Biomedical subjects

A Baici

Publications and source records attributed to A Baici.

At least 19 recordsLinked to original sources

PubMed2001

Alternative messenger RNA splicing and enzyme forms of cathepsin B in human osteoarthritic cartilage and cultured chondrocytes.

OBJECTIVE: In previous studies, we suggested that cathepsin B, which is present at sites of cartilage remodeling in osteoarthritis (OA), may act as an antagonist of cartilage repair, an enhancer of the action of metalloproteinases, and a mediator of cartilage neovascularization and mineralization. Alternative splicing of cathepsin B pre-messenger RNA (pre-mRNA) and/or mRNA overexpression is a plausible regulatory mechanism. In the present study, we investigated the abundance of cathepsin B transcripts and the properties of cathepsin B protein in normal and OA cartilage, osteophytes, and cultured chondrocytes. METHODS: Cathepsin B mRNA splice variants containing the full-length transcript (CB) and the variants lacking either exon 2 (CB[-2]) or lacking exons 2 and 3 (CB[-2,3]) were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot assays and were localized by in situ RT-PCR. Cathepsin B protein was analyzed by electrophoretic, Western blot, and chromatographic methods. RESULTS: The relative content of CB, CB(-2), and CB(-2,3) varied considerably in OA cartilage and osteophytes, with less variation in normal cartilage. The mean cathepsin B mRNA level was significantly higher in OA cartilage and osteophytes than in normal cartilage. Normal cultured chondrocytes attained cathepsin B mRNA levels similar to those in OA cartilage. Enzyme overexpression resulted in the secretion of procathepsin B, followed by activation to the proteolytically active form. CONCLUSION: The high levels of CB and CB(-2) are consistent with an overproduction of secreted procathepsin B in OA. Up-regulation of chondrocyte cathepsin B, which takes place at both the transcriptional and the translational level, suggests a leading role of the enzyme in the progression of OA.

Aged↗

PubMed2000

Cytoskeletal architecture and cathepsin B trafficking in human articular chondrocytes.

In the differentiated state, human articular chondrocytes exhibited modestly developed cytoskeletal components, while cells dedifferentiated by serial subcultures in vitro displayed a prominent cytoskeleton. Morphological changes, a well-developed actin cytoskeleton, and the presence of numerous intracellular organelles were characteristic features of the dedifferentiated chondrocyte phenotype. These properties correlated with the expression, biosynthesis, storage, and secretion of the cysteine peptidase, cathepsin B, a marker of the dedifferentiated chondrocyte phenotype and a potent mediator of cartilage catabolism in osteoarthritis. Both the actin cytoskeleton and microtubules were responsible for trafficking of cathepsin B between cellular compartments in chondrocytes. Despite the endosomes and lysosomes storing high amounts of mature cathepsin B, this enzyme could not be visualized in its active form within these organelles. However, enzymatically active cathepsin B was associated with polymerized tubulin, and was no longer detectable after disruption of the microtubules. This enzyme species possibly represents the mature cathepsin B form in transport vesicles, after cleavage of the inhibitory propeptide, on the way to a final target. These results suggest noteworthy parallels between osteoarthritic articular cartilage and the artificially dedifferentiated cell phenotype, including the expression of type I collagen, the expression of cathepsin B, a significant modification of the cytoskeleton, and the formation of abundant secretory vesicles. These similarities justify the use of chondrocyte cultures as models of the behavior of cartilage cells in osteoarthritis.

Antigens, CD↗

PubMed2000

The relative importance of cysteine peptidases in osteoarthritis.

OBJECTIVE: To assess the activity of cysteine peptidases in cultured human articular chondrocytes as well as in osteoarthritic (OA) cartilage and subchondral bone, and to interpret their relative importance in cartilage destruction and remodeling of the subchondral region. METHODS: Intracellular and secreted cysteine peptidase activity was measured in chondrocytes using fluorimetric assays, and enzymes were immunolocalized using monospecific antibodies. Enzyme histochemistry in normal and OA femoral heads was used to characterize enzymatic activity in full thickness samples containing cartilage and subchondral bone. The zonal distribution of cathepsin activity was measured in tissue slices of normal and OA femoral heads cut parallel to the joint surface, using fluorogenic substrates. RESULTS: Cathepsins B and L were localized by immunohistochemistry with lysosome-like structures in dedifferentiated chondrocytes. Free cysteine peptidase activity (i.e., not requiring prior activation), secreted and intracellularly stored by chondrocytes, was due to cathepsin B, while cathepsin L contributed a minor fraction of the total activity, and was seen only after activation at acidic pH. Histochemistry and activity measurements confirmed cathepsin B as the major, active cysteine peptidase in OA cartilage, particularly at sites where matrix neosynthesis took place. However, free cathepsin L and/or cathepsin K activity was found subchondrally in association with cathepsin B in osteophytes, in zones undergoing bone remodeling, and at sites of inflammation. CONCLUSION: Cathepsin B, not cathepsin L or cathepsin K, is a candidate for articular cartilage catabolism in OA. While cathepsin K is the major osteoclastic cysteine peptidase, cathepsin L and cathepsin B may also participate in the remodeling processes of bone as well as in bone erosion by inflammatory cells.

Adult↗

PubMed1999

Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases.

The tissue inhibitors of matrix metalloproteinases (MMPs), TIMP-1 and TIMP-2, are also angiogenesis inhibitors. Cathepsin B and MMPs are found at sites of neovascularization in pathologies such as cancer and osteoarthritis. Treatment of TIMP-1, TIMP-2, and of a mixture of both inhibitors from human articular chondrocytes with cathepsin B resulted in their fragmentation, whereby they lost their MMP-inhibitory and anti-angiogenic activities. Our data suggest that, besides directly participating in tissue destruction, cathepsin B can be harmful for two further reasons: it raises the activity of the MMPs also in the absence of mechanisms up-regulating these enzymes, and it stimulates angiogenesis. This is a prerequisite for blood vessel invasion in a variety of pathological situations of which cancer and osteoarthritis are prominent examples.

Cartilage, Articular↗

PubMed1998

Characterization of human matrilin-3 (MATN3).

We have isolated a cDNA clone for human matrilin-3 from a cartilage-specific cDNA library. The polypeptide predicted from the nucleotide sequence of this clone shared 83% identity with matrilin-3 from mouse and 61% with that from chicken. It was composed of 486 amino acid residues that were arranged in seven domains: a signal peptide, a von Willebrand factor A domain, four EGF repeats, and an alpha-helical region. The gene for human matrilin-3 (MATN3) was assigned to chromosome region 2p24-p23. The corresponding mRNA of 2.8 kb was expressed in every type of cartilage investigated thus far. It was also produced in vitro by primary chondrocytes isolated from articular cartilage. However, dedifferentiated chondrocytes of the third passage did not express it at all. Matrilin-3 might therefore serve as a marker for the differentiation state of chondrocytes.

Amino Acid Sequence↗

PubMed1998

The axonally secreted serine proteinase inhibitor, neuroserpin, inhibits plasminogen activators and plasmin but not thrombin.

Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig-kappa-variable region, an Ig-kappa enhancer, and the exon encoding the Ig-kappa constant region (C kappa) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C kappa. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.

Amino Acid Sequence↗

PubMed1998

Parallel pathways in the folding of a short-term denatured scFv fragment of an antibody.

BACKGROUND: Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (VH and VL) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated. RESULTS: Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin. CONCLUSIONS: The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerization reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.

Immunoglobulin Fragments↗

PubMed1997

Differential expression of mRNAs for endopeptidases in phenotypically modulated ('dedifferentiated') human articular chondrocytes.

Human articular chondrocytes modulated away from their original phenotype by serial subcultures in monolayer differentially express mRNAs for endopeptidases. The mRNAs for the cathepsins B and L are extremely low in differentiated cells, but are soon expressed in parallel with the loss of the differentiated state. In contrast, the mRNA for collagenase-1 is strongly expressed by differentiated chondrocytes and declines rapidly following phenotypic modulation. The mRNA for stromelysin-1 and the tissue inhibitor of metalloproteinases-2 is high and does not appreciably change after modulation. Chondrocyte activation induced by alteration of its original phenotype leads to the expression of endopeptidases in a way that markedly differs from that induced by cytokines. The results are relevant to cartilage catabolism in osteoarthritis and suggest a prominent role of fibroblastic metaplasia on the part of the chondrocytes as a mechanism of expressing catabolic endopeptidases.

Adult↗

PubMed1997

The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system.

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK ATP; (2) DnaJ-triggered conversion of peptide x DnaK x ATP (T state) to slowly-acting, high-affinity peptide x DnaK x ADP x P(i) (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T --> R and R --> T conversion were 0.04 s(-1) and 1.0 s, respectively. The clearly rate-limiting T --> R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T --> R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.

Adenosine Triphosphatases↗

PubMed1997

Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper.

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.

Amino Acid Sequence↗

PubMed1997

Electrostatic interactions between human leukocyte elastase and sulfated glycosaminoglycans: physiological implications.

The influence of ionic strength and composition on the binding and inhibition of human leukocyte elastase by glycosaminoglycans with variable degree and position of sulfation was investigated. The kinetic mechanism of inhibition had a hyperbolic, mixed-type character with a competitive component that was promoted by low ionic strength, reduced by phosphate ions, and which also depended on the substrate and glycosaminoglycan structure. Enzyme binding was a cooperative phenomenon that varied with ionic strength and composition. The inhibition patterns correlated with the cationic character of elastase and with the distribution of arginines on its molecular surface, most notably with residues located in the vicinity of the substrate binding region. The order of affinity for elastase binding was chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate, iduronate-containing derivatives being superior with respect to the glucuronate-containing counterparts. Additional sulfation at both the 4- and 6- positions or at the N- and 4-positions of the N-acetylgalactosamine moiety decidedly improved the inhibitory efficiency. The results highlight a fundamental physiological role of enzyme-glycosaminoglycan interactions. In the azurophil granule of the human polymorphonuclear neutrophil, elastase and other enzymes are bound to a matrix of chondroitin 4-sulfate because this is the only glycosaminoglycan that simultaneously offers good binding for enzyme compartmentalization together with prompt release from the bound state at the onset of phagocytosis.

Enzyme Inhibitors↗

PubMed1996

Antileukoprotease inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative function in desquamation.

The stratum corneum chymotryptic enzyme (SCCE) has been previously purified from human stratum corneum and resembles a chymotryptic serine endopeptidase involved in physiological detachment of corneocytes from human stratum corneum. From human stratum corneum two inhibitory activities of SCCE could be extracted. These were due to serine protease inhibitors already known to be present in human epidermis, antileukoprotease (secretory leukocyte protease inhibitor) and elafin (skin-derived antileukoprotease). The Inhibition of SCCE by antileukoprotease shows a hyperbolic, mixed type inhibition with an equilibrium dissociation constant of 63 n. Antileukoprotease also inhibits detachment of corneocytes from human plantar callus in vitro almost completely (>96%). In addition, elafin was shown to be a weak inhibitor for SCCE activity, and elafin significantly reduces the shedding of corneocytes. Thus, antileukoprotease, which is known to be produced by human keratinocytes, is likely to be the major physiological inhibitor of SCCE in the epidermis. It seems to be involved in the regulation of desquamation under physiological and pathophysiological conditions.

Aprotinin↗

PubMed1996

pH-dependent hysteretic behaviour of human myeloblastin (leucocyte proteinase 3).

Human myeloblastin (leucocyte proteinase 3) showed a very slow approach to the steady-state velocity when the pH was rapidly increased from 3.2 to 7.0. The kinetic mechanism of this hysteretic process was interpreted as a slow conformational change of myeloblastin from an inactive form at acidic pH to the active form at neutral pH. The transition between the two enzyme forms could occur spontaneously in the absence of substrates with a first-order rate constant of 0.0033 s-1. In the presence of peptide substrates activation occurred more rapidly: the observed rate constant was linearly dependent upon the substrate concentration and contained a contribution of the spontaneous as well as of the substrate-dependent process, whose second-order rate constant was characteristic of the particular substrate. This pH-dependent phenomenon of hysteresis on the part of myeloblastin, that is not manifested by the closely related leucocyte elastase, may have a physiological control function during phagocytosis by damping the rate of interconversion between enzymically inactive and active enzyme conformations.

Humans↗

PubMed1996

Human myeloblastin (leukocyte proteinase 3): reactions with substrates, inactivators and activators in comparison with leukocyte elastase.

Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.

Enzyme Activation↗

PubMed1995

Kinetics of folding of leucine zipper domains.

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Comparison of chondroitin sulphate composition of femoral head articular cartilage from patients with femoral neck fractures and osteoarthritis and controls.

The glycosaminoglycan (GAG) and uronic acid (UA) composition of human hip articular cartilage from patients with femoral neck fractures [assumed osteoporosis (OP); n = 12], from patients with osteoarthritis (OA; n = 12) and from normal controls (n = 9) was determined. Full depth tissue samples from the control and OP groups were analysed from the superior, inferior, anterior and posterior regions, while the OA tissue was from cystic (tissue growing on top of cystic bone lesions) and osteophytic regions, from normal and fibrillated resident cartilage and from regions immediately adjacent to eburnated bone. The total sulphated GAG and UA content was reduced in the inferior region of control cartilage compared to the other regions and the values of all regions of the assumed OP group. Cystic regions and OA cartilage adjacent to the bone also showed lower GAG and UA levels than the other regions. The ratios of chondroitin 6-sulphate (C6S) to chondroitin 4-sulphate (C4S) indicated a similar pattern in the different regions of controls and the patient group with femoral neck fracture (OP group). The cystic and osteophytic cartilage of the OA group exhibited lower C6S/C4S ratios than any other region. The levels of dermatan sulphate (DS) in the cartilage of all regions of the OP and control groups were very similar and low, while the tissues of the OA group contained significantly higher amounts, particularly the cartilage from osteophytes. The previously presumed compositional similarity between normal aged and osteoporotic articular hip cartilage was essentially confirmed in a comparative analysis. Significant changes in GAG and UA composition of OA cartilage from distinct regions was also recorded.

Aged↗

PubMed1995

Cathepsin B in osteoarthritis: zonal variation of enzyme activity in human femoral head cartilage.

OBJECTIVES: To determine the quantitative topographical distribution of cathepsin B in human femoral head cartilage by measuring the zonal variation of enzyme activity in specimens taken from various anatomical regions of normal and osteoarthritic (OA) tissues, and to correlate this parameter with the severity of the OA lesions. METHODS: OA articular cartilage was obtained at surgery for total hip replacement and control cartilage obtained at postmortem. Cylinders of full thickness cartilage with underlying bone were retrieved with a biopsy trephine. Sections of cartilage were produced by cryocutting the tissue as slices parallel to the articular surface and assayed for cathepsin B with a specific, highly sensitive fluorogenic substrate. The severity of the OA lesions was graded according to the histopathological-histochemical method of Mankin. RESULTS: Zonal cathepsin B activity of normal cartilage was uniform and low in all regions of the femoral head. In apparently intact OA cartilage and in severely degraded tissue the zonal distribution and the amounts of enzyme were similar to control values. At sites with active disease, cathepsin B activity was much greater than in controls and its irregular zonal distribution correlated with tissue degeneration, hypercellularity, or cloning of chondrocytes as determined histochemically. Particularly high enzyme levels were observed at sites with regenerating cartilage, where some zonal peaks attained 20-fold activity with respect to controls. CONCLUSION: Cathepsin B may play a role in sustaining the chronicity of OA, not as an initiator, but rather as a perpetuator of the disease and as an antagonist of regeneration.

Adult↗

PubMed1995

Cathepsin B in osteoarthritis: cytochemical and histochemical analysis of human femoral head cartilage.

OBJECTIVE: To localise the cysteine endopeptidase cathepsin B in chondrocytes and cartilage from normal and osteoarthritic (OA) human femoral heads in order to provide qualitative information on its cellular expression and distribution at possible sites of action. METHODS: OA articular cartilage was obtained at surgery for total hip replacement; control cartilage was obtained at postmortem. Chondrocytes were isolated by sequential enzymatic digestion and cathepsin B analysed by immunocytochemistry and activity staining with a fluorogenic substrate. Lysosomes were visualised by fluorescence microscopy after staining of living cells with acridine orange. Using a histochemical reaction, enzyme activity was measured in cryosections of full thickness cartilage. RESULTS: Chondrocytes from normal cartilage contained very few lysosomes and only a minor cell population was cathepsin B positive. A high proportion of chondrocytes from active OA cartilage contained a large number of lysosomes and an excess of cathepsin B in intracellular organelles; the enzyme was stored in an active form. In this respect, OA chondrocytes closely resembled normal cells that had been phenotypically modulated by serial subcultures. No cathepsin B activity could be detected by histochemistry in either chondrocytes or matrix of normal cartilage. While apparently intact and severely degraded OA cartilage was also cathepsin B negative, tissue at sites of active destruction and, particularly, at repair sites was highly positive. CONCLUSION: The presence and the particular distribution of active cathepsin B in OA cartilage at 'more involved' sites suggest a pathological role for this enzyme in sustaining and perpetuating cartilage degradation. While other stimuli may also be responsible for cathepsin B expression in OA chondrocytes, the similarity with artificially modulated cells indicates fibroblastic metaplasia as a plausible mechanism.

Adult↗