Vascular Oxidases in Migration

描述(由申请人提供)：血管平滑肌细胞(VSMC)迁移在血管成形术和动脉粥样硬化病变形成后的新生内膜形成中至关重要。在上一次资助期间，我们发现VSMC在血小板衍生生长因子(PDGF)刺激下的迁移需要产生活性氧(ROS)，而ROS是介导肌动蛋白细胞骨架重组的丝氨酸/苏氨酸激酶-1(PDK1)酪氨酸磷酸化的中介。PDK1进而磷酸化p21激活的蛋白激酶-1(PAK1)，这两种蛋白都是迁移所必需的。然而，细胞迁移是一个多步骤的过程，包括片状脂膜的形成，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在这些步骤中的每个步骤中的作用尚不清楚。在这个授权期，我们建议研究导致ROS形成的机制和调节迁移早期步骤的下游分子靶标。实现4个具体目标。首先，我们将研究PDGF刺激的细胞中ROS的来源，重点是NAD(P)H氧化酶NOX1和NOX4。我们将使用RNA沉默技术来研究这两种重要的氧化酶在片状脂溢症和局灶性粘连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介导的片状脂肪形成的信号过程。第三，我们将研究NOX4和PDK1在介导局灶性黏附激酶激活和帕西林的磷酸化中的作用，而这反过来又调节焦点接触向局灶性黏附的转化。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体内迁移模型中的作用。迁移将由颈动脉结扎或股钢丝损伤诱导，并使用形态计量学技术。总之，这些目标将为NAD(P)H氧化酶如何介导VSMC迁移从而形成病变提供新的见解。这些信息可能导致开发新的治疗策略，这些策略可以仔细和具体地针对疾病启动过程中的关键事件。

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.