Rho kinase in immune-mediated atherosclerosis

描述（由申请人提供）：虽然Rho激酶（ROCK）以其在调节细胞骨架蛋白中的作用而闻名，但近年来ROCK的几种其他功能也已被鉴定。这些功能之一是免疫反应和炎症。由于其独特的调节肌动蛋白-肌球蛋白细胞骨架等重要细胞功能的能力，ROCK 很可能会影响各种白细胞功能。事实上，我们最近已经证明 ROCK 的抑制会影响某些巨噬细胞的功能。例如，我们的数据表明巨噬细胞的迁移和基质侵袭是由 ROCK 介导的。由于 ROCK 能够影响与动脉粥样硬化过程密切相关的巨噬细胞功能，因此 ROCK 很可能在免疫介导的动脉粥样硬化中发挥重要作用。在目标 1 中，我们将通过仅在巨噬细胞中使用 ROCK 缺陷小鼠的巨噬细胞来检验 ROCK 影响巨噬细胞运输的假设。我们将使用体外和体内方法来证明 ROCK 缺陷对巨噬细胞的粘附、迁移和基质侵入特性的影响。我们还将确定调节 ROCK 介导的巨噬细胞趋化性的信号传导机制。此外，我们将使用体外和体内方法来表征 ROCK 在细胞外基质重塑中的作用。在目标 2 中，我们将研究 ROCK 在巨噬细胞的两种特性中的作用，这两种特性对于动脉粥样硬化形成至关重要。通过培养来自上述小鼠骨髓的巨噬细胞，我们将检查巨噬细胞在有或没有ROCK的情况下吞噬脂质并成为泡沫细胞的能力。我们还将研究 ROCK 在脂质负载细胞脂质流出中的作用。机理研究将尝试识别影响脂质负荷的 ROCK 介导的分子。将要研究的巨噬细胞的另一个特性是 ROCK 在调节这些细胞被激活的能力中的作用。此外，还将评估活化的巨噬细胞执行其促炎功能的能力。在目标 3 中，巨噬细胞中特异性缺乏 ROCK 的小鼠将与易发生动脉粥样硬化的 LDLR-/- 小鼠杂交。这些小鼠的动脉粥样硬化程度和动脉粥样硬化病变的形态将与未缺乏ROCK的小鼠的病变进行比较。对这些小鼠病变的检查将揭示巨噬细胞特异性 ROCK 在动脉粥样硬化中的作用。这些研究的成功完成可能表明 ROCK 参与了几种动脉粥样硬化过程，并可能导致开发旨在抑制巨噬细胞中 ROCK 以对抗动脉粥样硬化的疗法。

DESCRIPTION (provided by applicant): Although Rho kinase (ROCK) is most well known for its role in regulating cytoskeletal proteins, several other functions of ROCK have been identified in recent years. One of these functions is in immune response and inflammation. Because of its unique ability to regulate such vital cellular functions as actin- myosin cytoskeleton, it is likely that ROCK will influence various leukocyte functions. Indeed, we have shown recently that inhibition of ROCK affects certain macrophage function. For example, our data suggest that migration as well as matrix invasion of macrophages are mediated by ROCK. Because of its ability to affect macrophage functions that are intimately involved in the atherogenic process, it is likely that ROCK plays an important role in immune-mediated atherosclerosis. In aim 1, we will test the hypothesis that ROCK influences macrophage trafficking by using macrophages from mice with ROCK deficiency only in the macrophage. We will use both in vitro and in vivo approaches to demonstrate the effect of ROCK deficiency on the adhesive, migratory and matrix invasive properties of macrophages. We will also identify the signaling mechanisms that regulate ROCK-mediated macrophage chemotaxis. In addition we will characterize ROCK's role in extracellular matrix remodeling using both in vitro and in vivo approaches. In aim 2, we will investigate the role of ROCK in two properties of macrophages that are essential to atherogenesis. By culturing macrophages derived from bone marrow of the above mice, we will examine the ability of the macrophages to phagocytose lipid and become foam cells in the presence or absence of ROCK. We will also examine the role of ROCK in lipid efflux from lipid-loaded cells. Mechanistic studies will attempt to identify the ROCK-mediated molecules that affect lipid loading. The other property of macrophages that will be studied is the role of ROCK in modulating the ability of these cells to become activated. Furthermore, the ability of the activated macrophages to perform their proinflammatory function will be assessed. In aim 3, mice lacking ROCK specifically in macrophages will be crossed with the atherosclerosis-prone LDLR-/- mice. The extent of atherosclerosis and the morphology of atherosclerotic lesions in these mice will be compared to lesions in mice that have no deficiency of ROCK. Examination of lesions in these mice will reveal the role of macrophage-specific ROCK in atherosclerosis. Successful completion of these studies will likely implicate ROCK in several atherogenic processes and may lead to development of therapies aimed at inhibiting ROCK in macrophages to combat atherosclerosis.