Macrophage Pericellular Proteinase Cascade

描述(申请人提供)：基质降解蛋白水解酶的合成和/或激活在单核细胞和巨噬细胞在组织中的迁移中起着至关重要的作用。其中最重要的是尿激酶型纤溶酶原激活物(UPA)-纤溶酶原系统。纤溶酶结合并降解纤维蛋白和细胞外基质(ECM)的几种成分。此外，纤溶酶激活基质金属蛋白酶家族中的几个成员，这些成员负责降解胶原和细胞外基质的其他成分。尽管早期观察到与细胞表面的结合赋予纤溶酶原激活和保护纤溶酶免受抑制的动力学优势，但膜结合的纤溶酶的生物学在很大程度上仍未被探索，它作为慢性炎症性疾病的治疗靶点的作用仍未得到检验。这项建议中描述的实验的总体目的是验证一种假设，即巨噬细胞表面的纤溶酶结合部位是其细胞周蛋白酶级联的主要调节因子，而阻断其表面的纤溶酶(原)结合是减少巨噬细胞聚集及其在慢性炎症性疾病中的后遗症的有效策略。我们提出了两个互补的策略：(1)利用定义的纤溶酶原结合位点(Annexin II)缺陷的小鼠，我们将直接确定纤溶酶结合位点在巨噬细胞激活基质金属蛋白酶-9、降解细胞外基质和依赖单核细胞趋化蛋白-L的体外细胞外基质迁移中的作用(特定目标1)。此外，我们还将利用硫代乙酸酯诱导的腹膜炎和异物诱导的肉芽肿模型，以及Apo E-/-IAnx II-/-小鼠的巨噬细胞募集对动脉粥样硬化病变的募集(特定目标2)，来确定膜联蛋白II缺乏对巨噬细胞募集的影响。(2)我们将确定一段失活的纤溶酶原细胞结合片段在体外阻断巨噬细胞对基质金属蛋白酶-9的激活、细胞外基质的降解和单核细胞趋化蛋白-1依赖的细胞外基质的迁移，以及体内巨噬细胞在硫代乙醇酸盐诱导的腹膜炎和异物诱导的肉芽肿模型中的治疗效果(特异性目标3)。 我们相信，拟议的体外和体内实验结果将为以纤溶酶结合位点为靶点的新的治疗策略提供基础，以调节慢性炎症、组织重塑和动脉粥样硬化。

DESCRIPTION (provided by applicant): The synthesis and/or activation of matrix degrading proteinases play an essential role in the migration of monocytes and macrophages through tissue. Foremost among these proteinases is the urokinase type plasminogen activator (uPA)-plasminogen system. Plasmin binds to and degrades fibrin and several components of the extracellular matrix (ECM). In addition, plasmin activates several members of the family of matrix metalloproteinases (MMP), which are responsible for the degradation of collagen and other components of the ECM. Despite earlier observations that binding to the cell surface imparts a kinetic advantage to plasminogen activation and protection of plasmin from inhibition, the biology of membrane-bound plasmin remains largely unexplored, and its role as a therapeutic target in chronic inflammatory diseases remains untested. The overall purpose of experiments described in this proposal is to test the hypothesis that plasmin binding sites on the surface of macrophages are the primary regulator of their pericellular proteinase cascade, and blocking plasmin(ogen) binding to their surface is an effective strategy to reduce macrophage accumulation, and its sequelae in chronic inflammatory diseases. Two complimentary strategies are proposed: (1) Utilizing mice deficient in a defined plasminogen binding site (annexin II), we will directly determine the role of plasmin binding sites in macrophage activation of MMP-9, degradation of ECM and MCP-l-dependent migration through ECM in vitro (Specific Aim 1). Also, we will determine the effect of annexin II deficiency on macrophage recruitment in vivo utilizing thioglycollate-induced peritonitis and foreign body-induced granuloma models and recruitment to atherosclerotic lesions in Apo E-/- IAnx II-/- mice (Specific Aim 2). (2) We will determine the therapeutic effectiveness of an inactive cell-binding fragment of plasminogen to block macrophage activation of MMP-9, ECM degradation and MCP-1- dependent migration through ECM in vitro, as well as macrophage recruitment in vivo in the thioglycollate-induced peritonitis and foreign body-induced granuloma models (Specific Aim 3). We believe that the results of proposed in vitro and in vivo experiments will provide a basis for novel therapeutic strategies targeting plasmin-binding sites to modulate chronic inflammation, tissue remodeling and atherosclerosis.