Abstract
Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). CCR2 has been recognized as an important potential target in OA: CCR2, its ligands CCL12 (a.k.a. MCP-5) and CCL2 (a.k.a. MCP-1, the major human circulating ligand), are significantly increased in both rodent models of OA and in humans with OA. In addition, they have been shown to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up. Using the destabilization of medial meniscus (DMM) murine model of injury-induced OA, we demonstrated that targeting the CCR2 signaling at early OA stages had beneficial effects on OA progression and that there was a disconnect between structure and pain that has been noted in human OA. We therefore hypothesize that alterations in joint mechanics upregulate the CCR2 pathway in cartilage and bone in a time-dependent manner altering the structural organization of both tissues, ultimately leading to OA and pain. In the present study, by using a temporal and tissue specific inactivation of CCR2 in cartilage, we determine the role of CCR2 signaling in-vivo at sequential times during injury-induced OA in order to establish the contribution of CCR2 to the development of whole joint degeneration. Methods: We obtained an inducible tissue-specific CCR2 deletion in chondrocytes by combining chondrocyte-specific expression of CreER with Tamoxifen (Tam) injections in CCR2flx/flxeGFP mice crossed with Aggrecan-CreERT2 (CCR2AggERKO). The Cre-mediated recombination deletes CCR2 exon3, generating a functional knockout, and activates the expression of eGFP under the CCR2 locus. Therefore, recombination can be validated by GFP expression. CCR2AggERKO and CCR2flx/flx mice were injected with Tamoxifen (40μg/g) for 5 days and subjected to DMM 1 week after the last injection. Mice were euthanized at 2, 4, 8 and 12 weeks after DMM and knees were prepared for histological evaluation (Safranin-O/Fast Green, H&E) and immunohistochemical (IHC) analyses with anti-GFP, anti-Collagen10 and anti-osterix antibodies). Results: We successfully inactivated the CCR2 receptor in the cartilage tissue, as shown by the intense GFP expression in the cartilage tissues of CCR2AggERKO mice compared to controls. The expression of CCR2 in other tissues is not altered by the recombination. Histological evaluation shows that deletion of CCR2 in cartilage tissue decreases articular cartilage structure score and Safranin-O staining score at 4, 8 and 12 weeks post-surgery compared to controls. In addition, at both 8 and 12 weeks post surgery, protein levels of Collagen 10 are significantly higher in DMM-CCR2AggERKO mice while almost absent in DMM-controls, were the whole cartilage is replaced by bone. Conclusions: Our results show that we were able to selectively inhibit the CCR2 expression in chondrocytes during OA progression, while its expression was unchanged in other joint tissues. We demonstrated that the CCR2 deletion in cartilage slows cartilage damage at the mild, moderate and severe OA stage compared to controls. Other studies are currently in progress in our lab to determine whether CCR2 deletion in cartilage indirectly affect the bone tissue as well. Behavioral pain analyses are also in progress to determine whether CCR2 cartilage deletion alleviate pain responses.
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