Copper transport protein and inflammatory angiogenesis

描述（由申请人提供）：本授权的总体目标是阐明铜转运蛋白“抗氧化剂1（Atox 1）”和“NADPH氧化酶”之间的新联系，参与炎症性血管生成。缺血性疾病是世界范围内发病率和死亡率的主要原因。血管新生是缺血后重要的修复过程，其机制主要依赖于血管生成、炎症反应和活性氧。铜（Cu）是一种必需的微量营养素，参与生理修复过程，如伤口愈合和血管生成，以及各种病理生理学，包括肿瘤生长、动脉粥样硬化和炎性疾病。由于过量的铜是有毒的，细胞内铜的生物利用度是紧密的， 由Cu转运蛋白如Cu伴侣Atox 1控制。我们的实验室提供了第一个证据表明Atox 1作为铜依赖性转录因子调节铜诱导的细胞生长。此外，我们是最早证明来源于NADPH氧化酶（Nox）的ROS在内皮细胞（EC）的血管生成信号传导以及出生后缺血性损伤的血管生成中起重要作用的研究之一。然而，铜转运蛋白在炎症性血管生成中的作用及其与Nox的联系是完全未知的。我们的初步数据表明，Atox 1缺陷小鼠血管生成和炎症细胞的招聘由于减少在缺血组织中的内皮活性氧的生产受损。骨髓（BM）重建表明，Atox 1在EC中，而不是BM细胞，是需要缺血后血管重建。基于新的初步数据，我们假设Atox 1的功能作为一种新的调节Nox的转录调控p47 phox以及激活Rac 1;两者都是关键的细胞溶质组分的Nox，在铜依赖的方式。这反过来又促进了与EC中的炎症和血管生成反应相关的ROS依赖性信号传导，这有助于响应于缺血性损伤的新血管形成。Aim 1将专注于确定Atox 1在调节NADPH氧化酶和ROS依赖性炎症和血管生成信号传导中的作用，以及以铜依赖性方式在EC中的功能。目的2将集中于确定Atox 1如何参与NADPH氧化酶的激活，通过转录调节p47 phox和激活Rac 1通过结合Rac 1结合支架蛋白IQGAP 1在EC中以铜依赖的方式。目的3将重点关注使用Atox 1-/-小鼠的后肢缺血模型，通过以铜依赖性方式调节损伤组织中的活性氧产生、血管生成和炎症细胞募集来确定Atox 1在体内新血管形成中的功能作用。将进行骨髓移植、体内活体显微镜和生物发光成像、高度创新的体外和体内Cu成像分析。我们的研究将提供新的见解铜转运蛋白及其调节剂作为治疗血管生成和炎症依赖性缺血性心血管疾病的潜在治疗靶点。

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.