Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan.

Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.

Human cartilage lysozyme.

The lysozyme content of human cartilage was measured by incubation of lyophilized, powdered cartilage in a variety of buffers and salt solutions, and the factors controlling the binding of lysozyme within cartilage were studied. Lysozyme was extracted from hyaline cartilage by buffers of pH greater than 9.0 by solutions 1 M in monovalent cations, and by solutions 0.12-0.40 M in divalent cations. The ability of cations to extract lysozyme from cartilage agreed with their known affinities for binding to chondroitin sulfate. The total extractable lysozyme content of five samples of human costal cartilage ranged from 1.45 to 3.36 mug lysozyme per mg of cartilage; for five samples of hyaline cartilage from peripheral joints the range was 0.80-3.03 mug lysozyme per mg of cartilage. Cartilage incubated in excess exogenous lysozyme could bind 0.053 equivalents of lysozyme per equivalent of chondroitin sulfate. Fibrocartilage and synovium from knee joints yielded no detectable lysozyme, despite the fact that synovium, a tissue rich in lysosomes, contained measurable quantities of beta-glucuronidase. Lysozyme extraction from cartilage was not augmented by incubation with streptolysin S. When incubation was carried out with mild extraction techniques, lysozyme extraction from cartilage tended to parallel uronic acid release, both as a function of time and from one specimen to another. The active material as lysozyme. Lysozyme occurs in human hyaline cartilage as a counterion to polyanionic glycosaminoglycans. Carextracted from cartilage met five criteria for identification tilage lysozyme appears to be extracellular and nonlysosomal. Degradation of cartilage may contribute to the increased serum and synovial fluid lysozyme levels often present in patients with rheumatoid arthritis.

Composition of cartilage from lysozyme-deficient rabbits.

Costal and auricular cartilage obtained from mutant rabbits exhibiting lysozyme deficiency has been found to be identical to similar tissue from control animals in a variety of biochemical parameters. These data seriously question the putative role of lysozyme as a structural component of cartilage.

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen.

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.

Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase.

Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCl), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCl also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCl. HOCl (5 microM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106-01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50-400 microM) and chemically modified tetracycline, CMT-1 (100-400 microM), significantly inhibited collagenase activity 50-90% and 40-80%, respectively, in the presence of 5 microM HOCl. Concentrations of 6-25 microM doxycycline and 10-50 microM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCl2 or 50 microM ZnCl2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCl and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.

Superoxide dismutase and catalase as therapeutic agents for human diseases. A critical review.

The list of human and animal diseases for which oxygen radical scavenging therapy is being recommended continues to grow, based primarily on inferential evidence suggesting a potential role for oxygen-derived free radicals in various types of pathophysiology. Some distinct advances in pharmacologic manipulation of protein scavengers have been made which could ultimately greatly enhance the use of these reagents as drugs, as well as some innovative techniques for drug delivery (direct injection via endoscopy, iontophoresis). Unfortunately, most of the therapeutic reports in the literature, almost all of which are based on usage of standard (native) SOD and/or catalase, are still anecdotal and/or uncontrolled. A review of the human disease/treatment literature suggests that further tightening of the scientific design of such trials is still badly needed; hopefully better experimental design will be applied when products such as PEG conjugates or genetically engineered polymers are ready for testing.

Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: a direct spectrophotometric detection system based on degradation of deoxyribose.

Hydroxyl radical is produced secondarily after phagocytic cells have been stimulated to generate superoxide anion. The systems used most commonly for detection of cell-generated hydroxyl radical are often inconvenient for routine biomedical research. We have modified an assay used heretofore in cell-free systems, that is, the degradation of deoxyribose, and adapted it for use with neutrophils. The time and dose responses of the system, requirement for chelated iron, inhibition profiles with various scavengers, and correlation with superoxide production have been ascertained. The method correlated strongly with a standard but more cumbersome technique. Values for a normal population are provided. The method can readily be used to study the parameters of superoxide-hydroxyl radical conversion by cells in various disease or treatment states.

Influence of chemically modified tetracyclines on proliferation, invasion and migration properties of MDA-MB-468 human breast cancer cells.

Chemically modified tetracyclines (CMTs) are promising anti-cancer agents. In this study, we found that CMT-3 and CMT-8 showed dose-dependent cytotoxicities in MDA-MB-468 human breast cancer cells. Moreover, both CMT-3 and CMT-8 significantly inhibited in vitro cell migration and invasion at non-cytotoxic concentrations. Anti-invasion and migration potentials of the CMTs were associated with an increased expression of E-cadherin/catenins (alpha, beta and gamma-catenin) and tumor suppressor BRCA1. In addition, CMT-3 and CMT-8 abolished or reduced spontaneous and HGF/SF-induced cell invasion and migration in U-373 MG human glioblastoma cells. Our current finding is the first demonstration that CMT-3 and CMT-8 can activate the function of invasion suppressor molecules associated with the suppression of breast cancer cell invasion and migration. Thus, clinical application of CMTs may provide potential benefit for suppression of breast cancer growth, invasion and metastasis.

Excessive matrix metalloproteinase activity in diabetes: inhibition by tetracycline analogues with zinc reactivity.

Diabetes mellitus in rats is characterized by excessive activity of several matrix metalloproteinases (MMPs), notably collagenase(s) and gelatinase(s), in skin, gingiva, and other tissues. A number of tetracyclines (TCs), both antimicrobial compounds as well as chemically modified non-antimicrobial TC analogues (CMTs) are known to possess potent inhibitory activity against these enzymes. Three conventional antimicrobial TCs and six CMTs were used in this study. In vitro, doxycycline was shown to possess higher inhibitory capacity (i.e. lower IC(max)) against diabetic rat skin collagenase than either minocycline or tetracycline HCl. Addition of excess zinc partially reversed the proteinase inhibition by TCs. In vivo, using rats made diabetic with streptozotocin (STZ), oral administration of various TCs led to decreased weight loss and substantial reductions in the activity of both skin collagenase and skin gelatinase (primarily MMP-9, 92 kDa) without affecting blood glucose. Using an in vitro spectrophotometric technique, the Zn(++) reactivity of several CMTs was assessed and found to be positively related to the potency of these compounds as MMP inhibitors. One particular CMT (CMT-5, pyrazole analogue), which is neither antimicrobial nor capable of binding metal cations, did not inhibit the MMPs. TCs have potential utility in management of diabetic complications mediated by excessive activity of MMPs.

Rapid skeletal turnover and hypercalcemia associated with markedly elevated interleukin-6 levels in a young black man.

A 24-year-old black man presented with diffuse musculoskeletal pain and shotty lymphadenopathy. Laboratory studies revealed hypercalcemia and hyperphosphatemia, very high serum alkaline phosphatase activity, diffuse but intense uptake of radionuclide on a bone scan, urinary N-telopeptide excretion 30 times the upper limit of normal, and serum interleukin-6 100 times the upper limit of normal. An extensive workup for etiologies of the disorder was negative. A bone biopsy revealed intense osteoclastic resorption coupled with rapid bone formation and/or remodeling. This case appears to represent a new entity. Treatment with bisphosphonates produced symptomatic and biochemical improvement.

In vitro sensitivity of the three mammalian collagenases to tetracycline inhibition: relationship to bone and cartilage degradation.

There are at least nine tetracycline (TC) analogs (both antimicrobial and nonantimicrobial) with documented capacity to inhibit, both in vitro and in vivo, the connective tissue degrading activity of matrix metalloproteinases (MMPs). Of the three MMPs that can degrade native helical collagens, MMP-13 (initially identified as rat osteoblast and human breast cancer collagenase, and now known to also be expressed by human cartilage and bone cells) is the most sensitive to TC inhibition (IC50 values in vitro generally less than 1 microgram/mL); the TCs inhibit both the collagenolytic as well as the gelatinolytic activity of this enzyme. The IC50 for MMP-8 (neutrophil collagenase) in vitro ranges from 15 to 86 micrograms/mL depending on assay conditions and choice of TC, whereas inhibition of the fibroblast enzyme (MMP-1) generally requires levels in excess of 200 micrograms/mL (except for CMT-3). The TC compounds that are highly effective against MMP-13 in vitro are also highly inhibitory of glycosaminoglycan release from interleukin-1-stimulated cartilage explants in culture. The current data correlate well with: (i) literature values for TC inhibition of bone resorption by isolated osteoclasts; (ii) inhibition by TCs of avian tibial resorption in organ culture; and (iii) the dramatic ability of TCs to inhibit bone destruction in many rat models (rats have only MMP-8 and MMP-13, and no MMP-1). By carefully selecting a TC-based MMP inhibitor and controlling dosages, it should be possible to inhibit pathologically excessive MMP-8 and/or MMP-13 activity, especially that causing bone erosion, without affecting the constitutive levels of MMP-1 needed for tissue remodeling and normal host function; in this regard, three newly developed CMTs (especially CMT-8, and, to a lesser extent, CMT-3 and -7) appear to be most effective.

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Oxygen radicals, inflammation, and arthritis: pathophysiological considerations and implications for treatment.

A vast amount of circumstantial evidence implicates oxygen-derived free radicals, especially superoxide and hydroxyl radical (and to lesser extent, hydrogen peroxide), as mediators of inflammation and/or tissue destruction in inflammatory and arthritic disorders. The substrates for radical generation, namely properly stimulated phagocytic cells, transition metal catalysts, and (to a limited extent) ischemia, are all amply present, although there is no particular rheumatic disease in which a consistent abnormality of radical generation has been identified. These radical species can clearly degrade hyaluronic acid, modify collagen and perhaps proteoglycan structure and/or synthesis, alter and interact with immunoglobulins, activate enzymes and inactivate their inhibitors, and possibly participate in chemotaxis. In most situations, however, there is ample scavenging ability to detoxify these radicals before they hit their target, and many rheumatic disease drugs can decrease their production and/or effects. Despite the apparent sufficiency of natural scavengers and the lack of direct evidence that oxygen radicals are pathogenetically important, substantial pharmaceutical effort is still being made to develop free radical scavengers as therapeutic agents. Although individual free radicals die out quickly, rheumatologic interest in them has been sustained for nearly two decades.

Connective tissue lysozyme in health and disease.

As the lysozyme story continues to unfold, rheumatic disease is one area where the study of this fascinating protein will be most important. The special biochemical features of lysozyme--its hexosaminidase function, its ability to bring about transglycosylation, its homology to alpha-lactalbumin, and its cationic nature--suggest that the connective tissues may prove to be the key to the understanding of the function of lysozyme. As methods for its accurate measurement become standardized, better data on the activity of the enzyme in various tissues and body fluids, in both health and disease, will be forthcoming. As additional studies are done to ascertain which of the hypothetical functions attributed to lysozyme are of significance in vivo, it will be the student of the connective tissues and the diseases thereof who can be expected to profit most from an udnerstanding of the role of lysozyme in mammalian biology.

Thirty-six years in the clinic without an MMP inhibitor. What hath collagenase wrought?

Vertebrate collagenase was discovered in 1962, and within a few short years, several inhibitors had been identified. At one time or another, virtually every major drug company has had an MMP inhibitor program, but in 1999, there is only one such product on the market. With a potential market for lifelong therapy in rheumatoid arthritis, osteoarthritis, periodontal disease, osteoporosis, and cancer, this is certainly puzzling. The problem is that the chemistry appears to have outstripped the biology. In vitro, there are many inhibitors with nanomolar or picomolar efficacy, but in vivo efficacy in animal models does not always follow. There is also a conceptual problem regarding broad-spectrum vs. highly specific inhibitors. Designing human trials to demonstrate MMP inhibition and clinical efficacy is a daunting problem, especially if one seeks to distinguish anti-MMP activity from anti-inflammatory effect. Adult periodontal disease may be the best available human disease model for development of an MMPI.

Inhibition of epiphyseal cartilage collagenase by tetracyclines in low phosphate rickets in rats.

Drugs in the tetracycline family can inhibit mammalian tissue collagenase both in vitro and in vivo by a mechanism that is independent of antibiotic action. The epiphyseal cartilages of rachitic rats contain extremely high levels of collagenase (CGase), and we have used this model to study further the phenomenon of tetracycline inhibition of tissue CGase. Rickets was induced in rats by phosphate/vitamin D deficiency and parameters of gross bone morphology, bone chemistry, and serum chemistry were evaluated in both rachitic and nonrachitic animals with and without treatment with oral tetracyclines (TETs). Minocycline (or doxycycline) partially suppressed the appearance of many of the expected changes in the rachitic animals, including gross bone hardness, growth plate widening, long bone length, suppression of weight gain, and decreased bone ash content. The effects were dose dependent and were associated with marked suppression of the enhanced CGase activity. Examination of collagen breakdown products by SDS-PAGE documented that the rachitic enzyme behaved like other mammalian collagenases including in vitro inhibition with minocycline 10-20 micrograms/ml and with a nonantibiotic tetracycline. No evidence of TET osseous toxicity was noted, and, in fact, administration of TET to nonrachitic animals had a mildly favorable effect on growth and development. TET suppression of CGase can be demonstrated in a well defined model system and this form of pharmacologic enzyme inhibition can be a useful probe for delineating the role of the enzyme in connective tissue pathology.

A non-antibacterial chemically-modified tetracycline inhibits mammalian collagenase activity.

Tetracyclines (including the semi-synthetic analogues, minocycline and doxycycline) are considered useful adjuncts in periodontal therapy because they suppress Gram-negative periodontopathogens. Recently, these antibiotics were found to inhibit mammalian collagenase activity, a property which may also be of therapeutic value. It has been suggested that the anti-collagenase properties of the tetracyclines are independent of their antibiotic efficacy. To advance this hypothesis further, we chemically converted tetracycline hydrochloride to its non-antimicrobial analogue, de-dimethylaminotetracycline. This chemically-modified tetracycline (CMT), although no longer an effective antibiotic, was found to inhibit the in vitro activity of collagenase from partially purified extracts of human rheumatoid synovial tissue and rachitic rat epiphysis. In a preliminary in vivo study, pathologically-excessive collagenase in skin and gingiva was induced by rendering adult male rats diabetic, and the oral administration of CMT to these rats significantly reduced the excessive collagenase activity in both tissues. Moreover, CMT administration did not affect the severe hyperglycemia in these rats but did prevent, at least in part, the diabetes-induced loss of body weight, skin weight, and skin collagen mass; these effects suggest a lack of toxicity in this animal model. A proposed clinical advantage of CMT over conventional tetracyclines, in the treatment of diseases characterized by excessive collagenolytic activity, is the lack of development of antibiotic-resistant micro-organisms during prolonged use. However, the consideration of clinical trials to support this hypothesis must await further laboratory and extensive toxicity tests.

Effect of agarose variability on the measurement of lysozyme activity.

Lysozyme assays are often performed by a diffusion technique utilizing agarose gels impregnated with substrate organisms (lysoplates), but the results differ greatly from those obtained with spectrophotometric or immunologic techniques. We have investigated the effect of agarose composition on the lysoplate assay utilizing 10 different gels varying in ionic parameters. Standard curves generated with purified human lysozyme solutions were parallel, but the diameters of the zones of lysis varied inversely with gel sulfate content. The different agaroses had variable effects on determinations of normal serum lysozyme, and the results obtained on any given gel agreed with neither those found on other gels nor with independent assay in another system. The lysoplate assay should be utilized only in those laboratories that can obtain uniform agarose preparations and extensively calibrate normal ranges for their gels.

Inhibition of human proteases: from target identification to therapy. 23-24 April 1998, Boston, MA, USA.

This two-day meeting, sponsored by the Cambridge Healthtech Institute, was attended by around 70 scientists, primarily from the pharmaceutical industry, with a small number from academia. Most of the industry representatives were from smaller companies engaged in drug development; virtually no multinational companies were represented. Four main topics were covered: target identification and pathophysiology; structural analysis and drug design; screening for proteinase inhibitors; and, clinical development. The target identification session was by far the strongest. Only two posters were shown and podium presentations predominated. Several speakers dealt in depth with the chemistry of their company's line of potential inhibitors; a detailed description of the numerous possible chemical modifications is beyond the scope of this review.

Degradation of hyaluronic acid by polymorphonuclear leukocytes.

Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate.