Copper transport protein and inflammatory angiogenesis

描述（由申请人提供）：本次资助的总体目的是阐明铜转运蛋白“抗氧化剂1（Atox1）”和参与炎症血管生成的“NADPH氧化酶”之间的新联系。缺血性疾病是全世界发病和死亡的主要原因。新血管形成是响应缺血的重要修复过程，这取决于血管生成、炎症和活性氧（ROS）。铜 (Cu) 是一种必需的微量营养素，参与伤口愈合和血管生成等生理修复过程，以及肿瘤生长、动脉粥样硬化和炎症性疾病等各种病理生理学。由于过量的铜是有毒的，因此细胞内铜的生物利用度严格限制 由 Cu 转运蛋白（例如 Cu 伴侣 Atox1）控制。我们的实验室提供了第一个证据，证明 Atox1 作为 Cu 依赖性转录因子来调节 Cu 诱导的细胞生长。此外，我们是最早证明源自 NADPH 氧化酶 (Nox) 的 ROS 在内皮细胞 (EC) 的血管生成信号传导以及响应缺血性损伤的出生后血管生成中发挥重要作用的人之一。然而，铜转运蛋白在炎症血管生成中的作用及其与 Nox 的联系尚不清楚。我们的初步数据表明，Atox1 缺陷小鼠由于缺血组织中内皮 ROS 产生减少，血管生成和炎症细胞募集受损。骨髓 (BM) 重建表明 EC 细胞（而非 BM 细胞）中的 Atox1 是缺血后血运重建所必需的。基于新的初步数据，我们假设 Atox1 通过 p47phox 的转录调节以及激活 Rac1 作为 Nox 的新型调节剂。两者都是 Nox 的关键细胞质成分，以 Cu 依赖性方式。这反过来又促进与 EC 中炎症和血管生成反应相关的 ROS 依赖性信号传导，从而有助于响应缺血性损伤的新生血管形成。 Aim1 将重点关注 Atox1 在调节 NADPH 氧化酶和 ROS 依赖性炎症和血管生成信号以及 EC 中铜依赖性功能中的作用。目标 2 将重点确定 Atox1 如何通过 p47phox 的转录调控参与 NADPH 氧化酶激活的分子机制，以及如何通过与 EC 中的 Rac1 结合支架蛋白 IQGAP1 以 Cu 依赖性方式结合来激活 Rac1。目标 3 将重点确定 Atox1 在体内新生血管形成中的功能作用，通过使用 Atox1-/- 小鼠后肢缺血模型，以 Cu 依赖性方式调节受损组织中的 ROS 产生、血管生成和炎症细胞募集。将进行骨髓移植、活体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.