Doxycycline is a commonly used broad-spectrum antibiotic. Recently, it has been shown that it also inhibits the activity of mammalian collagenases and gelatinases, an activity unrelated to its antimicrobial efficacy. In this study, we show that doxycycline not only inhibits MMP-8 and MMP-9 (gelatinase B) activity, but also the synthesis of MMPs in human endothelial cells. Doxycycline (50 microM) completely inhibited the phorbol-12-myristate-13-acetate (PMA)-mediated induction of MMP-8 and MMP-9, as measured by Western blotting and gelatin zymography, respectively. The inhibition was also observed at the mRNA level. No effect was observed on the expression of MMP-2 and of the MMP inhibitors TIMP-1 and TIMP-2. Chemically modified tetracyclines (CMTs) showed an inhibition similar to that of doxycycline, albeit less efficient. These observations demonstrate that endothelial cells display a specific regulation of MMPs, which may have implications for the pharmaceutical interaction in angiogenesis and angiogenesis-related diseases.

Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease. * Corresponding author. cultured on modified 5% sheep blood agar (Trypticase soy agar base supplemented with 0.5% yeast extract, 5 ,ug of hemin per ml, and 5 ,ug of menadione per ml) (56) grown at 37°C in an anaerobic chamber (Forma Scientific, Marietta, Ohio) for 48 h. Estimates of cell numbers were made by measuring turbidity with a spectrophotometer at 480 nm and comparing with known standards obtained by counting P. gingivalis cell dilutions in a Petroff-Hauser chamber. P. gingivalis 2561, used for purified antigen preparation, was grown according to procedures previously described (45, 46). Animals. Male germfree Sprague-Dawley rats (Taconic Farms, Germantown, N.Y.) were kept under gnotobiotic conditions in plastic cages with stainless steel tops in positive-pressure plastic inflatable film isolators (Standard Safety Equipment Co., Palatine, Ill.). Each group of eight animals was kept in a separate isolator. Food pellets (diet L-485; Teklad, Madison, Wis.) and water were available ad libitum. Cage bedding consisted of 1/4-in. (1 in. = 2.54 cm) granules (Bed o' Cobs; Anderson, Inc., Maumee, Ohio) and was changed twice a week to minimize trauma to periodontal tissues from impaction of hair and bedding. All equipment, food, bedding, and water were sterilized by autoclaving. Materials entering the isolator were sprayed with 2% peracetic acid. Fecal and surface swab samples from the isolators were cultured once a week by using both aerobic and anaerobic incubation conditions. Experimental design. Six groups of eight rats each were used. Group A was sham-immunized, uninfected animals used as negative controls, and group B was P. gingivalis sham-immunized, infected animals used as a positive control for infection. The other four groups (C to F) were immunized with selected P. gingivalis antigens and subsequently monoinfected with P. gingivalis 381. Group C was immunized with heat-killed whole cells; group D was immunized with highly purified 43-kDa fimbrial protein from P. gingivalis; group E was immunized with highly purified 75-kDa cell surface component from P. gingivalis; and group F was immunized with a cell surface protein fraction which included the 43-kDa protein, the 75-kDa protein, and other proteins extracted during the purification process.

The concentrations of doxycycline and 4-de-dimethylaminotetracycline required to inhibit 50% of collagenase activity were found to be 15 to 30 ,uM for human neutrophil and gingival crevicular fluid collagenases. Fibroblast collagenase was relatively resistant to inhibition by tetracyclines; the 50% inhibitory concentrations of doxycycline and 4-de-dimethylaminotetracycline were 280 and 510 ,uM, respectively.Interstitial collagenases (EC 3.4.24.7) are considered to be key initiators of collagen degradation during the progression of inflammatory diseases such as rheumatoid arthritis, corneal ulceration, and periodontal diseases. Elevated tissue levels of collagenase have been detected in these inflammatory diseases characterized by excessive collagen degradation. Both the amount of the enzyme and its conversion to an active form, possibly mediated by the action of proteinases and/or reactive oxygen metabolites, are increased during inflammation. Although the cellular origin(s) of collagenases in these diseases remains unclear, resident fibroblasts and epithelial cells as well as infiltrating leukocytes (neutrophils and macrophages) are considered potential sources of the enzymes (1,4,14,(23)(24)(25). Fibroblast-type interstitial collagenase (matrix metalloproteinase 1 or MMP-1), which is also produced by epithelial cells and monocyte/macrophages, and neutrophil interstitial collagenase (MMP-8) are distinct gene products and differ in their immunologic characteristics and substrate specificities (7,12,24). In addition, the physiological inhibitors a2-macroglobulin and tissue inhibitor of metalloproteinases have been found to inhibit the fibroblast collagenase more efficiently than the neutrophil collagenase (3, 27).Recently, Golub et al. (9) discovered a new, nonantimicrobial property of tetracyclines-an ability to inhibit the activity of interstitial collagenases from a variety of cellular and tissue sources. This effect has been confirmed by other investigators (4, 15), Moreover, a chemical modification of the tetracycline molecule that eliminates its antimicrobial efficiency does not result in a loss of its ability to inhibit collagenase (10). The specificity of the effect was partially addressed in a study showing that the tumor cell-derived type IV collagenase/gelatinase (MMP-2) can also be inhibited by tetracyclines (29). However, the ability of these drugs to inhibit different types of interstitial collagenases ' has not yet been investigated. We report here the differential susceptibility of human neutrophil and fibroblast interstitial collagenases to inhibition by a commercial antimicrobial tetracycline, doxycycline (DOXY) and by a chemically modified nonantimicrobial tetracycline (4-de-dimethylaminotetracycline or CMT-1) (10). Furthermore, we addressed * Corresponding author. the cellular source of collagenase in the inflammatory exudate of the human periodontal pocket (also called the gingival crevicular fluid) by using tetracycline inhibition as a probe.4-Aminophenylmercuric acetate and phenylmethylsul...

Tetracyclines have recently been shown to inhibit the activity of some but not all mammalian matrix metalloproteinases believed to mediate periodontal destruction. However, the specificity of this effect, which could have significant therapeutic implications for different periodontal diseases, has not been examined in detail. Doxycycline and 4de‐dimethylaminotetracycline (CMT‐1) have been tested in vitro for their ability to inhibit human neutrophil and fibroblast interstitial collagenases and collagenase in human gingival crevicular fluid (GCF). The GCF samples were obtained from systemically healthy and insulin‐dependent diabetic adult Periodontitis patients and from localized juvenile Periodontitis (UP) patients. The concentrations of these 2 tetracyclines required to inhibit 50% of the collagenase activity (IC50) were found to be 15 to 30 μM for human neutrophil collagenase and for collagenase in GCF of systemically healthy and diabetic adult Periodontitis patients. These concentrations approximate the tetracycline levels observed in vivo during treatment with these drugs. In contrast, human fibroblast collagenase and GCF collagenase from UP patients were both relatively resistant to tetracycline inhibition; the IC50 for doxycycline and CMT‐1 for these 2 sources of collagenase were 280 and 500 μM, respectively. Based on these and other findings, we propose the following: 1) that systemic levels of tetracycline may inhibit connective tissue breakdown by inhibiting neutrophil collagenase; 2) that tetracyclines do not inhibit fibroblast‐type collagenase, which may help explain their lack of effect on normal connective tissue remodeling; 3) that tetracycline inhibition of collagenases may serve to identify the cellular origin of the enzyme; and 4) that tetracyclines can also prevent the oxidative activation of latent human procollagenases. With regards to therapy, the anti‐collagenase property of tetracyclines may be an effective adjunct in targeting tissue breakdown in systemically healthy and diabetic adult Periodontitis patients. However, in juvenile Periodontitis the anticollagenase property of tetracyclines may be less important than the antimicrobial activity of the drug because of the relative resistance of fibroblast‐type collagenase to tetracycline inhibition. J Periodontol 1993; 64:82–88.