Oxidized-LDL, LOX-1 and angiogenesis

描述(由申请人提供)： 血管生成是胚胎发育和伤口修复所必需的生理过程，定义为新血管的形成。血管生成也是各种病理事件的重要组成部分，如组织缺血、癌症、糖尿病视网膜病变和包括动脉粥样硬化在内的慢性炎症状态。导致血管生成的关键事件之一是超氧阴离子和过氧化氢等活性氧物种(ROS)的产生，它们在生理和病理生理状态中发挥关键作用。LOX-1是一种凝集素样氧化型低密度脂蛋白受体，负责内皮细胞对氧化型低密度脂蛋白的结合和摄取。研究表明，LOX-1的激活本身可以刺激ROS的形成，并启动一系列氧化还原敏感的信号事件。我们推测，低浓度的氧化型低密度脂蛋白(ox-LDL)可激活内皮细胞中的LOX-1，诱导低水平的ROS释放，并启动血管生成反应。我们在这项建议中的具体目标是：目的1：研究氧化型低密度脂蛋白(OX-LDL)对血管生成的影响-这些研究将在人冠状动脉内皮细胞、血管生成的主动脉环模型以及野生型和LOX-1基因敲除(KO)小鼠中进行。目的#2：明确氧化低密度脂蛋白诱导血管生成反应的机制-这些研究将涉及一些特定的抑制剂，LOX-1抗体，针对LOX-1的siRNA，以及LOX-1的上调，并将涉及最新的分子生物学方法。此外，为了确定氧化低密度脂蛋白的作用机制，将利用微阵列技术识别在血管生成过程中上调和下调的基因。该建议的优点是：1.PI在血管生成和LOX-1研究方面有很强的背景。2.PI拥有LOX-1 KO小鼠。3.是否有训练有素、精通研究大部分方面的分子生物学家。4.PI的一名团队成员在微阵列技术方面拥有丰富的经验。意义：了解新生血管形成的基础可能会在动脉粥样硬化的基础上提供新的信息，特别是在不稳定病变的发展中。从这项研究中获得的信息也可能与癌症的发展有关，癌症的生长依赖于新生血管。 公共卫生相关性： 叙述动脉硬化(也称为动脉粥样硬化)是心脏病发作的原因。随着动脉粥样硬化覆盖区域的增长，会形成新的小血管；这一过程被称为“血管生成”。血管生成在一定程度上是由被称为活性氧物种(ROS)的氧气分子引起的。我们的工作表明，ROS刺激一种称为LOX-1的氧化胆固醇受体。拟议的实验旨在测试低浓度氧化胆固醇激活LOX-1，诱导低水平ROS释放和启动血管生成的概念。相反，高浓度的ROS导致大量LOX-1表达和细胞损伤。为了确认LOX-1在血管生成中的作用，我们将使用缺乏LOX-1的特殊小鼠。在第二组实验中，我们将通过检测基因的变化来研究血管生成的潜在机制。这项研究的信息对于了解动脉粥样硬化和某些依赖血管生成的癌症的发展可能很重要。

DESCRIPTION (provided by applicant): Summary: Angiogenesis, defined as formation of new blood vessels, is a physiological process necessary for embryonic development and wound repair. Angiogenesis is also an important component of various pathologic events such as tissue ischemia, cancer, diabetic retinopathy, and chronic inflammatory states including atherosclerosis. Among the key events leading to angiogenesis is generation of reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide which play a key role in physiological and pathophysiological states. LOX-1, a lectin-like ox-LDL receptor, is responsible for binding and uptake of ox-LDL in endothelial cells. It has been well documented that the activation of LOX-1 itself can stimulate the formation of ROS and initiate a cascade of redox-sensitive signaling events. We postulate that oxidized LDL (ox-LDL) at low concentrations activates LOX-1 in endothelial cells, induces low levels of ROS release and initiates the angiogenic response. Our specific aims in this proposal are: Aim # 1: To study the effect of oxidized LDL (ox-LDL) on angiogenesis- These studies will be done in human coronary artery endothelial cells, in an aortic ring model of angiogenesis, as well as in wild-type and LOX-1 knock out (KO) mice. Aim # 2: To define the mechanisms of ox-LDL-induced angiogenic response- These studies will involve a number of specific inhibitors, LOX-1 antibody, siRNA against LOX-1, and upregulation of LOX-1, and will involve state-of-art molecular biology approaches. Further, to define the mechanism of the effects of ox-LDL, microarray technology will be utilized to identify genes that are up-regulated and down-regulated during angiogenesis. The strengths of the proposal are: 1. The PI has strong background on angiogenesis and LOX-1 research. 2. The PI has LOX-1 KO mice in his possession. 3. Availability of well trained molecular biologist well-versed in most aspects of the research. 4. One of the PI's team members has extensive experience in microarray technology. Significance: Understanding of the basis of formation of neovasculature may provide novel information on the basis of atherogenesis, especially in the development of unstable lesions. Information gained from this study may also have relevance in the development of cancers which are dependent of neovasculature for their growth. PUBLIC HEALTH RELEVANCE: Narrative Hardening of the arteries (also known as atherosclerosis) is the cause of heart attacks. As the area covered with atherosclerosis grows, there is formation of small new blood vessels; a process called "angiogenesis". Angiogenesis is caused in part by oxygen-derived molecules called reactive oxygen species (ROS). Our work has shown that ROS stimulate a receptor for oxidized cholesterol called LOX- 1. The proposed experiments are designed to test the concept that oxidized cholesterol at low concentration activates LOX-1, induces low levels of ROS release and initiates angiogenesis. In contrast, high concentration of ROS causes much LOX-1 expression and cell injury. To confirm the role of LOX-1 in angiogenesis, we will use specialized mice that lack the LOX-1. In the second set of experiments, we will study the underlying mechanisms of angiogenesis by examining changes in genes. Information from this study may be important in understanding the development of atherosclerosis and certain cancers that are dependent of angiogenesis.