Rho kinase in immune-mediated atherosclerosis

描述（由申请人提供）：尽管Rho激酶（ROCK）以其调节细胞骨架蛋白的作用而闻名，但近年来已经发现了ROCK的其他一些功能。其中一个功能是免疫反应和炎症。由于其独特的调节诸如肌动蛋白-肌球蛋白细胞骨架等重要细胞功能的能力，ROCK可能会影响各种白细胞功能。事实上，我们最近已经表明，抑制ROCK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。由于其能够影响巨噬细胞功能，而巨噬细胞功能与动脉粥样硬化过程密切相关，因此ROCK可能在免疫介导的动脉粥样硬化中发挥重要作用。在目的1中，我们将通过只在巨噬细胞中使用ROCK缺乏小鼠的巨噬细胞来验证ROCK影响巨噬细胞运输的假设。我们将采用体外和体内两种方法来证明ROCK缺乏对巨噬细胞的粘附、迁移和基质侵袭特性的影响。我们还将确定调控rock介导的巨噬细胞趋化的信号机制。此外，我们将利用体外和体内方法表征ROCK在细胞外基质重塑中的作用。在目的2中，我们将研究ROCK在巨噬细胞的两种特性中的作用，这两种特性对动脉粥样硬化的发生至关重要。通过培养来自上述小鼠骨髓的巨噬细胞，我们将检测巨噬细胞在存在或不存在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在脂质负载细胞的脂质外排中的作用。机制研究将试图确定影响脂质负荷的rock介导分子。巨噬细胞的另一个将被研究的特性是ROCK在调节这些细胞活化能力中的作用。此外，活化的巨噬细胞执行其促炎功能的能力将被评估。在目的3中，巨噬细胞中特异性缺乏ROCK的小鼠将与动脉粥样硬化易感的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不缺乏ROCK的小鼠的病变进行比较。检查这些小鼠的病变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会暗示ROCK在几个动脉粥样硬化过程中，并可能导致开发旨在抑制巨噬细胞ROCK以对抗动脉粥样硬化的治疗方法。

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.