Protease inhibitors decrease rabbit cartilage degradation after meniscectomy.

In vitro proteoglycan (PG) synthesis and release were measured on cartilage removed from rabbit knees within 1 week of meniscectomy. Three days following partial lateral meniscectomy, 72% of the femurs and 82% of the tibias had visible ulcers. Cartilage from the weight-bearing areas incorporated 2.0-2.9 times more 35S-sulfate in vitro than cartilage from the opposite, unoperated knees. 3H-thymidine incorporation was 2.5-3.4 times higher for surgical than control groups. 35S-sulfate incorporation by the surgical group was inhibited by 22% in the presence of 10(-4) M U24522, an inhibitor of rabbit chondrocyte metalloprotease (CMP). 3H-thymidine incorporation by the surgical group was inhibited by 28% by 10(-4) M U24522. In vitro PG release from cartilage removed 2 days after surgery was 1.6-3.7 times higher for the surgical than the control group. PG release by the surgical group after 22 h of incubation was reduced to the control level by three CMP inhibitors, U24278, U24279, and U24522. PG release by cartilage from the nonsurgical group was also reduced by these compounds at 22 h. These results suggest that both the anabolic and catabolic processes that are stimulated by surgery can be isolated in vitro and that CMP may be involved in the catabolic process.

Degradation of rat chondrosarcoma proteoglycans by a neutral metalloprotease from rabbit chondrocytes.

Rat chondrosarcoma proteoglycan aggregate with radiolabeled core protein was digested with a chondrocyte metalloprotease (CMP) or clostripain (CP) at neutral pH. The rates of product formation and the sizes and antigenicities of the products were studied using column chromatography and monoclonal antibodies. Sixteen percent of [35S]methionine label and 17-18% of [3H]serine label in core protein were freed from glycosaminoglycan bound peptides by 50 U/ml (760 micrograms/ml) of CP or 10 micrograms/ml (estimated) of CMP in 180 min. The CP reaction was almost complete at 5 minutes while the CMP reaction proceeded slowly from 5 to 180 min. The chondroitin-sulfate rich fragments were smaller after CP than CMP treatment. The 180 min CMP digest contained protein that migrated in 2 peaks on Sepharose CL6B. These two peaks corresponded to the peaks where hyaluronic acid binding region produced by CP and link protein migrate. Metalloenzyme inhibitors inhibited CMP with IC50s of 5 x 10(-5)M, 1 x 10(-3)M, and 80 micrograms/ml for phenanthroline, EDTA, and alpha 2-macroglobulin, respectively.

Proteoglycan degradation by a chondrocyte metalloprotease. Effects of synthetic protease inhibitors.

Synthetic inhibitors of a chondrocyte metalloprotease (CMP) were assessed for potency. Proteoglycan core protein was used as substrate. The IC50 values were between 2 X 10(-6) and 7 X 10(-6) M for two types of inhibitors, thiol tripeptides and N-carboxyalkyl peptides. Hydroxamic acid peptides were more potent, with IC50 values of 3.2 X 10(-8) to 6.0 X 10(-8) M. These results confirm inhibitory concentrations reported using a proteoglycan-polyacrylamide bead assay. The slopes of the dose-response curves for the thiol compounds were steeper than the slopes for the other two types of compounds. All of the culture media tested inhibited CMP to some extent. Some media also interfered with inhibitor activity. In Ham's F10 nutrient medium, minimum CMP inhibition occurred, and all four hydroxamic acid peptides retained their activity for 1-2 days at 37 degrees. One thiol peptide compound assayed lost activity in 1 hr in thiocyanate-treated serum. All four hydroxamic acid peptides assayed retained activity in thiocyanate-treated serum after 3 days at 37 degrees. The hydroxamic acid peptides may provide a way to block endogenous CMP activity in vivo and to assess the role of CMP in normal and experimentally altered cartilage. They are more potent than other known CMP inhibitors. They retain activity in culture media and serum conditions used for in vivo and in vitro tests of CMP activity and toxicity.

Effect of synthetic metalloprotease inhibitors on cartilage autolysis in vitro.

The role of chondrocyte metalloprotease (CMP) in mediating cartilage autolysis was studied. Proteoglycan (PG) release and synthesis by rabbit articular cartilage explants were measured. After a 1-day preculture in control medium, 3.3 X 10(-6) M retinoic acid (RET) treatment for 1 day stimulated PG release several fold. RET also caused a large decrease in PG synthesis that returned to the control level after a 3-day recovery period. The effect on PG synthesis was observed at serum levels of 5 and 0.05%. The effect of RET on PG release required protein synthesis, inasmuch as it was lost in cultures maintained in media without amino acids or in a low volume of media. Interleukin-1 (IL-1) and lipopolysaccharide (LPS) treatment for 2 days also stimulated PG release. More PG was released after RET than after IL-1 or LPS, and only RET produced an effect that was evident by day 1. The amount of CMP that produced the same size effect on PG release as these stimulators was below the detection level of PG protease assays. Three potent CMP inhibitors reduced RET-, IL-1- and LPS-stimulated PG release to control levels. These inhibitors did not block another action of RET on chondrocytes, namely the inhibition of PG synthesis by RET immediately after treatment. The inhibitors did not act by reducing cell viability, because recovery of the rate of PG synthesis 3 days post-treatment occurred in inhibitor-treated cultures. These studies suggest that CMP is involved in cartilage autolysis that is stimulated by RET and IL-1.