Endothelial Cell VCAM-1 Signal Transduction

描述(由申请人提供)：这项计划的长期目标 建议了解血管细胞黏附分子-1(VCAM-1)在内皮细胞中的信号转导的分子机制。中断 VCAM-1基因会导致胚胎死亡。VCAM-1表达于 易形成动脉粥样硬化病变部位的内皮细胞和 血管内皮细胞在动脉粥样硬化、感染和同种异体移植中的作用 拒绝。VCAM-1与白细胞结合促进白细胞渗入 纸巾。然而，对信号转导的机制知之甚少。 在内皮细胞中通过VCAM-1。VCAM-1信号转导的研究受阻 通过诱导VCAM-1表达的信号的并发症和通过 其他内皮细胞黏附分子与血管内皮细胞的同时相互作用 白细胞。为了绕过研究VCAM-1信号的这些问题，我们 开发并鉴定了构成表达的内皮细胞系 VCAM-1，而不是其他已知的黏附分子。利用这些内皮细胞 和原代内皮细胞培养，我们证明了VCAM-1 刺激内皮细胞NADPH氧化酶催化的反应性生成 氧物种(ROS)。这种ROS的产生是内皮细胞所必需的 形态改变和内皮细胞促进白细胞迁移。 另一份报告表明，VCAM-1的抗体交联激活了一种 三磷酸肌醇的钙通量和磷脂酶释放。然而，它是 不知道这些信号对内皮细胞功能有什么影响。它有 已证实磷脂酶代谢产物刺激NADPH氧化酶 中性粒细胞。这一信息使我们假设VCAM-1激活 磷脂酶/钙/蛋白激酶C通路激活内皮细胞 NADPH氧化酶产生ROS调控内皮细胞肌动蛋白 重组和白细胞迁移。根据报告，低水平的 ROS激活基质金属蛋白酶(MMPs)，抑制磷脂酶，VCAM- 1-刺激ROS的产生可能通过这些信号传递内皮细胞形状变化的信号 酵素。为了解决我们的假设，我们将使用生化、遗传和 药理学方法来确定信号级联是否涉及 钙流量(目标1)、磷脂酶活性(目标2)和蛋白激酶C 血管细胞黏附分子-L刺激NADPH氧化酶活性所需的活性(目标3) 在内皮细胞中。然后，我们将确定VCAM-1刺激的NADPH ROS产生的氧化酶激活MMPs(目标4)并调节磷酸酶 在局部内皮细胞形状改变期间的活性(目标5)。它是 预计拟议中的实验将提供对 血管细胞黏附分子-I调控内皮细胞信号转导的机制。 这一信息可能为设计调制的治疗提供新的靶点 动脉粥样硬化、感染或移植期间的血管功能。

DESCRIPTION (provided by the applicant): The long term objective of this proposal is to understand molecular mechanisms for vascular cell adhesion molecule- 1 (VCAM- 1) signal transduction in endothelial cells. Disruption of the VCAM- 1 gene results in embryonic death. VCAM- 1 is expressed on endothelial cells in sites predisposed to atherosclerotic lesion formation and by endothelial cells during atherosclerosis, infection, and allograft rejection. VCAM- 1 binds to leukocytes to promote leukocyte extravasation into tissues. However, little is known regarding mechanisms for signal transduction in endothelial cells via VCAM- 1. Study of VCAM- 1 signaling has been impeded by complications from signals for induction of VCAM- 1 expression and by simultaneous interactions of other endothelial cell adhesion molecules with leukocytes. To circumvent these problems in studying VCAM- 1 signaling, we developed and characterized endothelial cell lines that constitutively express VCAM-1 and not other known adhesion molecules. Using these endothelial cells lines and primary endothelial cell cultures, we demonstrated that VCAM- 1 stimulates endothelial cell NADPH oxidase-catalyzed production of reactive oxygen species (ROS). This production of ROS is required for endothelial cell shape changes and for endothelial cell promotion of leukocyte migration. Another report indicates that antibody cross linking of VCAM- 1 activates a calcium flux and phospholipase release of inositol triphosphate. However, it is not known what effects these signals have on endothelial cell function. It has been established that phospholipase metabolites stimulate NADPH oxidase in neutrophils. This information led us to hypothesize that VCAM- 1 activation of a phospholipase/calcium/protein kinase C cascade activates endothelial cell NADPH oxidase production of ROS for the modulation of endothelial cell actin restructuring and leukocyte migration. Based on the reports that low levels of ROS activate matrix metalloproteinases (MMPs) and inhibit pbosphatases, VCAM- 1-stimulated ROS production may signal endothelial cell shape changes via these enzymes. To address our hypothesis, we will use biochemical, genetic, and pharmacological approaches to determine whether a signaling cascade involving a calcium flux (Aim 1), phospholipase activity (Aim 2), and protein kinase C activity (Aim 3) is required for VCAM-l stimulation of NADPH oxidase activity in endothelial cells. Then, we will determine whether VCAM-1 -stimulated NADPH oxidase production of ROS activates MMPs (Aim 4) and modulates phosphatase activity (Aim 5) during localized endothelial cell shape changes. It is anticipated that the proposed experiments will provide new insight into the mechanisms by which VCAM- I regulates endothelial cell signal transduction. This information may provide novel targets for therapies designed to modulate vascular function during atherosclerosis, infection, or transplantation.