Doxycycline Inhibition of Cartilage Matrix Degradation^a

Abstract

Tetracyclines decreased collagenase activity in several animal as well as in the synovial tissues and fluid of seven patients with rheumatoid arthritis who had received minocycline prior to joint replacement. The effects of doxycycline were tested in an in vitro system in which the cartilage of embryonic avian tibias was degraded during culture in the absence of serum6,'; during culture, cartilage matrix loss occurs in two phases with proteoglycan loss preceding collagen loss. The phase of collagen loss is accompanied by the release of active collagenase and gelatinase into the conditioned media.To test the potential of doxycycline to inhibit degradation of cartilage during culture, tibias from 12-day chicken embryos were cultured for 30 days with 5, 20, or 40 kg/mL of doxycycline (Sigma). Control tibias were cultured under identical conditions but without doxycycline. Following the 30-day culture, conditioned media were examined to detect matrix metalloproteinase activity, collagenase, and gelatinase using gelatin zymograms.* The conditioned media were also analyzed for hydroxyproline content9 and sulfated glycosaminoglycan and lactate dehydrogenase activity (Sigma Diagnostic Kit-LD No. 34O-UV).l1 The tibias were processed for histologic observation after being fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with safranin 0 and fast green to observe proteoglycan loss.'*In the presence of all three concentrations of doxycycline, cartilage was not resorbed, whereas control cartilage was completely degraded as previously described.6 In the gelatin zymograms, collagenase activity was identified by two bands at an M , of 57,000 and 50,000 on culture days 18-30 as previously described'; collagenase activity in the conditioned media of the doxycycline-treated tibias was not detectable on gelatin zymograms. Gelatinase is constitutively produced by the cultures and appears on the zymograms as 66,000 and 62,000 cleared bands. Gelatinase activity was present throughout the culture in the presence of 5 kg/mL of doxycycline. At both 20 and 40 pg/mL of doxycycline, gelatinase was undetectable by culture day 18. Collagen degradation, evaluated as hydroxyproline content in the conditioned media, was reduced at all doses of doxycycline, whereas proteoglycan degradation was inhibited in a dose-dependent manner. Lactate dehydrogenase in the conditioned media of the control cultures showed peak activity (18,000 units/ tibia) on culture day 10 and returned to baseline (400 unitdtibia) by culture day 14. Doxycycline reduced peak lactate dehydrogenase activity in a dose-dependent fashion. At higher concentrations of doxycycline the cartilage length was increased

A novel mechanism of action of tetracyclines: Effects on nitric oxide synthases

“…The concentrations of doxycycline that inhibited NO production in our studies are comparable to those required for the inhibition of matrix metalloproteases (19,(30)(31)(32)(33). In those studies, doxycycline at 20-50 g͞ml inhibited the activity of proteolytic enzymes such as collagenase and gelatinase, blocked proteoglycan degradation, reduced the cell death associated with proteoglycan loss, and augmented cartilage growth (31). Effects of Doxycycline and Minocycline on iNOS in Murine Macrophages.…”

“…Therefore, we first examined whether doxycycline or minocycline could block human OA-NOS activity in ex vivo conditions. Generally accepted pharmacologically relevant concentrations were selected for this study based on previous reports (19,(30)(31)(32). OA-affected cartilage was obtained from patients with advanced OA undergoing knee replacement surgery.…”