Regulation of Vascular Smooth Muscle Cell Migration

描述（申请人提供）：血管平滑肌细胞（VSMC）的迁移在血管生成、再狭窄和动脉粥样硬化等血管生理和病理生理功能中起着关键作用。细胞在三维（3D）基质中的迁移是一种比传统的二维（2D）细胞迁移更密切相关的组织模型系统。然而，对三维矩阵中VSMC迁移的控制机制研究甚少。我们将使用3D迁移系统和VSMC作为体外模型系统来确定调节VSMC迁移的机制。肌球蛋白II是肌肉收缩的关键运动成分，在各种类型的细胞运动中起着重要作用。尽管对肌凝蛋白II运动在VSMC收缩中的作用有大量的研究，但对肌凝蛋白II运动如何在三维矩阵中控制和调节VSMC迁移的细节知之甚少。在脊椎动物中，肌球蛋白II分子的功能是由其调控轻链（MLC）的磷酸化决定的。因此，人们认为MLC的磷酸化是调控许多细胞运动的关键因素。除了MLC磷酸化参与细胞运动外，非肌肉肌球蛋白IIA （NMIIA）和IIB （NMIIB）亚型已被证明在哺乳动物成纤维细胞的细胞迁移调节中起不同的作用。最近，我们提出了一个新的模型，其中肌凝蛋白II以消极和积极的方式控制细胞迁移，这是由cPKC1和ZIP激酶介导的。因此，以下具体目标将被解决：具体目标1)确定MLC磷酸化控制肌动球蛋白细胞骨架重组和调节VSMC在3D胶原基质中的迁移的机制。将进行分析以确定血小板衍生生长因子（PDGF）介导的VSMC迁移过程中MLC磷酸化发生的时间和地点，并研究cPKC1和ZIP激酶在这一过程中的作用。2)确定非肌肉肌球蛋白IIA （NMIIA）和IIB （NMIIB）亚型在三维矩阵中调控VSMC迁移的方向性和收缩性中的作用和机制。RNA干扰（RNAi）方法将用于检查不同的NMII亚型在三维胶原基质中VSMC迁移中的作用。我们的研究将为动脉粥样硬化过程中VSMC的运动行为提供高度相关的重要基础，动脉粥样硬化被认为是心血管疾病如心脏病发作和中风的最关键的潜在原因。此外，本文提出的研究对于理解血管生成和伤口愈合等血管发育过程中VSMC迁移的基本机制具有重要意义。

DESCRIPTION (provided by applicant): Migration of vascular smooth muscle cells (VSMC) plays a key role in both the physiological and the pathophysiological vascular function such as angiogenesis, restenosis and atherosclerosis. Migration of cells in three-dimensional (3D) matrix is a more closely related model system to tissue than conventional two-dimensional (2D) cell migration. However, there is very little research on the mechanisms controlling VSMC migration in 3D matrix. We will use a 3D migration system and VSMC as an in vitro model system for identifying the mechanisms that regulate VSMC migration. It is well established that myosin II is the crucial motor component of muscle contraction and plays a fundamental role in various types of cellular movement. Whereas there are abundant studies of the myosin II motor in VSMC contraction, little is known of the details of how the myosin II motor controls and is regulated in VSMC migration in 3D matrix. In vertebrate, the function of myosin II molecules is dictated by phosphorylation of its regulatory light chain (MLC). Therefore, it has been thought that the phosphorylation of MLC is a key factor in the regulation of numerous cellular movements. In addition to the involvement of MLC phosphorylation in cell motility, non-muscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms have been shown to serve different roles in the regulation of the cell migration in mammalian fibroblasts. Quite recently, we have proposed a new model where myosin II controls cell migration in a negative and positive fashion, which are mediated by cPKC1 and ZIP kinase. Thus, the following Specific Aims will be addressed: Specific Aim 1) To determine the mechanism by which MLC phosphorylation controls the reorganization of the actomyosin cytoskeleton and regulates VSMC migration in the 3D collagen matrix. Analyses will be performed to determine when and where MLC phosphorylation occurs during platelet-derived growth factor (PDGF)-mediated VSMC migration, and to investigate the roles of cPKC1 and ZIP kinase in this process. Specific Aim 2) To determine the roles and mechanisms of non-muscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms in regulating directionality and contractility in VSMC migration in the 3D matrix. RNA interference (RNAi) approaches will be used to examine the roles of different NMII isoforms in VSMC migration in the 3D collagen matrix. Our studies will provide an important foundation of high relevance to motile behavior of VSMC during atherogenesis, which is considered the most critical underlying cause of vascular disease such as heart attack and stroke. Moreover, the studies proposed here will be important for understanding the basic mechanisms by which VSMC migration is achieved in vascular development such as angiogenesis and wound healing. PUBLIC HEALTH RELEVANCE: The proposed project focuses on the regulatory mechanisms of the cellular protein known as myosin II during cell migration. Migration of the vascular smooth muscle cells (VSMC) associated with blood vessels is a fundamental process in the development of atherosclerosis, a major cause of heart attacks and other cardiovascular diseases. Understanding the precise role of myosin II during VSMC migration will help to improve the current understanding of stable blood vessel formation-an essential step toward the treatment of deadly heart disease.