Vascular Oxidases in Migration

描述（由申请方提供）：血管平滑肌细胞（VSMC）迁移在血管成形术后新生内膜形成和动脉粥样硬化病变形成中至关重要。在最后一次资助期间，我们发现VSMC迁移对血小板源性生长因子（PDGF）的反应需要产生活性氧（ROS），ROS介导磷酸肌醇依赖性激酶-1（PDK1）的酪氨酸磷酸化，PDK1是一种丝氨酸/苏氨酸激酶，介导肌动蛋白细胞骨架重组。PDK1反过来磷酸化p21激活激酶-1（PAK1），这两种激酶都是迁移所必需的。然而，细胞迁移是一个多步骤的过程，包括形成片状伪足，在细胞前部产生局灶性粘连，细胞体收缩，以及在后缘溶解局灶性粘连。ROS和PDK1在这些步骤中的作用尚不清楚。在此期间，我们建议研究导致ROS形成的机制以及调节迁移早期步骤的下游分子靶点。将实现四个具体目标。首先，我们将研究PDGF刺激细胞中ROS的来源，重点是NAD（P）H氧化酶Nox 1和Nox 4。我们将使用RNA沉默技术来研究这两种重要氧化酶在板状伪足和粘着斑形成中的单独和联合作用。其次，我们将研究导致Nox1激活和PDK1介导的板状伪足形成的信号传导过程。第三，我们将研究Nox 4和PDK 1在介导粘着斑激酶激活和桩蛋白磷酸化中的作用，桩蛋白磷酸化反过来调节粘着斑向粘着斑的转化。最后，我们将使用独特的转基因和基因敲除动物（平滑肌特异性nox1-或过氧化氢酶过表达和nox1-/-小鼠）来研究Nox1和ROS在VSMC迁移体内模型中的作用。迁移将通过颈动脉结扎或股线损伤诱导，并随后使用形态测量技术。总之，这些目标将为NAD（P）H氧化酶如何介导VSMC迁移和损伤形成提供新的见解。这些信息可能会导致新的治疗策略的发展，可以仔细和具体针对疾病开始的关键重要事件。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) migration is critically important in neointimal formation following angioplasty and atherosclerotic lesion formation. During the last grant period, we showed that VSMC migration in response to platelet-derived growth factor (PDGF) requires the production of reactive oxygen species (ROS), which mediate the tyrosine phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), a serine/threonine kinase that mediates actin cytoskeletal reorganization. PDK1, in turn, phosphorylates p21- activated kinase-1 (PAK1), and both kinases are required for migration. However, cell migration is a multistep process that involves formation of lamellipodia, creation of focal adhesions at the front of the cell, contraction of the cell body, and dissolution of focal adhesions at the trailing edge. The role of ROS and PDK1 in each of these steps remains unclear. In this grant period, we propose to investigate the mechanisms leading to ROS formation and the downstream molecular targets that regulate the early steps in migration. 4 specific aims will be accomplished. First, we will investigate the sources of ROS in PDGF-stimulated cells, focusing on the NAD(P)H oxidases Nox1 and Nox4. We will use RNA silencing techniques to investigate the separate and joint roles of these 2 important oxidases in formation of lamellipodia and focal adhesions. Second, we will investigate the signaling processes leading to Nox1 activation and PDK1- mediated lamellipodia formation. Third, we will investigate the role of Nox4 and PDK1 in mediating focal adhesion kinase activation and phosphorylation 'of paxillin, which in turn regulate the conversion of focal contacts to focal adhesions. Finally, we will use unique transgenic and knockout animals (smooth muscle specific nox1- or catalase-overexpressors and nox1-/- mice) to investigate the role of Nox1 and ROS in an in vivo model of VSMC migration. Migration will be induced by carotid ligation or femoral wire injury and followed using morphometric techniques. Together, these Aims will provide new insight into how NAD(P)H oxidases mediate VSMC migration and therefore lesion formation. Such information may lead to the development of new therapeutic strategies that can be carefully and specifically targeted to the critically important events in disease initiation.