Rho kinase in immune-mediated atherosclerosis

描述(由申请人提供)：虽然Rho Kinase(ROCK)在调节细胞骨架蛋白中的作用最为人所知，但近年来也发现了ROCK的其他几种功能。这些功能之一是免疫反应和炎症。由于ROCK具有调节肌动蛋白-肌球蛋白细胞骨架等重要细胞功能的独特能力，因此很可能会影响多种白细胞功能。事实上，我们最近已经证明，抑制岩石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入侵是由岩石介导的。由于岩石能够影响与动脉粥样硬化形成过程密切相关的巨噬细胞功能，因此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，我们将使用岩石缺乏的小鼠的巨噬细胞只在巨噬细胞中检验岩石影响巨噬细胞运输的假设。我们将使用体外和体内两种方法来证明岩石缺乏对巨噬细胞的黏附、迁移和基质侵袭特性的影响。我们还将确定调节岩石介导的巨噬细胞趋化的信号机制。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，我们将研究岩石在巨噬细胞的两种特性中的作用，这两种特性对动脉粥样硬化的形成至关重要。通过培养上述小鼠骨髓来源的巨噬细胞，我们将检测巨噬细胞在有或没有ROCK的情况下吞噬脂肪并成为泡沫细胞的能力。我们还将研究ROCK在脂质负载细胞的脂质外流中的作用。机械学研究将试图确定影响脂质负荷的岩石介导的分子。将被研究的巨噬细胞的另一个特性是岩石在调节这些细胞的激活能力方面所起的作用。此外，还将评估激活的巨噬细胞执行其促炎功能的能力。在目标3中，缺乏巨噬细胞特异性ROCK的小鼠将与易患动脉粥样硬化的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没有岩石缺乏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岩石在动脉粥样硬化中的作用。这些研究的成功完成可能会将岩石牵涉到几个动脉粥样硬化的形成过程中，并可能导致旨在抑制巨噬细胞中的岩石以对抗动脉粥样硬化的疗法的开发。

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.