Rho kinase in immune-mediated atherosclerosis

描述（由申请人提供）：尽管Rho激酶（ROCK）在调节细胞骨架蛋白中的作用最为人们所熟知，但近年来已鉴定出ROCK的几种其他功能。这些功能之一是免疫反应和炎症。由于其独特的能力，以调节这些重要的细胞功能，如肌动蛋白-肌球蛋白细胞骨架，这是可能的，ROCK将影响各种白细胞功能。事实上，我们最近已经表明，抑制ROCK影响某些巨噬细胞功能。例如，我们的数据表明，巨噬细胞的迁移以及基质侵入是由ROCK介导的。由于其影响巨噬细胞功能的能力，密切参与动脉粥样硬化的过程中，它可能是ROCK在免疫介导的动脉粥样硬化中发挥重要作用。在目的1中，我们将通过使用来自仅在巨噬细胞中具有ROCK缺陷的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，迁移和基质侵袭特性的影响。我们还将确定调节ROCK介导的巨噬细胞趋化性的信号机制。此外，我们将使用体外和体内方法表征ROCK在细胞外基质重塑中的作用。在目标2中，我们将研究ROCK在动脉粥样硬化形成所必需的巨噬细胞的两种特性中的作用。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ROCK存在或不存在下吞噬脂质并变成泡沫细胞的能力。我们还将研究ROCK在脂质负载细胞的脂质流出中的作用。机制研究将试图确定ROCK介导的分子，影响脂质负荷。将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，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，将巨噬细胞中特异性缺乏ROCK的小鼠与动脉粥样硬化倾向性LDLR-/-小鼠杂交。将这些小鼠中动脉粥样硬化的程度和动脉粥样硬化病变的形态与不缺乏ROCK的小鼠中的病变进行比较。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将可能涉及ROCK在几个致动脉粥样硬化的过程，并可能导致旨在抑制巨噬细胞中的ROCK以对抗动脉粥样硬化的治疗的发展。

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.