Macrophage Pericellular Proteinase Cascade

描述（由申请人提供）：基质降解蛋白酶的合成和/或激活在单核细胞和巨噬细胞通过组织的迁移中起重要作用。这些蛋白酶中最重要的是尿激酶型纤溶酶原激活剂(uPA)-纤溶酶原系统。纤溶蛋白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（MMP）家族的几个成员，这些成员负责胶原蛋白和其他ECM成分的降解。尽管早期的观察表明，结合到细胞表面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仍在很大程度上未被探索，其作为慢性炎症性疾病的治疗靶点的作用仍未得到验证。本实验的总体目的是验证巨噬细胞表面的纤溶酶结合位点是其细胞周围蛋白酶级联的主要调节因子，阻断纤溶酶（原）与其表面的结合是减少慢性炎症性疾病中巨噬细胞积聚及其后遗症的有效策略。我们提出了两种互补的策略：(1)利用缺乏特定纤溶酶原结合位点（膜联蛋白II）的小鼠，我们将直接确定纤溶酶结合位点在巨噬细胞MMP-9激活、ECM降解和mcp -l依赖的体外ECM迁移中的作用（Specific Aim 1）。此外，我们将利用巯基乙酸盐诱导的腹膜炎和异物诱导的肉芽肿模型，确定膜联蛋白II缺乏对体内巨噬细胞募集的影响，以及Apo E-/- IAnx II-/-小鼠对动脉粥样硬化病变的募集（Specific Aim 2）。(2)我们将确定一种失活的纤溶酶原细胞结合片段在体外阻断巨噬细胞MMP-9激活、ECM降解和MCP-1依赖性迁移的治疗效果。以及巨噬细胞在巯基乙酸盐诱导的腹膜炎和异物诱导的肉芽肿模型中的体内募集（Specific Aim 3）。

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.