Role of CCR2 in osteoarthritis

项目摘要 尽管有证据表明，趋化因子在骨性关节炎关节中增加，但很少有研究研究它们在骨性关节炎和骨关节炎中的作用。 他们推动联合毁灭的机制。这项提案的目标是确定CC- 趋化因子受体2(CCR2)是骨性关节炎关节损伤和疼痛的重要介质。我们的初步研究和 其他人最近的工作表明，抑制CCR2可能会减少骨性关节炎的结构损伤和疼痛。如果这是真的， 这将是办公自动化管理方面的重大突破。然而，还需要做更多的工作来证实这些 并确定在OA过程中抑制CCR2的最佳时间。我们的数据来自一个社区- 基于人类骨性关节炎的队列研究表明，较高的基线血清CCL2水平(与CCR2结合) 受体)与5年随访的放射性骨性关节炎的进展有关。使用不稳定的中间体 半月板(DMM)小鼠创伤性骨性关节炎模型，我们发现CCL12(功能性的 HCCL2)的同源物，这是一种我们之前发现在联合发育中至关重要的CCR2配体，在 早期骨性关节炎的关节软骨和骨。关节软骨的骨关节炎改变(软骨丢失，MMP13增加 水平)和骨骼(骨硬化、骨赘)和疼痛行为通过全身阻断 CCR2，如果在DMM后前8周内给药，共4周。然而，如果治疗持续8周或 更重要的是，在抑制疼痛的同时，它对随后的结构进展不那么有效，这表明它与疼痛脱节 结构和疼痛之间的关系已经在人类骨性关节炎中被注意到。这些数据表明，CCR2信号具有 细胞和阶段在骨性关节炎中的特定作用，在早期阶段具有有益的影响。我们假设 损伤引起的关节力学改变在一段时间内上调软骨和骨骼中的CCR2通路- 依赖的方式改变了两个组织的结构组织，最终导致骨性关节炎和疼痛。我们 建议在软骨损伤的连续时间确定失调的CCR2信号的作用-和 年龄诱发的骨性关节炎，以确定该组织在整个关节退变和骨性关节炎疼痛中的作用 (目标1)。我们还将评估成骨细胞表达CCR2是否会导致关节损伤和疼痛 在损伤诱导的骨性关节炎期间(目标2)。为了实现这一点，我们将获得可诱导的组织特异性CCR2缺失 在软骨细胞(AIM 1)和成骨细胞(AIM 2)中结合软骨细胞或成骨细胞特异性表达的 CCR2 FLOX/FLOX小鼠注射的CRER分别与Aggrecan-CRERT2或COL1α1CreER杂交。 因为由巨噬细胞和破骨细胞组成的髓样浸润可能会导致加速的骨性关节炎 重塑可能会反映骨损伤，间接地反映软骨的动态平衡，我们也会有条件地 通过将CCR2 FLOX/FLOX小鼠与LysMERCre杂交来灭活巨噬细胞/破骨细胞中的CCR2(目标3)。我们会 还要评估每个组织选择性CCR2缺失改变的潜在途径。此外，通过使用体外培养 在原代培养的人软骨细胞中，我们将研究CCR2与其共同激活的信号通路。 多种配体刺激基质金属蛋白酶的产生。

PROJECT ABSTRACT Despite evidence that chemokines are increased in OA joints, few studies have examined their role in OA and the mechanism by which they promote joint destruction. The goal of this proposal is to determine if the CC- chemokine receptor 2 (CCR2) is a critical mediator of joint damage and pain in OA. Our preliminary studies and recent work by others suggest CCR2 inhibition may reduce both structural damage and pain in OA. If this is true, it would be a major breakthrough in the management of OA. However, more work is needed to confirm these findings and determine the optimal time to inhibit CCR2 during the OA process. Our data from a community- based cohort study of human OA shows that higher baseline serum levels of human CCL2 (which binds CCR2 receptor) correlates with progression of radiographic OA at 5-years follow up. Using the destabilized medial meniscus (DMM) murine model of injury-induced OA, we found that expression of CCL12 (the functional homologue of hCCL2), a CCR2 ligand we have previously found to be critical in joint development, increases in articular cartilage and bone during early OA. OA changes in articular cartilage (cartilage loss, increased MMP13 levels) and bone (bone sclerosis, osteophytes) and pain behavior were ameliorated by systemic blockade of the CCR2, if given for 4wks total within the first 8wks after DMM. However, if treatment was maintained for 8wks or more it was less effective on subsequent structural progression while still inhibiting pain, suggesting a disconnect between structure and pain that has been noted in human OA. These data suggest that CCR2 signaling has cellular and stage specific roles in OA, with beneficial effects when targeted at early stages. We hypothesize that injury-induced 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. We propose to determine the role of dysregulated CCR2 signaling in cartilage at sequential times during injury- and age-induced OA, in order to establish the contribution of this tissue to the whole joint degeneration and OA pain (Aim 1). We will also evaluate whether osteoblast expression of CCR2 contributes to joint damage and pain during injury-induced OA (Aim 2). To accomplish this, we will obtain an inducible tissue-specific CCR2 deletion in chondrocytes (Aim 1) and osteoblasts (Aim 2) by combining chondrocyte- or osteoblast-specific expression of CreER with Tam injections in CCR2flox/flox mice crossed with Aggrecan-CreERT2 or Col1α1CreER, respectively. Because the myeloid infiltration consisting of macrophages and osteoclasts may cause accelerated OA bone remodeling that may reflect on bone damage and, indirectly, on cartilage homeostasis, we will also conditionally inactivate CCR2 in macropahges/osteoclasts (Aim 3) by crossing CCR2flox/flox mice with LysMERCre. We will also evaluate potential pathways altered by each tissue-selective CCR2 deletions. In addition, by using in-vitro human primary chondrocyte cultures, we will study the signaling pathways by which activation of CCR2 with its multiple ligands stimulates MMP production.