondary structure as shown by circular dichroism spectroscopy. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn2+-binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, do not bind Zn2+ with high affinity.

Immune responses to cartilage link protein and the G1 domain of proteoglycan aggrecan in patients with osteoarthritis.

OBJECTIVE: To determine whether patients with osteoarthritis (OA) express cellular immunity to cartilage link protein (LP) and the G1 globular domain of proteoglycan (PG) aggrecan, and whether immunity to the G1 domain is influenced by the removal of keratan sulfate (KS). METHODS: LP and the G1 globular domain of PG were isolated from human and/or bovine cartilage and used in proliferation assays with peripheral blood lymphocytes (PBL) from 42 patients with OA and 40 healthy control subjects. RESULTS: Patients with OA expressed a higher prevalence of cellular immunity to human cartilage LP (42.4%) compared with the control group (13.3%). The prevalence of immune reactivity to bovine LP in patients with OA was lower (35.7%) compared with the immunity to human LP, but remained similar in the control group (13.8%). PBL from patients with OA exhibited low reactivity to the native G1 domain of bovine PG. However, removal of KS chains from the G1 globular domain resulted in increased cellular immune responses to the G1 domain in OA patients (45.8%) compared with the control group (7.7%). CONCLUSION: These results indicate the presence of immunity to cartilage-derived LP and the G1 globular domain of PG aggrecan in patients with OA and the inhibitory effect of KS chains on the G1 domain on the expression of this immunity in OA patients. This immune reactivity is commonly observed in patients with inflammatory joint disease and can experimentally induce arthritis. Thus, it may be involved in the pathogenesis of OA.

Evidence for altered synthesis of type II collagen in patients with osteoarthritis.

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA.

Induction of arthritis in BALB/c mice by cartilage link protein: involvement of distinct regions recognized by T and B lymphocytes.

Both type II collagen and the proteoglycan aggrecan are capable of inducing an erosive inflammatory polyarthritis in mice. In this study we provide the first demonstration that link protein (LP), purified from bovine cartilage, can produce a persistent, erosive, inflammatory polyarthritis when injected repeatedly intraperitoneally into BALB/c mice. We discovered a single T-cell epitope, located within residues 266 to 290 of bovine LP (NDGAQIAKVGQIFAAWKLLGYDRCD), which is recognized by bovine LP-specific T lymphocytes. We also identified three immunogenic regions in bovine LP that contain epitopes recognized by antibodies in hyperimmunized sera. One of these B-cell regions is found in the most species-variable domain of LP (residues 1 to 36), whereas the other epitopes are located in the most conserved regions (residues 186 to 230 and 286 to 310). The latter two regions contain an AGWLSDGSVQYP motif shared by the G1 globulin domain of aggrecan core protein, versican, neurocan, glial hyaluronan-binding protein, and the hyaluronan receptor CD44. Our data reveal that the induction of arthritis is associated with antibody reactivities to B-cell epitopes located at residues 1 to 19. Together, these observations show that another cartilage protein, LP, like type II collagen and the proteoglycan aggrecan, is capable of inducing an erosive inflammatory arthritis in mice and that the immunity to LP involves recognition of both T- and B-cell epitopes. This immunity may be of importance in the pathogenesis of inflammatory joint diseases, such as juvenile rheumatoid arthritis, in which cellular immunity to LP has been demonstrated.

Cellular immunity to the G1 domain of cartilage proteoglycan aggrecan is enhanced in patients with rheumatoid arthritis but only after removal of keratan sulfate.

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) express cellular immunity to the purified G1 globular domain of cartilage proteoglycan (PG) aggrecan and whether it is influenced by the removal of keratan sulfate (KS) chains from the molecule. METHODS: The G1 globular domain of PG was purified from mature bovine articular cartilage, digested with keratanase, and used in proliferation assays with peripheral blood lymphocytes (PBL) isolated from 43 patients with RA, 11 patients with nonarticular rheumatism (NAR), including soft tissue rheumatism and mechanical back pain, and 13 healthy age- and sex-matched control subjects. RESULTS: Removal of KS chains from the G1 globular domain resulted in significantly increased prevalence and values of cellular immune responses to G1 in RA patients compared with the control and NAR groups. In the majority of RA patients, KS chains on G1 significantly inhibited its immune recognition by PBL. There was no significant effect of KS removal on the immunity to G1 in patients with NAR and in the healthy control group. CONCLUSION: These results reveal that immune reactivity to the G1 globular domain of the cartilage PG aggrecan is enhanced in patients with RA but only when KS chains are removed. Thus, KS chains inhibit immune responses to this domain of aggrecan. Since immunity to the G1 globular domain of aggrecan induces an erosive polyarthritis in BALB/c mice after removal of KS chains, immunity to the G1 globular domain, cleaved by proteases to remove KS chains, may play a role in the pathogenesis of RA.

Arthritis induced by proteoglycan aggrecan G1 domain in BALB/c mice. Evidence for t cell involvement and the immunosuppressive influence of keratan sulfate on recognition of t and b cell epitopes.

Our previous work showed that the proteoglycan aggrecan can induce erosive polyarthritis and spondylitis in BALB/c mice, and that the G1 domain of the proteoglycan aggrecan (G1) is the arthritogenic region. In this study, two T cell epitopes residing on G1 within residues 70-84 (peptide G5) and 150-169 (peptide G9) were identified using synthetic peptides and aggrecan-specific T cell lines. Two G1-specific T cell hybridomas exclusively responded to peptide G5. When the G5-specific T cell line was injected intraperitoneally into BALB/c mice, it induced acute inflammatory arthritis in joints, but only in those that had been injected with the epitope recognized by these T cells. Furthermore, we also demonstrate that the keratan sulfate chain(s) (KS) on G1 possess immunosuppressive properties with respect to T and B cell epitope recognition. T cell lines that recognize both G1 and peptide G5 show an increased response to G1 after KS is removed. Antibodies in hyperimmune sera of mice immunized with G1 show increased epitope recognition (quantitative and qualitative) after KS removal before immunization. These studies reveal that a T cell line specific to an epitope on the G1 domain of aggrecan, also recognizing a corresponding mouse G1 epitope, can induce arthritis by adoptive transfer and homing to the intraarticular epitope, thereby implicating T cells in arthritis development caused by immunity to the G1 domain of aggrecan. Moreover, the presence of KS on G1 can inhibit arthritis development by suppressing T and B cell epitope recognition.

Decorin-binding adhesins from Borrelia burgdorferi.

Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi. Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres. We demonstrated previously that B. burgdorferi is unable to bind collagen, but can bind the collagen-associated proteoglycan decorin and expresses decorin-binding proteins (Dbps). We have now cloned and sequenced two genes encoding the proteins, DbpA and DbpB, which have a similar structure, as revealed by circular dichroism (CD) spectroscopy of recombinant proteins. Competition experiments revealed a difference in binding specificity between DbpA and DbpB. Western blot analysis of proteinase K-treated intact B. burgdorferi and transmission electron microscopy studies using antibodies raised against recombinant Dbps demonstrated that these proteins are surface exposed. DbpA effectively inhibits the attachment of B. burgdorferi to a decorin substrate, whereas DbpB had a marginal effect, suggesting a difference in substrate specificity between the two Dbps. Polystyrene beads coated with DbpA adhered to a decorin-containing extracellular matrix produced by cultured skin fibroblasts, whereas beads coated with OspC did not. Taken together, these data suggest that Dbps are adhesins of the MSCRAMM (microbial surface component-recognizing adhesive matrix molecule) family, which mediate B. burgdorferi attachment to the extracellular matrix of the host.

Serum content of the C-propeptide of the cartilage molecule type II collagen in children.

OBJECTIVE: The C-propeptide of cartilage type II procollagen, together with the N-propeptide, are removed from newly synthesized procollagen during collagen fibril assembly in cartilage matrix. The presence and content of the C-propeptide reflect the synthesis of this molecule. Recently, we showed that serum levels of the C-propeptide are increased in adults with rheumatoid arthritis, pointing to increased synthesis of this molecule. In this study we examined its content in the sera of children to determine whether it changes during development. METHODS: Sera were obtained from 44 premature infants (cord blood), 75 children (0-18 years), 14 young adults (18-22 years) and 47 adults (35-60 years). The concentration of serum C-propeptide of type II procollagen was determined by a solution phase competitive inhibition radioimmunoassay which uses a polyclonal antiserum specific for the bovine and human C-propeptide. RESULTS: Compared with adults, concentrations of the C-propeptide of type II procollagen were significantly elevated in children of ages 0-14 years. Concentrations were constant until 10 years of age (premature infants: 14.5 +/- 1.4 ng/ml, mean +/- SE; 0-10 years: 13.6 +/- 1 ng/ml). In children of ages 10-14 years, during which the pubertal growth spurt is ordinarily observed, the mean concentration increased (10-14 years: 21.6 +/- 0.7 ng/ml) although not significantly due to the variation between individuals. Concentrations at all ages younger than 14 were significantly greater than those in older adolescents ages 14-18 (6.3 +/- 0.7 ng/ml), young adults (8.4 +/- 2.0 ng/ml) and adults (5.7 +/- 0.4 ng/ml). Serum concentrations did not show significant differences with respect to sex, but varied from child to child at any given age. CONCLUSIONS: The measurement of this circulating C-propeptide may be of use in studying the biochemical and physiological bases of changes in cartilage turnover in children, and abnormalities thereof.

Immunity to cartilage link protein in patients with juvenile rheumatoid arthritis.

OBJECTIVE: To determine whether children with juvenile rheumatoid arthritis (JRA) express cellular immunity to cartilage link protein. METHODS: Link protein was purified from human fetal epiphyseal and bovine adult nasal cartilage. It was used in proliferation assays with the peripheral blood lymphocytes isolated from 54 children with JRA and 22 nonarthritic controls. RESULTS: Patients with JRA expressed a significantly higher prevalence of cellular proliferation to human link protein compared with the control group, independent of whether they had pauciarticular, polyarticular, or systemic disease. In the case of bovine link protein, significant differences were only noted for the polyarticular group. There was significant correlation between the immunity to bovine and to human link proteins. Furthermore, immune responses to both molecules correlated with measures of joint disease activity. CONCLUSION: These results suggest that immunity to link protein may play a role in the pathogenesis of JRA.

Contents and distributions of the proteoglycans decorin and biglycan in normal and osteoarthritic human articular cartilage.

The study was designed to determine the contents and distributions of the proteoglycans decorin and biglycan in adult human femoral condylar cartilage and whether these may change in osteoarthritis. New radioimmunoassays were established using peptides representing the amino-terminal 21 amino acid sequence of each proteoglycan (to which a tyrosine was added for radioiodination) and antibodies in a rabbit antiserum raised to both these molecules. Cartilage was extracted with 4 M guanidine HCl to determine total content, and extracts were analyzed by chromatography to determine molecular sizes. Frozen sections were cut parallel to the articular surface and were extracted to determine distribution within the tissue. Gel chromatography on Sepharose CL-2B under dissociative conditions revealed molecules with a partition coefficient of 0.7-0.75 in both normal and osteoarthritic cartilage. In normal adult cartilage, the mean contents of the core proteins of biglycan and decorin were calculated to be approximately 0.34 and 0.48 mg per gram wet weight, respectively. These represented molar contents similar to that of aggrecan. In osteoarthritic cartilage, there were no overall significant changes in the content and distribution of these molecules. There was, however, considerable individual variation in both distribution and content. Analyses indicated that there was a trend in osteoarthritic cartilage toward a loss of biglycan and decorin from the more superficial layers of intact cartilage, where both these molecules are normally more concentrated. This was accompanied by maintenance of proteoglycan content deeper in the cartilage, regardless of the degree of degeneration.

Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins.

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca] x [PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 microgram/ml (0.01 microM)] whereas OC was of lower potency [IC50 = 6.1 micrograms/ml (1.1 microM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane.

Partial-thickness defects evolving in mature articular cartilage do not heal spontaneously. This type of defect was created in the articular cartilage of adult rabbits and Yucatan minipigs, and the effects of chondroitinase ABC or trypsin, fibrin clots, and mitogenic growth factors on the healing process were examined histologically at intervals ranging from one to forty-eight weeks. The effect of chondroitinase ABC or trypsin was examined initially. Articular cartilage contains macromolecules, including proteoglycans, which render the surfaces of this tissue, and of partial-thickness defects within it, antiadhesive. Chondroitinase ABC digests the glycosaminoglycan chains of cartilage proteoglycans, and trypsin degrades their core proteins. To test the hypothesis that mesenchymal cells may be prevented from adhering to and migrating over the surfaces of partial-thickness defects by proteoglycans, we removed a superficial layer of these macromolecules from the surface of the defect with use of one of these enzymes. The treatment evoked an increase in the coverage of the defect surface with mesenchymal cells; when combined with the local application of a mitogenic growth factor (basic fibroblast growth factor, transforming growth factor-beta 1, epidermal growth factor, insulin-like growth factor-1, or growth hormone), the coverage was more extensive but mesenchymal cells did not extend into and completely fill the volume of the defect. When the surface of the defect was treated with chondroitinase ABC and the cavity of the defect was filled with a fibrin clot to furnish a matrix or scaffolding for the migration of cells therein, there was migration and proliferation of cells throughout the volume of the defect but at a low population density. Mesenchymal cells remodeled the deposited fibrin matrix, which was replaced by a loose fibrous connective tissue. When defects that had been treated with chondroitinase ABC were filled with a fibrin clot containing a mitogenic growth factor, mesenchymal cells filled the entire cavity of the defect, and the density of the cells was greatly increased, particularly when transforming growth factor-beta 1 was used. Histological studies revealed a continuous layer of mesenchymal cells extending from the synovial membrane across the superficial tangential zone of normal articular cartilage into the defect, indicating that the cells that were recruited for the repair process were of synovial origin. At forty-eight weeks, the entire cavity of the defect remained filled with a fibrous connective tissue.

Effect of link protein concentration on articular cartilage proteoglycan aggregation.

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6-8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.

Immunity to the G1 globular domain of the cartilage proteoglycan aggrecan can induce inflammatory erosive polyarthritis and spondylitis in BALB/c mice but immunity to G1 is inhibited by covalently bound keratan sulfate in vitro and in vivo.

Earlier work from this laboratory showed that the human proteoglycan aggrecan from fetal cartilages can induce a CD4+ T cell-dependent inflammatory polyarthritis in BALB/c mice when injected after removal of chondroitin sulfate chains. Adult keratan sulfate (KS)-rich aggrecan does not possess this property. We found that two CD4+ T cell hybridomas (TH5 and TH14) isolated from arthritic mice recognize bovine calf aggrecan and the purified G1 domain of this molecule, which also contains a portion of the interglobular domain to which KS is bound. These hybridoma responses to G1 are enhanced by partial removal of KS by the endoglycosidase keratanase or by cyanogen bromide cleavage of core protein. KS removal results in increased cellular uptake by antigen-present cells in vitro. After removal of KS by keratanase, G1 alone can induce a severe erosive polyarthritis and spondylitis in BALB/c mice identifying it as an arthritogenic domain of aggrecan. The presence of KS prevents induction of arthritis presumably as a result of an impaired immune response as observed in vitro. These observations not only identify the arthritogenic properties of G1 but they also point to the importance of glycosylation and proteolysis in determining the arthritogenicity of aggrecan and fragments thereof.

Adherence of Borrelia burgdorferi to the proteoglycan decorin.

Lyme disease is a tick-borne infection that can develop into a chronic, multisystemic disorder. The causative agent, Borrelia burgdorferi, is initially deposited by the tick into the host dermis, where it associates with collagen fibers, replicates, and eventually disseminates to other tissues. We have examined the adherence of the spirochete to different components of the collagen fiber and demonstrated that decorin, a proteoglycan which decorates collagen fibers, can support the attachment of B. burgdorferi. No significant direct attachment to isolated type I or III collagens could be detected. Attachment of the spirochetes to decorin was highly specific, and the process could be inhibited by soluble decorin but not by various unlabeled, unrelated components. B. burgdorferi also bound soluble 125I-labeled decorin in a time- and concentration-dependent manner. Spirochete binding of soluble 125I-labeled decorin required intact proteoglycan and could not be inhibited by either isolated core protein or glycosaminoglycan chain. B. burgdorferi expresses two decorin-binding proteins with apparent molecular masses of 19 and 20 kDa as revealed in a Western blot (immunoblot)-type assay. Our results indicate that decorin may mediate the adherence of B. burgdorferi to collagen fibers in skin and other tissues.

N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage.

In the hyaluronan binding region (HABR) peptide of aggrecan, there is a marked increase in the level of keratan sulfate (KS) during aging. To determine the sites of KS attachment, KS-containing peptides were prepared from HABRs from immature and mature bovine articular cartilage by digestion with trypsin or papain followed by carbohydrate analysis and peptide sequencing. KS is attached to Thr42 within loop A in mature, but not in immature, HABR. Within loop B KS is N-linked to Asn220 in both HABRs, but in the immature HABR the chains are shorter. Asn314 in loop B' of mature HABR is substituted either with a KS chain or with an oligosaccharide of the complex type. In immature HABR this site does not carry KS. In the interglobular domain, 2 threonine residues within the sequence TIQTVT are substituted in both calf and steer, and in steer further substitution occurs within the sequence NITEGEA, which contains a major catabolic cleavage site (Sandy, J., Neame, P.J., Boynton, R., and Flannery, C.R. (1991) J. Biol. Chem. 266, 8683-8685). The extreme polydispersity of mature HABR was investigated by preparing four subfractions of increasing molecular size which had essentially the same protein core, i.e. Val1-Arg367 or Val1-Arg375. The smaller species lacked the KS chains attached to loop A. These results show that KS substitution occurs within each of the disulfide-bonded loops of the HABR, that the KS may be either N- or O-linked, and that variations in the addition of KS are responsible for the polydispersity of mature HABR.

Inhibition of glycogenin-catalyzed glucosyl and xylosyl transfer by cytidine 5'-diphosphate and related compounds.

The self-glucosylation of beef kidney glycogenin was inhibited by the following pyrimidine nucleotides and nucleotide sugars, listed in order of decreasing effectiveness: CDP-glucose, CDP, UDP-xylose, UDP-N-acetylglucosamine, UDP-galactose, UDP, CTP, CDP-choline, UDP-glucuronic acid, beta-S-UDP-glucose, and CMP. In contrast, the purine nucleotide sugars, ADP-glucose and GDP-glucose, were essentially ineffective, as was the pyrimidine nucleoside, cytidine. UDP-Xylose may be utilized by glycogenin as an alternative sugar donor instead of UDP-glucose (Rodén, L., Ananth, S., Campbell, P., Manzella, S., and Meezan, E. (1994) J. Biol. Chem. 269, 11509-11513) and therefore presumably inhibited the glucosyl transfer reaction by being a competitive substrate. Like glucosyl transfer, xylosyl incorporation into glycogenin was also inhibited effectively by CDP. On the other hand, UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26) was not affected by CDP, nor was it inhibited by UDP-glucose. Addition of CDP or UDP-glucose to reaction mixtures containing both enzymes therefore made it possible to assay xylosyltransferase EC 2.4.2.26 reliably without the extensive product characterization that is otherwise necessary. The CDP effect on glycogenin further allowed the development of an improved procedure for the purification of this enzyme, in which specific elution of an affinity matrix (UDP-glucuronic acid-agarose) was carried out with CDP as the eluant.

Cartilage and bone metabolism in rheumatoid arthritis. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism.

Serum concentrations of specific cartilage and bone molecules reflecting tissue turnover were measured in two well-defined patient groups with early rheumatoid arthritis with distinctly different disease outcome to see if early differences in their levels are prognostic of the rate of joint destruction. Compared with a matched normal population, increased concentrations of cartilage oligomeric matrix protein (COMP) were found in all patients who developed rapid hip joint destruction. In contrast, levels of a putative marker of cartilage aggrecan synthesis, the chondroitin sulfate epitope 846, were increased only in patients with slow joint destruction. Levels of bone sialoprotein (BSP) were increased in both groups, as were levels of the C-propeptide of type II procollagen (CPII), a marker of collagen II synthesis. The increased concentrations of the 846 epitope in patients with slow joint destruction suggest increased aggrecan synthesis. The low levels of the 846 epitope in patients with rapid joint destruction, concomitant with elevated levels of CPII, suggest a selective increase in collagen synthesis. The elevated BSP levels indicate an increased bone turnover in both groups. Thus elevated serum levels of COMP may indicate an unfavorable prognosis for rapid joint destruction, whereas elevated 846 epitope indicates a more favorable prognosis.

Specific effects of glycosaminoglycans in an analog of extracellular matrix that delays wound contraction and induces regeneration.

Previous studies have shown that a simple analog of extracellular matrix delays wound contraction and, if seeded with keratinocytes, inhibits scar synthesis by inducing partial regeneration of the dermis and the epidermis in full-thickness skin wounds in the guinea pig. The active extracellular matrix analog was selected from a large number of copolymers of type I collagen and chondroitin-6-sulfate differing in average pore diameter and degradation rate. However, these previous studies did not provide information on the potential role of the glycosaminoglycan component of this collagen-glycosaminoglycan matrix. The present study focuses on the effect of substitution of chondroitin-6-sulfate by other glycosaminoglycans or with the corresponding proteoglycans. The three substituents of chondroitin-6-sulfate studied were dermatan sulfate, decorin (a proteoglycan-containing dermatan sulfate chain), and aggrecan (a proteoglycan in which 90% of the glycosaminoglycan component was chondroitin-6-sulfate). Each test matrix was grafted on full-thickness skin wounds in the guinea pig, and the wound contraction kinetics were followed. Previous studies have strongly suggested that delay in onset of contraction is necessary for regeneration. Substitution of chondroitin-6-sulfate by either dermatan sulfate or decorin increased the delay in wound contraction by the greatest increment. However, the difference between substitution either by the dermatan sulfate chains or by the corresponding proteoglycan was not significant. Substitution of chondroitin-6-sulfate by aggrecan did not affect the activity of the extracellular matrix analog. These results suggest that the glycosaminoglycan component of the proteoglycan, rather than the protein core, is responsible for the increment of activity. It is speculated that the morphogenetic activity of the extracellular matrix analog resides in its putative ability to neutralize transforming growth factor-beta, leading thereby to downregulation of the inflammatory response in the wound bed.

Age-related changes in cartilage proteoglycans: quantitative electron microscopic studies.

Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules.