Objective: To explore the involvement of protease‐activated receptor 1 (PAR‐1) and PAR‐2 in the pathologic processes of osteoarthritis (OA) and to identify the cells/tissues primarily affected by ablation of PAR‐1 or PAR‐2 in mice.Methods: OA was induced in the joints of wild‐type (WT), PAR‐1^+/+, PAR‐1^−/−, and PAR‐2^−/− mice by destabilization of the medial meniscus (DMM), and scores of histologic features (cartilage aggrecan loss and erosion, subchondral bone sclerosis, osteophytes, and synovitis) were compared at 1, 4, and 8 weeks post‐DMM. The effects of PAR ablation on cartilage degradation and chondrocyte metalloproteinase expression/activity were studied in cultures of mouse femoral head tissue with or without interleukin‐1α (IL‐1α). At 1 week post‐DMM, synovial expression of cytokines and metalloproteinase genes was measured by reverse transcription–polymerase chain reaction, and populations of inflammatory cells were quantified by flow cytometry.Results: Deletion of PAR‐2, but not that of PAR‐1, in mice significantly delayed the progression of cartilage damage and inhibited subchondral bone sclerosis following DMM. There was no inhibitory effect of PAR‐1 or PAR‐2 ablation on IL‐1α–induced cartilage degradation or chondrocyte metalloproteinase expression/activation. A low but significant level of synovitis persisted in mice subjected to DMM compared to that in control mice subjected to sham surgery, but no differences between the genotypes were seen 4 or 8 weeks post‐DMM. One week after DMM, increased synovial expression of proinflammatory cytokines and metalloproteinase genes, along with increased levels of CD4+ T cells, inflammatory monocytes, and activated macrophages, were seen in all genotypes. However, there was a significant reduction in the percentage of activated macrophages in PAR‐2^−/− mice compared to PAR‐1^−/− and WT mice.Conclusion: Deletion of PAR‐2, but not that of PAR‐1, results in a significant decrease in DMM‐induced cartilage damage. The chondroprotection in PAR‐2^−/− mice appears to occur indirectly through modulation of extracartilaginous events such as subchondral bone remodeling and synovial macrophage activation, rather than through alteration of chondrocyte catabolic responses.

Objective: To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG‐2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene‐knockout model.Methods: Maturational changes were evaluated histologically in the knees of 3‐, 6‐, and 12‐week‐old wild‐type (WT) mice and Hspg2^{Δ3−/Δ3−} mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2^{Δ3−/Δ3−} mice. Changes in cartilage expression of FGF‐2, FGF‐18, HSPG‐2, FGF receptor 1 (FGFR‐1), and FGFR‐3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin‐1α (IL‐1α), FGF‐2, and FGF‐18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified.Results: No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2^{Δ3−/Δ3−} mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA‐induced loss of chondrocyte expression of FGF‐2, FGF‐18, and HSPG‐2 occurred in both genotypes. Expression of FGFR‐1 after OA induction was maintained in WT mice, while FGFR‐3 loss after OA induction was significantly reduced in Hspg2^{Δ3−/Δ3−} mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL‐1α–stimulated cartilage. However, IL‐1α–induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2^{Δ3−/Δ3−} mice. Cartilage GAG release in either the presence or absence of IL‐1α was unaltered by FGF‐2 in both genotypes. In cartilage cultures with FGF‐18, IL‐1α–stimulated GAG loss was significantly reduced only in Hspg2^{Δ3−/Δ3−} mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA.Conclusion: Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR‐3 and increased FGF signaling.

Objective To investigate the role of activated protein C (APC) in cartilage degradation. Methods Chondrocyte expression of protein C, endothelial protein C receptor (EPCR), and thrombomodulin (TM) were evaluated by reverse transcription–polymerase chain reaction (RT-PCR). APC was immunolocalized in developing joints and in osteoarthritic (OA) cartilage from humans. The effect of APC on aggrecan and collagen degradation was examined in explant cultures of ovine cartilage in control cultures and in cultures stimulated with interleukin-1α (IL-1α), tumor necrosis factor α (TNFα), or retinoic acid (RetA), using colorimetric assays and Western blotting. Chondrocyte expression of matrix metalloproteinases (MMPs), ADAMTS, and tissue inhibitor of metalloproteinases (TIMPs) was measured by RT-PCR. MMP-2 and MMP-9 activity was evaluated by gelatin zymography and MMP-13 by fluorogenic assay. Results Positive cellular immunostaining for APC was found at sites of MMP activity in developing joints and in OA, but not normal, cartilage. Chondrocytes expressed messenger RNA for protein C, EPCR, and TM, with the latter 2 levels increased by IL-1α and TNFα stimulation. APC augmented aggrecan release and initiated collagen breakdown in IL-1α–treated and TNFα-treated cartilage, but not in normal or in RetA-treated cartilage. APC-stimulated aggrecan and collagen breakdown were due to MMP activity but were not associated with modulation of MMP, ADAMTS, or TIMP expression. APC resulted in MMP-13 activation in cartilage cultures. APC could not directly activate proMMP-13, but it was associated with increased MMP-2 and MMP-9 activity. Conclusion APC may be a relevant activator of MMPs in cartilage and may play a role in progressive cartilage degradation in arthritis.