The kallikrein-kinin system in endothelial progenitor cell homing in arthritis

描述（由申请人提供）： 滑膜新生血管在类风湿性关节炎（RA）的发病机制中起着关键作用，并且是炎性细胞浸润、滑膜细胞扩张和血管翳形成所必需的。了解控制新生血管过程的机制是重要的，因为它将确定关键分子作为特异性抑制剂和受体拮抗剂的药理学阻断的靶点。最近，人们注意到内皮祖细胞（EPCs）被募集到发炎的滑膜并参与关节炎滑膜新生血管形成。然而，EPC归巢的机制尚不清楚，以下重要问题需要解决：在关节炎的炎症条件下，EPCs是如何调节的？EPC归巢到炎症滑膜的细胞和分子机制是什么？内皮祖细胞如何分化为新血管？它们在多大程度上促进滑膜新生血管形成？临床和实验性关节炎与血浆激肽释放酶-激肽系统（KKS）的体内活化相关。缓激肽是KKS的活化产物，通过其受体B1 R和B2 R介导关节炎的炎症反应。申请人最近发现EPCs表达B2 R，缓激肽刺激EPCs的跨内皮迁移。在R 03的建议中，中心假设是通过释放缓激肽激活KKS，刺激关节炎中EPCs的滑膜募集。这一假设是创新的，因为在滑膜新血管形成中，作为炎症主要介质的KKS的作用尚不清楚。在拟定的研究中，将使用动物模型、细胞生物学、药理学、生物化学和分子方法。该假设将通过以下两个具体目标进行测试：具体目标1将确定KKS的激活是否刺激EPC归巢到发炎的滑膜和参与关节炎中的滑膜新血管形成。申请人已成功地从刘易斯大鼠中分离出EPC并检测了植入EPC的滑膜募集。将通过使用血浆激肽释放酶抑制剂、抗激肽原抗体和激肽原缺陷动物模型来检查KKS活化与EPC介导的新血管形成的关联。具体目标2将定义缓激肽受体B1 R和B2 R在内皮祖细胞滑膜募集中的作用。将在膝关节灌注的大鼠模型中评价缓激肽对EPC的滑膜募集的影响。申请人还将测试激肽受体的上调是否增强缓激肽刺激的EPC归巢。将通过使用特异性拮抗剂或激肽受体的基因沉默来确定每种激肽受体在EPC的滑膜募集中的作用。这些拟议的调查不仅将启动进一步研究的细胞和分子机制的EPC归巢和分化的关节炎，但也将提高深入了解的病理生理作用的KKS在滑膜新生血管。 公共卫生相关性： 滑膜新生血管是类风湿关节炎发病的关键。本研究旨在探讨血浆激肽释放酶-激肽系统对内皮祖细胞（EPCs）的调节作用，并确定EPCs对滑膜新生血管形成的作用。该项目将为滑膜新生血管形成机制提供新的见解，并将确定治疗关节炎的潜在治疗靶点。

DESCRIPTION (provided by applicant): Project Summary/Abstract Synovial neovascularization plays a critical role in the pathogenesis of rheumatoid arthritis (RA) and is required for inflammatory cell infiltration, expansion of synovial cells and pannus formation. Understanding the mechanisms that control neovascular process is important because it will identify key molecules as targets for pharmacologic blockade by specific inhibitors and receptor antagonists. Recently, it has been noted that endothelial progenitor cells (EPCs) are recruited to inflamed synovium and participate in synovial neovascularization in arthritis. The mechanism for EPC homing, however, remainsunknown and the following important issues need to be addressed: How are EPCs regulated under the inflammatory conditions in arthritis ? What are the cellular and molecular mechanisms for EPC homing to inflamed synovium ? How do EPCs differentiate into new blood vessels and to what extent do they contribute to synovial neovascularization ? Clinical and experimental arthritis is associated with in vivo activation of the plasma kallikrein-kinin system (KKS). Bradykinin, the activation product of the KKS, mediates inflammatory response in arthritis through its receptors B1R and B2R. The applicant has recently found that EPCs express B2R, and bradykinin stimulates transendothelial migration of EPCs. In this R03 proposal, the central hypothesis is that activation of the KKS, via the release of bradykinin, stimulates synovial recruitment of EPCs in arthritis. This hypothesis is innovative because in the synovial neovascularization, the role of the KKS, which is a primary mediator of inflammation, is unclear. In the proposed studies, animal models, cell biological, pharmacological, biochemical and molecular approaches will be used. The hypothesis will be tested through the pursuit of the following two specific aims: Specific Aim 1 will determine whether activation of the KKS stimulates EPC homing to inflamed synovium and participation in synovial neovascularization in arthritis. The applicant has succeeded in isolating EPCs from Lewis rats and detecting synovial recruitment of implanted EPCs. Association of the KKS activation with EPCs-mediated new vessel formation will be examined by using plasma kallikrein inhibitor, anti-kininogen antibody and kininogen deficient animal model. Specific Aim 2 will define the role of bradykinin receptors, B1R and B2R, in synovial recruitment of EPCs. The effect of bradykinin on synovial recruitment of EPCs will be evaluated in a rat model of knee joint perfusion. The applicant will also test whether upregulation of kinin receptors potentiates bradykinin-stimulated homing of EPCs. The role of each kinin receptor in synovial recruitment of EPCs will be determined by using specific antagonists or gene silencing of kinin receptors. These proposed investigations will not only initiate further studies of the cellular and molecular mechanisms for EPC homing and differentiation in arthritis, but will also improve in depth understanding of the pathophysiological role of the KKS in synovial neovascularization. PUBLIC HEALTH RELEVANCE: Project Narrative Synovial neovascularization is critical for the pathogenesis of rheumatoid arthritis. The proposed research examines the regulation of endothelial progenitor cells (EPCs) by plasma kallikrein-kinin system and determines the contribution of EPCs to synovial neovascularization. This project will provide novel insight into the mechanism of synovial neovascularization and will identify potential therapeutic targets for treatment of arthritis.