Endothelial Cell VCAM-1 Signal Transduction

描述（由申请人提供）：本项目的长期目标 我们的建议是了解血管细胞粘附的分子机制 分子-1（VCAM- 1）信号转导。破坏 VCAM- 1基因导致胚胎死亡。VCAM- 1表达于 易形成动脉粥样硬化病变部位的内皮细胞， 在动脉粥样硬化、感染和同种异体移植过程中由内皮细胞 排斥反应VCAM- 1与白细胞结合以促进白细胞外渗到 组织中然而，关于信号转导的机制知之甚少 通过VCAM- 1在内皮细胞中表达。VCAM- 1信号传导的研究受到阻碍 通过诱导VCAM- 1表达的信号的并发症， 其他内皮细胞粘附分子与 白细胞为了在研究VCAM- 1信号传导中规避这些问题，我们 开发并表征了组成性表达 VCAM-1而不是其他已知的粘附分子。利用这些内皮细胞 细胞系和原代内皮细胞培养，我们证明VCAM- 1 刺激内皮细胞NADPH氧化酶催化产生反应性 氧物种（ROS）。这种活性氧的产生是内皮细胞 形态改变和促进内皮细胞白细胞迁移。 另一份报告表明，VCAM- 1的抗体交联激活了一种免疫抑制剂。 三磷酸肌醇的钙流和磷脂酶释放。但据 尚不清楚这些信号对内皮细胞功能有什么影响。它有 已经确定磷脂酶代谢物刺激NADPH氧化酶， 中性粒细胞这一信息使我们假设，VCAM- 1的激活可能是由于VCAM- 1的激活导致的。 磷脂酶/钙/蛋白激酶C级联激活内皮细胞 NADPH氧化酶产生活性氧调节内皮细胞肌动蛋白 重组和白细胞迁移。根据报告， ROS激活基质金属蛋白酶（MMPs）并抑制磷酸酶、VCAM- 1-刺激的ROS产生可以通过这些信号传递内皮细胞形状变化 内切酶为了解决我们的假设，我们将使用生物化学，遗传学， 药理学方法，以确定是否涉及一个信号级联反应， 钙通量（Aim 1）、磷脂酶活性（Aim 2）和蛋白激酶C 活性（Aim 3）是VCAM-1刺激NADPH氧化酶活性所必需的 在内皮细胞中。然后，我们将确定VCAM-1刺激的NADPH ROS的氧化酶产生激活MMPs（Aim 4）并调节磷酸酶 活性（目的5）在局部内皮细胞形状的变化。是 预计拟议的实验将提供新的见解， VCAM-1调节内皮细胞信号转导的机制。 这些信息可能为设计用于调节 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.