Biomedical subjects

A H Plaas

Publications and source records attributed to A H Plaas.

15 recordsLinked to original sources

PubMed1995

Large and small proteoglycans of osteoarthritic and rheumatoid articular cartilage.

OBJECTIVE: To identify characteristic changes in large aggregating (aggrecan) and small proteoglycan (PG) populations in articular cartilages during osteoarthritis (OA) and rheumatoid arthritis (RA). METHODS: Aggrecan populations in guanidine extracts of femoral condylar cartilages of 46 OA and 8 RA patients who underwent total knee arthroplasty, as well as of 2 fetuses and 6 normal adults, were separated in agarose-polyacrylamide composite gels. Small PGs (biglycan, decorin, and fibromodulin) in the same extracts were analyzed in 12% polyacrylamide gels. Gels were stained or electrophoretically transferred and probed with antibodies to aggrecan epitopes and to small PGs. Epitope contents of the samples were also compared by inhibition radioimmunoassay. RESULTS: There were significant differences found among normal and diseased samples in their electrophoretic mobilities, band distributions, and antibody staining. OA and especially RA samples were heavily degraded, lacked certain aggrecan populations, and contained fewer keratan sulfate and chondroitin-6-sulfate epitopes compared with normal samples. Levels of chondroitin-4-sulfate and "fetal-type" epitopes were elevated in the OA samples compared with the normal ones. More core proteins of small PGs were found in diseased than in normal cartilages, but they were more heterogeneous in size and glycosaminoglycan substitution. CONCLUSION: There is extensive degradation of both large and small PGs in diseased cartilages, but a repair process does exist, especially in OA cartilages. Chondrocytes of diseased cartilages are able to synthesize fetal-type aggrecans. Small PGs are glycosylated differently in diseased cartilages than in normal ones.

Adult↗

PubMed1991

Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants.

The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with [35S]sulfate and [3H]proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of [3H]hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules. This study provides a framework for elucidating the biophysical mechanisms involved in the redistribution, catabolism, and loss of macromolecules during cartilage compression.

Animals↗

PubMed1990

Identification of the keratan sulfate attachment sites on bovine fibromodulin.

The small keratan sulfate-substituted proteoglycan (fibromodulin) from articular cartilage was shown to contain keratan sulfate linked to the core protein through N-glycosidic linkages to residues Asn-109, Asn-147, Asn-182, and Asn-272. Biosynthetic experiments with articular chondrocytes in the presence of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, demonstrated a specific inhibition of [35S]SO4 incorporation into fibromodulin. Under the same conditions no effect on the addition of keratan sulfate to the large aggregating proteoglycan was detected. Fibromodulin substituted with keratan sulfate was purified from bovine articular cartilage extracts by density gradient centrifugation, ion-exchange chromatography, and gel-permeation chromatography. Isolation of glycosylated peptides from tryptic digests of fibromodulin by ion-exchange chromatography and reversed-phase high performance liquid chromatography revealed four separate hexosamine-rich species, that were also immunoreactive with monoclonal antibody 5D4. Sequence analysis of these glycopeptides gave blank cycles at positions which corresponded to Asn followed by X-Ser/Thr in the sequence derived from cDNA (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D. (1989) EMBO J. 8, 2601-2604). Hence, all four Asn residues in the leucine-rich region of the fibromodulin core protein can serve as acceptor sites for keratan sulfate addition.

Amino Acid Sequence↗

PubMed1990

Effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in cartilage explants.

The effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in calf cartilage explants were examined. Pulse-chase experiments showed that conversion of low-affinity monomers to the high-affinity form (that is, to a form capable of forming aggregates with 1.6% hyaluronate on Sephacryl S-1000) occurred with a t1/2 of about 5.7 h in free-swelling discs at pH 7.45. Static compression during chase (in pH 7.45 medium) slowed the conversion, as did incubation in acidic medium (without compression). Both effects were dose-dependent. For example, the t1/2 for conversion was increased to about 11 h by either (1) compression from a thickness of 1.25 mm to 0.5 mm or (2) medium acidification from pH 7.45 to 6.99. Oscillatory compression of 2% amplitude at 0.001, 0.01, or 0.1 cycles/s during chase did not, however, affect the conversion. Changes in the hyaluronate-binding affinity of [35S]proteoglycans in these experiments were accompanied by no marked change in the high percentage (approximately 80%) of monomers which could form aggregates with excess hyaluronate and link protein. Since static tissue compression would result in an increased matrix proteoglycan concentration and thereby a lower intra-tissue pH [Gray, Pizzanelli, Grodzinsky & Lee (1988) J. Orthop. Res. 6, 777-792], it seems likely that matrix pH may influence proteoglycan aggregate assembly by an effect on the hyaluronate-binding affinity of proteoglycan monomer. Such a pH mechanism might have a physiological role, promoting proteoglycan deposition in regions of low proteoglycan concentration.

Animals↗

PubMed1989

Synthesis of small proteoglycans substituted with keratan sulfate by rabbit articular chondrocytes.

35S-Labeled proteoglycans produced by chondrocytes from immature and mature rabbits were fractionated on associative CsCl gradients. In all cultures, greater than 85% of the incorporated radioactivity was present in the A1 fraction (rho 1.60) as chondroitin sulfate/keratin sulfate-substituted aggregating proteoglycan monomer; the remainder was present in small proteoglycans in the A2, A3, and A4 fractions of low buoyant densities (rho 1.53, 1.45, 1.37, respectively). Detailed glycosaminoglycan analysis of the A2, A3, and A4 fractions showed dermatan sulfate-rich species were present throughout. However, in both immature and mature cultures, 30-45% of the glycosaminoglycans in the A3/A4 combined fractions were present as keratan sulfate, as shown by insensitivity to digestion with chondroitinase ABC, specific digestion with endo-beta-galactosidase, and reactivity with antibody 5D4. Immature and mature chondrocytes synthesized very similar amounts of the low buoyant density keratan sulfate proteoglycan on a per cell basis. Moreover, 51 and 37% of the total keratan sulfate produced by immature and mature chondrocytes, respectively, were present in the low buoyant density proteoglycan. Pulse-chase experiments indicated that the low buoyant density keratan sulfate was not derived from the large aggregating proteoglycan by proteolysis in the extracellular space. The small keratan sulfate proteoglycans appear to be present as a species distinct from the small dermatan sulfate proteoglycans in these cultures in that they can be separated on Q-Sepharose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent size (40-60 kDa), composition, and heterogeneity of the keratan sulfate proteoglycans suggest that they may be related to the small keratan sulfate proteoglycans of cornea.

Animals↗

PubMed1989

Biosynthetic response of cartilage explants to dynamic compression.

The biosynthetic response of calf articular cartilage explants to dynamic compression was examined over a wide range of amplitudes, waveforms, and frequencies. Glycosaminoglycan synthesis was assessed by 35S-sulfate incorporation, and amino acid uptake and protein synthesis were assessed by 3H-proline incorporation. Two culture chambers were designed to allow uniaxial radially unconfined compression and mechanical testing of cartilage disks: one chamber was used inside a standard incubator; the other was used with a mechanical spectrometer and allowed load and displacement to be monitored during compression. Dynamic stiffness measurements of 3-mm diameter disks identified a characteristic frequency [0.001 Hz (cycles/sec)] that separated low- and high-frequency regimes in which different flow and deformation phenomena predominated; e.g., at 0.0001-0.0001 Hz, significant fluid was exuded from cartilage disks, whereas at 0.01-1 Hz, hydrostratic pressure increased within disks. At the higher frequencies, oscillatory strains of only approximately 1-5% stimulated 3H-proline and 35S-sulfate incorporation by approximately 20-40%. In contrast, at the lower frequencies (a) compressions of less than 5% had no effect, consistent with the dosimetry of biosynthetic inhibition by static compression (approximately 25% compression caused a approximately 20% inhibition of radiolabel incorporation), and (b) higher amplitudes (cycling between disk thicknesses of 1.25 and 0.88-1.00 mm) stimulated 3S-sulfate incorporation by approximately 20-40%, consistent with the kinetics of response to a single 2-h compression and release. None of the compression protocols was associated with detectable alterations in (e.g., compression-induced depletion of) total glycosaminoglycan content. This study provides a framework for identifying both the physical and biological mechanisms by which dynamic compression can modulate chondrocyte biosynthesis. In addition, the culture and compression methodology potentially allows in vitro evaluation of clinical strategies of continuous passive motion therapy to stimulate cartilage remodeling.

Animals↗

PubMed1989

Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures.

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds.

Animals↗

PubMed1988

Biosynthesis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits.

Chondrocytes from immature and mature rabbits have been compared in biosynthetic studies with [3H] leucine and [35S]sulfate as precursors. The time course of incorporation of [3H]leucine into general protein, proteoglycan monomer core protein, and link protein and of [35S]sulfate into proteoglycan monomer has been examined. Proteoglycan monomer was isolated from the high buoyant density (p greater than 1.60) fractions of dissociative CsCl gradients and link protein by immunoprecipitation with antibody 8A4 followed by gel electrophoresis. Results based on the period of linear isotope incorporation showed that mature cells synthesize protein at about 40% of the rate of immature cells and both proteoglycan and link protein at about 20% of the rate of immature cells. The labeling rates obtained suggest that immature cells synthesize an approximate 1:1 molar ratio of link protein to proteoglycan monomer, and for mature cells this ratio is about 0.8:1. While cell layer retention of newly synthesized proteoglycan was markedly lower in mature relative to immature cell cultures, link protein retention was high in both immature and mature cultures; this finding provides an explanation for our previous observation (Plaas, A. H. K., and Sandy, J. D. (1984) Biochem, J. 220, 337-340) that link-free monomer accumulates in the medium of mature but not immature cultures. The link protein synthesized by both ages of cells and isolated from cell layer or medium was a single major species of apparent molecular mass 48-51 kDa. The results suggest that mature chondrocytes are less efficient than immature chondrocytes in the coordinated assembly of link-stabilized proteoglycan aggregates in this culture system.

Animals↗

PubMed1987

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures.

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.

Animals↗

PubMed1987

The biosynthetic response of the mature chondrocyte in early osteoarthritis.

Metabolic studies in early experimental osteoarthritis (OA) have shown that the rate of proteoglycan synthesis in the diseased articular cartilage may be markedly enhanced relative to normal; elevated rates of synthesis were however accompanied by increased release of new molecules from the tissue so that the response was apparently non-reparative. Described here are experiments with immature and mature chondrocytes in culture which show that aging of chondrocytes in vivo is accompanied by a marked fall in the capacity of these cells to synthesize link protein and to assemble a proteoglycan-rich matrix. It is suggested that poor deposition of proteoglycan by mature chondrocytes in OA may result from insufficient synthesis of link protein for stabilization of aggregates.

Aging↗

PubMed1986

The affinity of newly synthesized proteoglycan for hyaluronic acid can be enhanced by exposure to mild alkali.

The affinities for hyaluronic acid of newly synthesized proteoglycan from post-confluent rabbit chondrocyte cultures and purified bovine proteoglycan monomer were compared. In mixtures prepared at pH 6.8 the newly synthesized proteoglycan had the lower affinity; however, in mixtures incubated at pH 8.5 for 24 h before addition of hyaluronic acid, the newly synthesized proteoglycan exhibited a markedly higher affinity than the bovine monomer. The results suggest that proteoglycan secreted without associated link protein [Plaas, Sandy & Muir (1983) Biochem. J. 214, 855-864] has a low affinity for hyaluronate and that this may be increased during subsequent extracellular processing.

Alkalies↗

PubMed1986

Age-related changes in the kinetics of release of proteoglycans from normal rabbit cartilage explants.

The release of proteoglycans from explant cultures of articular cartilage from immature and mature rabbits has been studied with the following results. At both ages the tissue proteoglycan was released in two phases: an initial extensive release (day 0 to 3) and a period of slow release (day 4 to 15). The percentage released in the initial phase was, however, significantly greater for mature (55%) than immature (38%) explants. At both ages the newly synthesized proteoglycans (in vivo labeled) were also released in two kinetic pools. Thus, graphical analysis of release data readily resolved the disappearance curves into two linear components with in vitro half-lives of 1 day and 22 days. Again, the percentage in the short half-life pool was much greater for mature (70%) than immature (40%) explants. At both ages the initial release was largely chondrocyte-mediated since freeze-thawing the tissue before culture markedly reduced proteoglycan release. At both ages the released proteoglycans were smaller than equivalent preparations of extracted proteoglycans and they were much less capable of forming aggregates with hyaluronate. The results show that there are age-dependent changes in rabbit articular cartilage that increase the proportion of proteoglycans, both total and newly synthesized, that are susceptible to rapid chondrocyte-mediated catabolism in explant cultures.

Age Factors↗

PubMed1985

A sensitive assay for active link protein from cartilage.

A new assay for the activity of cartilage link protein is described. The method is based on the finding [Plaas, Sandy & Muir (1983) Biochem. J. 214, 855-864] that addition of link protein to [35S]sulphate-labelled proteoglycan aggregates from rabbit chondrocyte cultures resulted in the formation of link-stabilized aggregates. The percentage aggregate was found to be related linearly to the amount of purified bovine link protein added in the 20-120 ng range. The assay was used to monitor loss of link-protein activity during heat denaturation and to measure binding of link protein by purified proteoglycan monomer.

Animals↗

PubMed1984

Age-related decrease in the link-stability of proteoglycan aggregates formed by articular chondrocytes.

Chondrocytes were isolated from the articular cartilage of rabbits aged between 6 and 50 weeks and labelled with [35S]sulphate after 48 h in monolayer culture. The percentage of the total proteoglycan monomers synthesized by each culture that were present as link-stabilized aggregates was shown to be about 83% at 6, 9 and 12 weeks, 73% at 15 weeks, 48% at 30 weeks and 32% at 50 weeks. The proliferative activity of the cells in culture also decreased markedly with the age of the donor. The results suggest that aging of chondrocytes in vivo is accompanied by a decrease in their capacity for link-protein synthesis.

Aging↗

PubMed1983

Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture.

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.

Animals↗