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关节炎(RA)的发病机制中起重要作用，是炎症细胞浸润、滑膜细胞扩张和血管疙瘩形成所必需的。了解控制新生血管过程的机制很重要，因为它将确定关键分子是特定抑制剂和受体拮抗剂药物阻断的靶点。最近，人们注意到内皮祖细胞被招募到炎症的滑膜中，参与关节炎滑膜新生血管的形成。然而，内皮祖细胞归巢的机制仍不清楚，需要解决以下重要问题：在关节炎的炎症条件下，内皮祖细胞是如何调节的？EPC归巢至炎症滑膜的细胞和分子机制是什么？内皮祖细胞是如何分化为新血管的？它们在多大程度上促进了滑膜新生血管？临床和实验性关节炎与体内血浆激肽释放酶-激动素系统(KKS)的激活有关。缓激肽是KKS的活化产物，通过其受体B1R和B2R介导关节炎的炎症反应。申请人最近发现，内皮祖细胞表达B2R，缓激肽刺激内皮祖细胞跨内皮细胞迁移。在这个R03提案中，中心假设是通过释放缓激肽激活KKS，刺激关节炎中内皮祖细胞的滑膜募集。这一假设是创新的，因为在滑膜新生血管中，作为炎症的主要介质的KKS的作用尚不清楚。在拟议的研究中，将使用动物模型、细胞生物学、药理学、生化和分子方法。这一假设将通过追求以下两个特定目标来验证：特定目标1将确定KKS的激活是否刺激EPC归巢到炎症的滑膜并参与关节炎滑膜新生血管的形成。申请人已成功地从Lewis大鼠中分离出内皮祖细胞，并检测到植入的内皮祖细胞的滑膜募集。通过血浆激肽释放酶抑制物、抗激肽原抗体和激肽原缺乏动物模型研究KKS的激活与内皮祖细胞介导的新血管形成的关系。具体目标2将确定缓激肽受体B1R和B2R在内皮祖细胞滑膜募集中的作用。缓激肽对内皮祖细胞滑膜募集的影响将在大鼠膝关节灌流模型中进行评估。申请者还将测试激动素受体上调是否增强缓激肽刺激的内皮祖细胞归巢。每种激动素受体在内皮祖细胞滑膜募集中的作用将通过使用特定的激动素受体拮抗剂或基因沉默来确定。这些研究不仅将启动对关节炎EPC归巢和分化的细胞和分子机制的进一步研究，而且还将加深对KKS在滑膜新生血管中的病理生理学作用的理解。 公共卫生相关性： 滑膜新生血管在类风湿性关节炎的发病机制中起关键作用。这项拟议的研究检测了血浆激肽释放酶-激动素系统对内皮祖细胞的调节，并确定了内皮祖细胞在滑膜新生血管中的作用。这个项目将为滑膜新生血管的机制提供新的见解，并将确定治疗关节炎的潜在靶点。

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.