GCL-M Regulation of Angiogenesis

描述（由申请人提供）：血管生成是从预先存在的血管床中萌发出新的血管结构，并且由于各种刺激而发生。血管生成的常见介质是血管内皮生长因子（VEGF）。VEGF与其受体（VEGFR 2）结合后通过活性氧（ROS）依赖性机制起作用，使其能够启动血管生成所需的信号传导过程。谷胱甘肽（GSH）作为内皮细胞内抗氧化应激的初始防御。GSH的从头合成依赖于限速全酶谷氨酸半胱氨酸连接酶（GCL），其由调节亚基（GCL-M）和催化亚基（GCL-C）组成。由于ROS水平的增加，GSH被氧化以中和任何相关的压力。当GSH水平降低时，其他抗氧化防御机制被激活，其中包括E2相关因子-2（Nrf-2），它与包括GCL-M在内的几个基因上的DNA抗氧化反应元件（ARE）结合。我们的实验室最近发现GCL-M小鼠表现出增强的血管生成活性，这表明内皮细胞谷胱甘肽水平对血管生长有重要调节作用。文献中的先前观察和本文提供的初步数据导致了GCL-M通过控制由VEGF通过激活NRF-2诱导的组织ROS水平来介导血管生成的假设。为了验证这一假设，本项目的具体目标是：（1）确定GCL-M KO小鼠内皮细胞对通过VEGF刺激增加的ROS水平的反应的迁移和增殖表型。为此目的，一组GCL-M KO小鼠将植入含有VEGF的扩散盘以确定血管募集，另一组将结扎其股动脉并在21天的时间内测量血管生成。将检查来自各种模型的组织的GSH水平、内皮细胞密度和增殖以及ROS生成。(2)确定GCL-M通过激活Nrf-2介导血管生成的分子和细胞机制。为此目的，将从小鼠中收获内皮细胞，并在VEGF存在下用于Dunn细胞迁移室中，以确定运动性以及ROS产生的机制。研究中提出的基因（GCL-M）与许多血管异常有关。提高我们的抗氧化剂调节血管生成的知识将导致更好地了解许多血管异常与ROS水平增加。

DESCRIPTION (provided by applicant): Angiogenesis is the sprouting of new vascular structures from preexisting vascular beds and occurs due to various stimuli. A common mediator of angiogenesis is vascular endothelial growth factor (VEGF). VEGF after association with its receptor (VEGFR2) acts through a reactive oxygen species (ROS) dependent mechanism allowing it to initiate a signaling process necessary for angiogenesis. Glutathione (GSH) acts as an initial defense against oxidative stress within the endothelial cells. De novo synthesis of GSH depends on a rate limiting holoenzyme, glutamate cysteine ligase (GCL), consisting of a modulatory (GCL-M) and catalytic subunit (GCL-C). Due to increases in ROS levels, GSH is oxidized to neutralize any associated stresses. As reduced GSH levels are altered other anti-oxidant defense mechanisms are activated, among these are E2-related factor-2 (Nrf-2) which binds to DNA antioxidant response elements (ARE) on several genes including GCL-M. Our laboratory has recently discovered that GCL-M mice display enhanced angiogenic activity suggesting that endothelial cell glutathione levels critically regulate blood vessel growth. Previous observations in the literature and preliminary data provided herein led to the hypothesis that GCL- M mediates angiogenesis by controlling tissue ROS levels induced by VEGF through activation of NRF-2. To test this hypothesis the specific aims of this project are: (1) Determine the migratory and proliferative phenotype of GCL-M KO mouse endothelial cells in response to increased ROS levels through VEGF stimulation. For this purpose a group of the GCL-M KO mice will be implanted with a VEGF containing diffusion disk to determine vascular recruitment, and another group will have their femoral artery ligated and angiogenesis measured over a period of 21 days. Tissues from various models will be examined for GSH levels, endothelial cell density and proliferation, and ROS generation. (2) Determine the molecular and cellular mechanisms by which GCL-M mediates angiogenesis through the activation of Nrf-2. For this aim endothelial cells will be harvested from mice and used in Dunn cell migration chambers in the presence of VEGF to determine mechanisms of motility as well as ROS production. The gene proposed for study (GCL-M) has been linked to many vascular abnormalities. Enhancing our knowledge of antioxidant regulation of angiogenesis will lead to a better understanding of many vascular abnormalities associated with increased levels of ROS.