Mechano-Chemical Signaling in Endothelial Cell Caveolae

内皮细胞小窝中的机械化学信号传导

基本信息

批准号：
7052830
负责人：
VICTOR J. RIZZO
金额：
$ 25.63万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-17 至 2008-02-29
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The vascular endothelium has evolved a set of adaptive responses to the changing hemodynamic forces that continually challenge them. Inadequate or inappropriate adjustments to alterations in flow often result in pathophysiology such as hypertension and atherosclerosis. Although past research has deciphered many of the mechanisms involved in transducing hemodynamic forces into chemical signals within the cell, a key missing element is the mechanoreceptor purported to exist in the endothelial cell. By using a unique methodology that allows for the purification of luminal endothelial cell plasma membranes, we recently demonstrated that significant mechano-signaling can be initiated by enhancing fluid flow in situ in specialized invaginated, microdomains on the cell surface called caveolae. We hypothesize that caveolae represent unique structures through which endothelial cells can discriminate amongst changing hemodynamic forces. In order to test this hypothesis, we propose to manipulate cell surface density of caveolae through over-expression and anti-sense depletion of caveolin-1. These cells will be subjected to precisely defined patterns and magnitudes of physiological flow in a parallel plate apparatus and measured for established biochemical and morphological endpoints that characterize the temporal nature of the mechanotransduction response. Satisfying this aim will serve to spatially define mechanotransduction however, the mechanism of shear-induced signal propagation within caveolae remains unclear. Emerging evidence indicates that nitric oxide (NO) and/or reactive oxygen species (ROS) serve as import early responding second messengers that may participate in the mechanotransduction process. We propose that NO and/or ROS are generated within caveolae in response to sheer-stress and serve a key mechanotransducing second messengers. The following aims are proposed to test this hypothesis: i) characterize the role of nitric oxide (NO) as a mechano-signaling mediator in caveolae, ii) determine the spatial location of shear-stress induced superoxide formation specifically within the endothelial plasma membrane and iii) examine the function of ROS generation in the caveolae mediated acute mechanotransduction process. Molecular and pharmacological approaches will be used to investigate the specific contribution of shear stress induced NO and ROS to the mechanotransduction process. Results from these studies will extend our insight into the basic mechanisms by which endothelial cells respond to changes in fluid mechanical forces generated by flowing blood. Such information would greatly add to our understanding of both normal cardiovascular function and the pathophysiology seen in atherosclerosis.

描述（由申请人提供）：血管内皮细胞已经进化出一系列适应性反应，以适应不断变化的血流动力学力，这些力不断挑战它们。对血流变化的不充分或不适当的调整通常会导致高血压和动脉粥样硬化等病理生理学后果。尽管过去的研究已经破译了许多将血流动力学力转化为细胞内化学信号的机制，但一个关键的缺失元素是据称存在于内皮细胞中的机械感受器。通过使用一种允许纯化管腔内皮细胞质膜的独特方法，我们最近证明，可以通过增强细胞表面上称为小窝的专门内陷微域中的原位流体流动来启动重要的机械信号传导。我们假设小窝代表了独特的结构，内皮细胞可以通过该结构区分不断变化的血流动力学力。为了检验这一假设，我们建议通过caveolin-1的过度表达和反义缺失来操纵caveolae的细胞表面密度。这些细胞将在平行板装置中受到精确定义的生理流模式和大小的影响，并测量已建立的生化和形态学终点，这些终点表征了机械转导反应的时间性质。满足这一目标将有助于在空间上定义力转导，然而，小凹内剪切诱导信号传播的机制仍不清楚。新的证据表明，一氧化氮（NO）和/或活性氧（ROS）作为重要的早期响应第二信使，可能参与机械传导过程。我们认为，NO 和/或 ROS 是在小凹内响应纯粹应力而产生的，并充当关键的机械传导第二信使。提出以下目标来检验这一假设：i）表征一氧化氮（NO）作为小凹中机械信号传导介质的作用，ii）确定剪切应力诱导的超氧化物形成的空间位置，特别是在内皮质膜内iii) 检查 ROS 生成在小窝介导的急性机械转导过程中的功能。将使用分子和药理学方法来研究剪切应力诱导的 NO 和 ROS 对力转导过程的具体贡献。这些研究的结果将扩展我们对内皮细胞响应血液流动产生的流体机械力变化的基本机制的认识。这些信息将极大地增进我们对正常心血管功能和动脉粥样硬化病理生理学的理解。