Macrophages Modulate Osteoclast Activity through Plasmin Regulation

摘要 拔牙后适当的骨愈合是恢复功能的关键。并发症可能会导致感染， 延长了疼痛时间，减少了患者的治疗选择。在愈合过程中，巨噬细胞被招募到这个部位 炎症，在损伤后24小时达到高峰，并持续到第7天。此外，巨噬细胞 已证明存在支持骨形成和泡细胞吞噬(吞噬凋亡细胞)是 与骨愈合有关。破骨细胞吸收是骨质疏松症早期的另一个基本步骤 伤口愈合。接受抗吸收药物治疗的患者有患颌骨坏死(ONJ)的风险，而 随着拔牙，发生ONJ的可能性显著增加。按项目划分的初步数据 研究人员发现纤溶酶原激活物抑制物2(蛋白质：PAI2，基因： SerpinB2)在巨噬细胞暴露于凋亡的骨细胞时，而在暴露于非 骨细胞。PAI2是丝氨酸超家族的成员，它抑制纤溶酶原激活物，导致纤溶酶原激活物减少 纤溶酶浓度。纤溶酶在纤溶系统中具有确定的蛋白水解酶的作用。 纤溶酶还裂解细胞外基质中的底物，并与类骨层的降解有关 覆盖在骨表面，因此增加了对破骨细胞吸收的敏感性。这个项目的总体目标是 该项目旨在研究口腔创伤愈合过程中纤溶系统和骨免疫学的整合。它是 假设吞噬巨噬细胞产生抑制纤溶酶原激活的PAI2 导致骨吸收减少，延迟伤口愈合。 提出了两个目标：1)确定巨噬细胞产生PAI2的条件 2)确定PAI2缺乏对破骨细胞活性的影响以及 伤口愈合。为了实现这些目标，首先是一个允许巨噬细胞的体外共培养系统 将使用吞噬凋亡的骨髓细胞。这将是衡量增长的一个有价值的工具 PAI2基因和蛋白在受控环境中的变化。此外，我们将描述PAI2是否是 细胞内的或分泌的。下一步，我们将使用一个 小鼠基因敲除模型。我们预计，由于纤溶酶在破骨细胞中的作用，可能会对破骨细胞的活性产生影响 引发骨吸收。因此，我们将重点评估这些样本的破骨细胞指标 活动。了解骨恢复的微环境机制拓宽了我们对基础知识的认识 骨生物学和病理学，为未来的治疗干预提供早期靶点。完成后， 这项建议中描述的实验将是确定PAI2在骨生物学和骨生物学中的作用的新方法 动态平衡，为骨骼微环境和信号通路提供了新的机械性见解。在……里面 此外，这些研究将是建立科学经验的基础，这些经验将启动成功的 项目首席调查员的科学生涯。

Abstract Proper bone healing following tooth extraction is critical to restore function. Complications can lead to infection, prolonged pain, and fewer treatment options for patients. During healing, macrophages are recruited to the site of inflammation, which peaks 24 hours after injury and persists through day 7. Additionally, macrophage presence has been shown to support bone formation and efferocytosis (phagocytosis of apoptotic cells) is associated with bone healing. Osteoclastic resorption is another essential step in early stages of osseous wound healing. Patients treated with anti-resorptives are at risk for osteonecrosis of the jaw (ONJ), and the possibility of developing ONJ dramatically increases with tooth extraction. Preliminary data by the project investigator showed a significant up-regulation of plasminogen activator inhibitor 2 (protein: PAI2, gene: SerpinB2) in macrophages when they were exposed to apoptotic bone cells, and not when exposed to non- bone cells. PAI2, a member of the serpin superfamily, inhibits plasminogen activators resulting in reduced plasmin concentrations. Plasmin has an established role as a proteolytic enzyme in the fibrinolytic system. Plasmin also cleaves substrates in the extracellular matrix and is implicated in degradation of the osteoid layer covering the bone surface, thus increasing susceptibility to osteoclastic resorption. The overall goal of this project is to study the integration of the fibrinolytic system and osteoimmunology in oral wound healing. It is hypothesized that efferocytosing macrophages produce PAI2, which inhibits plasminogen activation resulting in decreased bone resorption and delayed wound healing. Two aims are proposed: 1) to identify the conditions of PAI2 production by macrophages in response to apoptotic osteoblast engulfment, and 2) to determine the effect of PAI2 deficiency on osteoclastic activity and wound healing. To accomplish these aims, first an in vitro co-culture system that allows for macrophage engulfment of apoptotic bone marrow cells will be used. This will be a valuable tool to measure increases in PAI2 gene and protein changes in a controlled environment. Furthermore, we will delineate whether PAI2 is intracellular or secreted. Next, we will determine the dependence of oral socket bone healing on PAI2 using a murine knock out model. We anticipate there to be an effect on osteoclast activity due to the role of plasmin in initiating bone resorption. Therefore, we will focus evaluation of these samples on measures of osteoclastic activity. Understanding bone microenvironmental mechanisms for recovery broadens our knowledge of basic bone biology and pathology, providing early targets for future therapeutic interventions. When completed, the experiments described in this proposal will be novel in establishing the role of PAI2 in bone biology and homeostasis, providing new mechanistic insights into the bone microenvironment and signaling pathways. In addition, these studies will be foundational in establishing scientific experiences that will launch a successful scientific career for the project principal investigator.