Copper transport protein and inflammatory angiogenesis

描述(由申请人提供)：这项资助的总体目标是阐明铜运输蛋白“抗氧化剂1(Atox1)”和“NADPH氧化酶”之间参与炎症血管生成的新联系。缺血性疾病是世界范围内发病率和死亡率的主要原因。血管新生是机体对缺血作出反应的重要修复过程，依赖于血管生成、炎症反应和活性氧(ROS)。铜是一种必需的微量营养素，参与伤口愈合、血管生成等生理修复过程，也参与肿瘤生长、动脉粥样硬化和炎症性疾病等多种病理生理过程。由于过量的铜是有毒的，细胞内铜的生物有效性是紧密的 由铜转运蛋白控制，如铜伴侣Atox1。我们的实验室首次提供了Atox1作为铜依赖的转录因子来调控铜诱导的细胞生长的证据。此外，我们还首次证明NADPH氧化酶(NOx)产生的ROS在内皮细胞(ECs)的血管生成信号和出生后的血管生成中发挥重要作用，以响应缺血损伤。然而，铜转运蛋白在炎性血管生成中的作用及其与NOx的联系还完全不清楚。我们的初步数据表明，由于缺血组织中内皮细胞ROS的产生减少，Atox1缺陷小鼠的血管生成和炎性细胞募集受到了损害。骨髓(BM)重建表明，缺血后血管重建需要ECs中的Atox1，而不是BM细胞中的Atox1。根据新的初步数据，我们假设Atox1通过转录调控p47Phox和激活rac1来作为一种新的NOx调节因子；两者都是NOx的关键胞浆成分，并以一种铜依赖的方式。这反过来促进了内皮细胞中与炎症和血管生成反应相关的ROS依赖的信号转导，从而促进了对缺血损伤的新生血管的反应。AIM1将致力于确定Atox1在调控NADPH氧化酶和ROS依赖的炎症和血管生成信号以及铜依赖的内皮细胞功能中的作用。目的2将重点研究Atox1如何通过转录调控p47Phox参与NADPH氧化酶的激活，以及通过铜依赖的方式与内皮细胞中rac1结合的支架蛋白IQGAP1激活rac1的分子机制。目的3利用Atox1-/-小鼠后肢缺血模型，通过铜依赖的方式调节损伤组织中ROS的生成、血管生成和炎性细胞募集，以确定Atox1在体内新生血管中的功能作用。将进行骨髓移植、体内活体显微镜和生物发光成像、体外和体内高度创新的铜成像分析。我们的研究将为铜转运蛋白及其调节因子作为治疗血管生成和炎症依赖的缺血性心血管疾病的潜在治疗靶点提供新的见解。

DESCRIPTION (provided by applicant): The overall aim of this grant is to elucidate the novel linkage between copper transport protein "Antioxidant1 (Atox1)" and "NADPH oxidase" involved in inflammatory angiogenesis. Ischemic disease is a leading cause of morbidity and mortality in worldwide. Neovascularization is an important repair process in response to ischemia, which depends on angiogenesis, inflammation and reactive oxygen species (ROS). Copper (Cu), an essential micronutrient, is involved in physiological repair processes such as wound healing and angiogenesis as well as in various pathophysiologies including tumor growth, atherosclerosis and inflammatory diseases. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu transport proteins such as Cu chaperone Atox1. Our laboratories provided the first evidence that Atox1 functions as a Cu-dependent transcription factor to regulate Cu-induced cell growth. Furthermore, we are one of the first to demonstrate that ROS derived from NADPH oxidase (Nox) play an important role in angiogenic signaling in endothelial cells (ECs) as well as postnatal angiogenesis in response to ischemic injury. However, the role of Cu transport proteins in inflammatory angiogenesis and its linkage with Nox are entirely unknown. Our preliminary data suggest that Atox1 deficient mice have impaired angiogenesis and inflammatory cell recruitment due to decrease in endothelial ROS production in ischemic tissues. Bone marrow (BM) reconstitution indicates that Atox1 in ECs, but not BM cells, is required for post-ischemic revascularization. Based on new preliminary data, we hypothesize that Atox1 functions as a novel regulator for Nox by transcriptional regulation of p47phox as well as activating Rac1; both are critical cytosolic components of Nox, in a Cu-dependent manner. This in turn promotes ROS-dependent signaling linked to inflammatory and angiogenic responses in ECs, which contributes to neovascularization in response to ischemic injury. Aim1 will focus on establishing a role of Atox1 in regulating NADPH oxidase and ROS-dependent inflammatory and angiogenic signaling and function in ECs in a Cu-dependent manner. Aim 2 will focus on identifying molecular mechanisms of how Atox1 is involved in activation of NADPH oxidase through transcriptional regulation of p47phox and activating Rac1 via binding to a Rac1-binding scaffold protein IQGAP1 in ECs in a Cu-dependent manner. Aim 3 will focus on determining the functional role of Atox1 in neovascularization in vivo by regulating ROS production, angiogenesis and inflammatory cell recruitment in injured tissues in a Cu-dependent manner using hindlimb ischemia model with Atox1-/- mice. Bone marrow transplantation, in vivo intravital microscopy and bioluminescence imaging, highly innovative Cu imaging analysis in vitro and in vivo will be performed. Our study will provide novel insight into Cu transport protein and their regulators as potential therapeutic targets for treatment of angiogenesis- and inflammation-dependent ischemic cardiovascular diseases.