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. 2009 Jan 5;68(6):1051–1058. doi: 10.1136/ard.2007.086884

Figure 3.

Figure 3

Inhibition of aggrecanase activity by treatment of osteoarthritic chondrocytes and cartilage tissue with hyaluron (HA)2700. (A) Inhibition of interleukin (IL)1α-stimulated aggrecanase activity in chondrocytes treated with HA2700. Osteoarthritic chondrocytes were stimulated with IL1α (1 ng/ml) and treated with HA2700 (2.5 mg/ml), KB-RR7785 (1 μM), tissue inhibitor of metalloproteinases 3 (TIMP3) (100 nM), HAoligo (250 μg/ml) or buffer alone (None) in the presence of porcine aggrecan (100 μg/ml). Culture media were subjected to immunoblotting using aggrecan neoepitope-specific antibody (2 μg/ml). (B) Inhibition of IL1α-stimulated expression of ADAMTS4 and aggrecanase activity with small interfering RNA (siRNA) for ADAMTS4. Chondrocytes were treated with siRNA for ADAMTS4 (TS4) or non-silencing siRNA (NS), and expression of ADAMTS4 mRNA and protein was determined by RT-PCR and immunoblotting (IB) with ADAMTS4 antibody (0.5 μg/ml; 250-4F7). Inhibition of aggrecanase activity was evaluated by immunoblotting with aggrecan neoepitope antibody (2 μg/ml). (C) Inhibition of IL1α-stimulated aggrecanase activity in osteoarthritic cartilage tissue treated with HA2700. Cartilage tissue slices were treated for 5 days with buffer alone (None), HA2700 (2.5 mg/ml), KB-R7785 (1 μM), non-silencing siRNA (NS) or siRNA for ADAMTS4 (TS4) in the presence of IL1α (1 ng/ml), and then aggrecanase activity was detected by immunoblotting with aggrecan neoepitope antibody. Cont, cartilage tissue without IL1α treatment; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.