Acetyl-L-carnitine and alpha-lipoic acid supplementation of aged beagle dogs improves learning in two landmark discrimination tests.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.

A matrix metalloproteinase proenzyme activator produced by articular cartilage.

Matrix metalloproteinases (MMPs) are involved in connective tissue turnover under physiological and pathological conditions. MMP activity is regulated by the requirement for zymogen activation. This report describes a proMMP-3 activator produced by articular cartilage. The activator initiates a step-wise processing of proMMP-3 to generate an array of active species. Sequencing of activation intermediates demonstrated cleavage on the NH2-terminal side of certain basic residues in the MMP-3 propeptide. Metal ion chelators inhibited activator-dependent proteolysis, and activity was restored by low levels of ZnCl2. These catalytic properties suggest similarity to members of the insulinase superfamily of metalloendopeptidases with in vitro specificity for single arginine or paired basic processing sites in a variety of prohormones. Dibasic sites also exist in the propeptides of several MMPs including proMMP-3. This is the first report that cartilage produces a potent MMP proenzyme activator, opening the possibility of a novel intrinsic activation pathway for catabolic processes in this avascular tissue.

Matrix metalloproteinase digestion of aggrecan in human cartilage tumours.

Substantial experimental and clinical evidence suggests that the catabolism of extracellular matrix components is a prerequisite for invasive and metastatic behaviour of solid tumours. Chondrosarcomas are malignant cartilaginous tumours that most commonly arise in bone, and the large aggregating proteoglycan aggrecan is a major component of the extracellular matrix of these tumours. Matrix metalloproteinases (MMPs) have been implicated in tumour invasiveness. The purpose of this study was to determine whether MMPs play a role in aggrecan catabolism in cartilage tumours. In order to detect aggrecan digestion products resulting from in vivo cleavage at the MMP site, protein extracts from human articular cartilage and from various cartilage tumours were analysed by Western blot using an antibody to the FVDIPEN neoepitope generated by MMP cleavage. Examination of cartilage extracts revealed a trend of increasing aggrecan digestion at the MMP site with age. One hyaline chondrosarcoma and three osteochondromas lacked detectable aggrecan fragments with the carboxy terminal FVDIPEN neoepitope. Two osteochondromas gave weak signals. However, all chondrosarcomas with degenerating extracellular matrix or with a myxoid component exhibited strong FVDIPEN immunoreactivity. These results demonstrate that, in contrast to the benign cartilage tumour osteochondroma, human chondrosarcomas contain abundant aggrecan degradation products resulting from cleavage in vivo at the MMP site in the interglobular domain. These data support the concept that MMPs participate in the degradation of extracellular matrix in chondrosarcoma, allowing the neoplastic chondrocytes to escape local confinement, migrate, and invade neighbouring and remote tissues.

Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible autocrine/paracrine role in pathogenesis.

The interleukin-1 (IL-1) cytokines stimulate the synthesis of degradative enzymes in joint tissues and may play a role in the pathological joint destruction in osteoarthritis (OA). In this study, we have used immunohistochemistry and Western blot analysis to identify IL-1 in human OA cartilage. IL-1 alpha and IL-1 beta were evident in chondrocytes at the articular surface, as well as distributed throughout the cartilage. In many specimens, IL-1 beta but not IL-1 alpha was detected as a diffuse staining of the extracellular matrix especially surrounding superficial zone chondrocytes. Although chondrocyte-associated IL-1 alpha and IL-1 beta were detected in most specimens, cartilages exhibiting early osteoarthritic changes had the highest intensity of staining and the highest frequency of positive cells. Western blot analysis revealed intense immunoreactive bands corresponding to the 35 kDa precursor form of IL-1 alpha in all four chondrocyte lysates tested. The processed 18 kDa IL-1 beta species was present in only one of four chondrocyte lysates, and there was no clear evidence of precursor form within these cells. The results of this study indicate increased IL-1 alpha in cartilage showing early degenerative changes, suggesting an autocrine/paracrine role for this cytokine in OA pathogenesis.

Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of interleukin 1-mediated proteoglycan degradation in bovine articular cartilage explants by addition of sodium hyaluronate.

The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (IL-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to IL-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and IL-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend. Evaluation of alterations in protein (enzyme) release by IL-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of IL-1-induced proteins did not differ after introduction of hyaluronate into the culture system. Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with IL-1. Because IL-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of IL-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.

A plasminogen-related gene is expressed in cancer cells.

The breakdown of blood clots requires the fibrinolytic action of the serine proteinase plasmin, a two-chain polypeptide derived posttranslationally from its precursor zymogen, plasminogen. While investigating plasminogen gene expression in human extrahepatic tissues, a cDNA sequence was obtained which closely resembled the plasminogen cDNA, yet appeared to represent a distinct gene product. This sequence, which represents the transcript of the recently characterized plasminogen-related gene B, encodes a putative polypeptide of Mr 8800 and is expressed most prominently in malignant cancer cells.

Alternatively spliced annexin XI transcripts encode proteins that differ near the amino-terminus.

Annexins are a family of structurally related calcium-dependent phospholipid binding proteins. We recently described a new member of this family, bovine annexin XI [1]. Two kinds of cDNAs were identified corresponding to annexin XI mRNA variants A and B, which are generated by alternative splicing of identical primary transcripts. Annexin XI isoforms are predicted to differ at the amino-terminus, suggesting that they may have distinct biological roles.

Identification of a novel mammalian annexin. cDNA cloning, sequence analysis, and ubiquitous expression of the annexin XI gene.

Annexins (or lipocortins) are a family of at least 10 structurally related calcium- and phospholipid-binding proteins. Each protein consists of a conserved core domain having four (or eight) repeats of a segment approximately 70 amino acids in length and a nonconserved, usually short, amino-terminal domain. To date, amino acid sequences for eight distinct mammalian annexins have been predicted from cDNAs. This report describes an additional member of this family, bovine annexin XI, identified by cDNA cloning and sequence analysis. The 503-amino acid deduced protein consists of a core domain of four annexin repeats and a long amino-terminal domain rich in glycine, proline, and tyrosine. This novel annexin gene is expressed in a wide variety of tissues and isolated cells in culture.

Cartilage synthesizes the serine protease inhibitor PAI-1: support for the involvement of serine proteases in cartilage remodeling.

The work described here demonstrates the synthesis by human articular cartilage of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the serine protease tissue plasminogen activator (tPA). We also present data demonstrating an increase in PAI-1 messenger ribonucleic acid (mRNA) in chondrocytes exposed to the cytokine interleukin-1 (IL-1). Interestingly, this elevation of steady-state mRNA levels does not appear to result in an increase in synthesis of PAI-1 protein. Northern blot analysis reveals that of the two mRNA species (3.4 kb, 2.4 kb) previously reported for PAI-1, only the larger species (3.4 kb) appears to be synthesized by chondrocytes. Our data demonstrate the IL-1-stimulated production by cartilage of tissue plasminogen activator. We also show evidence for the presence of plasminogen in cartilage. A scheme is presented indicating the probable importance of the serine proteases (tPA and plasminogen) and PAI-1 in cartilage degradation.

Growth factors and articular cartilage.

Studies of articular cartilage over the decades have demonstrated a surprisingly brisk rate of synthesis of the matrix proteins which appears to vary considerably with metabolic, physicochemical and pathological state of the tissue. It has become evident that much of this activity is directed by low molecular weight protein mediators which act at specific receptor sites. Platelet derived growth factor (PDGF) is of limited action in normal cartilage, but insulin and its analogues, insulin growth factor-I and II are powerful stimulants of DNA synthesis. Basic fibroblast growth factor stimulates both DNA and protein synthesis and works synergistically with other factors. Transforming growth factor beta potentiates the action of the mitogens and enhances and regulates proteoglycan synthesis. These actions may be of special importance in osteoarthritis and lacerative injury to cartilage.

The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix.

The developing enamel matrix contains mostly amelogenins, which are hydrophobic proline-rich proteins. During amelogenesis, the amelogenins are presumably hydrolysed and removed from the enamel. Recently a number of metalloproteinases that may be important in amelogenesis have been identified in zymograms of the developing enamel matrix. In the present study an antibody specific for the matrix metalloproteinase collagenase activator protein (CAP) was characterized and used to identify this metalloproteinase in enamel. Immunoblotting showed that the CAP proteinase was present in the enamel matrix. Immunohistochemistry confirmed that the proteinase is localized in the enamel matrix, most specifically along the dentino-enamel junction. Purified CAP was found to hydrolyse amelogenin protein. Possible functions of the proteinase in the enamel matrix are discussed.

Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research.

Accurate estimation of cellular viability is important both in research and in aspects of orthopaedic clinical practice. We have been interested in the potential for flow cytometric application of fluorescein diacetate (FDA) in evaluating chondrocyte survival following cryopreservation of osteochondral allografts as well as in the assessment of sarcoma necrosis following preoperative chemotherapy. In order to evaluate the suitability of this method for cell viability assays, this study compared FDA with more traditional methodology (trypan blue, clonigenic assay, metabolic activity analysis, measurement of DNA synthesis, and histological assessment of necrosis). Both chondrocytes and sarcoma cells were exposed to various experimental injuries prior to viability analysis. Although it is evident from these experiments that FDA accurately reflects cell survival after physical injury, it underestimates the effect of chemotherapy on cell reproductive potential in vitro. However, FDA is highly correlated with histological assessment of tumor viability after chemotherapy in vivo. It is apparent that the methodology chosen for determination of viability should be appropriate for the type of experimental injury and should analyze the cell function (i.e., metabolic activity or reproductive capacity) that is appropriate for the experimental model.

Regulation of cartilage remodeling by IL-1: evidence for autocrine synthesis of IL-1 by chondrocytes.

Evidence is presented for the synthesis of Interleukin-1 (IL-1) by joint synovial tissue and chondrocytes. Purified preparations of mouse and human recombinant forms of this factor stimulate the synthesis of a secretory protease by cartilage. The IL-1 stimulated chondrocyte protease is capable of converting latent collagenase to its active form. Other proteases such as trypsin and the mercurial aminophenyl mercuric acetate will not activate collagenase in the absence of this protease. Evidence is presented showing that chondrocytes synthesize IL-1 suggesting autocrine control of cartilage matrix turnover.

The role of proteases in the pathogenesis of osteoarthritis.

It is proposed that the cartilage contains enzymes which are responsible for the degradation of the principle components of the matrix, the proteoglycan and collagen. Measurement of acid, lysosomal bound proteases, or neutral proteases shows increases in proportion to the severity of the disease. Collagenase activity also increases in human osteoarthritic cartilage in the same manner. In an experimental model of osteoarthritis, induced by mechanical factors, enzymatic activity also increased. By inhibiting the enzyme activity with chelators, the arthritic process could be slowed.

The synthetic processes of articular cartilage.

The cells of cartilage are constantly remodeling the matrix in which they are suspended. The stimulus to initiate remodeling is probably the chondrocyte's response to physical and or chemical changes in the environment. Heat, pressure, friction, load, pH, and growth are examples of such factors, which, if altered, would have a dramatic effect on the cell's state of health. The mode of response by the chondrocyte is specific for a given stimulus. Elevated temperature, for example, switches on a set of genes, the heat shock genes, in chondrocytes. This results in the synthesis of a series of cellular protein that presumably in turn protects the cell from the injurious effects of heat. Load and pressure affect both the synthetic rate of matrix protein and the degradation rates of preexisting matrix. A number of low-molecular-weight proteins appear to be involved in anabolic and catabolic processes of cartilage. A protein recently isolated from synovium stimulates the synthesis of degradative enzymes in cartilage. This factor is probably involved in the remodeling process under normal physiologic conditions. More recently, it has been found in elevated levels under pathologic conditions such as in the synovial fluid of patients with rheumatoid and osteoarthritis. The mechanism by which this factor turns on the degradative pathway appears complex and is under investigation.

Expression of IL-1 genes in human and bovine chondrocytes: a mechanism for autocrine control of cartilage matrix degradation.

In this report we describe the presence of interleukin-1 activity in medium conditioned by bovine articular cartilage. Preparations partially purified by Sephacryl S200 chromatography (Mr 18000-25000) stimulate murine thymocyte proliferation in the lymphocyte activation factor assay. Furthermore, the factor(s) activate cartilage tissue to secrete a protease which is essential for the activity of purified synovial collagenase. We also demonstrate the presence of mRNA coding for IL-1 alpha and beta in human articular chondrocytes and conclude that the human monocytic and chondrocytic mRNAs are identical. Our results demonstrating cartilage expression of IL-1 genes suggest the possibility of an autocrine mechanism whereby chondrocyte production of matrix degrading proteases is initiated by chondrocyte derived IL-1.

Purification and characterization of collagenase activator protein synthesized by articular cartilage.

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.

Stimulation of the synthesis of collagenase activator protein in cartilage by a factor present in synovial-conditioned medium.

We have purified a low molecular weight protein from medium conditioned by calf synovium with physical and biological properties similar to the leukocyte cytokine interleukin 1 (IL-1). The factor is active in stimulating the synthesis (three- to fivefold) of collagenase activator protein (CAP) by the surface (1-2 mm) of articular cartilage while CAP synthesis in the deeper zones of articular cartilage is not affected. Recombinant mouse IL-1 and commercially available purified human IL-1 are also capable of stimulating cartilage to synthesize and secrete CAP. The synthesis of other proteins, including collagenase, appeared to be unaffected by either the synovial factors or the human and mouse IL-1.

Degradative enzyme systems in cartilage.

There appears to be a final common pathway in the pathogenesis of osteoarthritis, regardless of the initiating cause. This involves an increase in degradative enzymes that arise from the cartilage. Both proteoglycan- and collagen-degrading enzymes, active at a neutral pH, increase in proportion to the severity of the arthritis until a final end-stage state is reached. This increase in enzyme activity may be triggered by release of a synovial messenger protein similar to interleukin-1. It is suggested by studies in an animal model that inhibition of these enzymes could lead to treatment of osteoarthritis.