Role of Cu Transporter Proteins in Atherosclerosis

 描述（由申请人提供）： 动脉粥样硬化是由炎症、内皮细胞 (EC) 屏障功能障碍和氧化应激调节的，是主动脉瘤 (AA) 和退伍军人群体死亡的主要原因。铜 (Cu) 是一种必需营养素，与正常功能有关，而过量的铜会导致炎症性疾病，包括机制不明的动脉粥样硬化。由于过量的铜是有毒的，细胞内铜的生物利用度受到铜转运 ATP 酶 (ATP7A) 的严格控制，ATP7A 从胞质铜伴侣 Atox1 中获取铜，然后将铜输送到包括 ecSOD 在内的分泌性铜酶，或将铜输出到细胞外空间。我们的实验室报告说，血管 ATP7A-ecSOD 轴可以防止高血压和 1 型糖尿病的内皮功能障碍，并且当 Atox1 定位于细胞核时，它可以作为 Cu 依赖性转录因子发挥作用。然而，ATP7A 在动脉粥样硬化中的作用及其与核 Atox1 的关系尚不清楚。初步数据表明，与 ApoE-/- 小鼠相比，铜输出功能降低的 ATP7A 突变体 (ATP7Amut) 小鼠与 ApoE-/- 小鼠杂交会加速动脉粥样硬化病变，并且与 ApoE-/- 小鼠相比，具有过多的炎症细胞和血管通透性。相比之下，Atox1-/-/ApoE-/- 小鼠表现出动脉粥样硬化病变显着减少，表明 ATP7A 具有动脉粥样硬化保护作用，而 Atox1 则具有促动脉粥样硬化作用。值得注意的是，ATP7Amut 和 Atox1-/- 小鼠均表现出通过铜伴侣 Atox1 介导的铜酶赖氨酰氧化酶活性降低程度相似，这并不能解释 ATP7Amut/ApoE-/- 小鼠中动脉粥样硬化或 AA 的增强。基于 ATP7A 耗尽的 EC 的其他新的初步数据，我们将测试新的假设，即炎症导致的 Cu 输出蛋白 ATP7A 下调会增加细胞内的 Cu，从而刺激“核 Atox1”介导的 EC 屏障功能障碍和 ROS 依赖性炎症反应。这反过来又会促进炎症细胞过度募集，从而加速动脉粥样硬化和 AA。目标 1 将定义 ATP7A 对炎症诱导的内皮屏障功能障碍和 ROS 依赖性炎症粘附分子表达的保护作用，这些功能以 Cu 和 Atox1 依赖性方式促进 EC 中白细胞跨内皮迁移 (TEM)。目标 2 将通过关注 Atox1 的 Cu 依赖性转录因子功能来上调抑制 VE-钙粘蛋白的 miR125b 以及增加 ROS-NFkB 的 NADPH 氧化酶的 p47phox，从而确定炎症诱导的细胞内 Cu 积累如何促进内皮屏障功能障碍和 ROS 依赖性炎症反应。目标 3 将通过体内 Cu 和 Atox1 依赖性方式调节血管通透性和炎症来定义 ATP7A 对动脉粥样硬化和 AA 发展的保护作用。我们将使用 ATP7Amut、ATP7A 转基因 Atox1-/- 小鼠；或诱导型 EC 特异性 ATP7A 条件缺陷型小鼠与经 Cu 螯合剂或 LNA-抗 miR125b 处理的高脂饮食 ApoE-/- 小鼠杂交。此外，还将使用铜成像分析（64Cu标记、电感耦合等离子体质谱（ICP-MS）和同步加速器X射线荧光显微镜（XFM））、活细胞成像、活体活体显微镜、使用NFkB转基因报告小鼠的非侵入性生物发光成像。我们的研究将为铜转运蛋白 ATP7A 或核 Atox1 作为治疗动脉粥样硬化等血管炎症性疾病的潜在治疗靶点提供新的见解。

 DESCRIPTION (provided by applicant): Atherosclerosis, which is regulated by inflammation, endothelial cell (EC) barrier dysfunction, and oxidative stress, is the major cause of aortic aneurysm (AA), and mortality in Veteran population. Copper (Cu), an essential nutrient, is involved in normal function while excess Cu contributes to inflammatory diseases including atherosclerosis with unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu transporting ATPase (ATP7A) which obtains Cu from cytosolic Cu chaperone Atox1 and then delivers Cu to secretory Cu enzymes including ecSOD, or exports Cu to extracellular space. Our lab reported that vascular ATP7A-ecSOD axis protects against endothelial dysfunction in hypertension and type1 diabetes, and that Atox1 functions as a Cu-dependent transcription factor, when it localizes at nucleus. However, a role of ATP7A and its relationship with nuclear Atox1 in atherosclerosis is entirely unknown. Preliminary data suggest that ATP7A mutant (ATP7Amut) mice which have reduced Cu export function crossed with ApoE-/- mice accelerate atherosclerotic lesion and AA with excess inflammatory cells and vascular permeability vs. ApoE-/- mice. By contrast, Atox1-/-/ApoE-/- mice exhibit significant reduction of atherosclerotic lesion, suggesting that ATP7A is atheroprotective while Atox1 is proatherogenic. Of note, both ATP7Amut and Atox1-/- mice show the similar extent of reduced Cu enzyme lysyl oxidase activity mediated through Cu chaperone Atox1, which does not explain enhancing atherosclerosis or AA in ATP7Amut/ApoE-/- mice. Based on additional new preliminary data with ATP7A-depleted ECs, we will test the novel hypothesis that Cu exporter ATP7A downregulation by inflammation increases intracellular Cu that stimulates "nuclear Atox1"- mediated EC barrier dysfunction and ROS-dependent inflammatory responses. This in turn promotes excess inflammatory cell recruitment, which accelerates atherosclerosis and AA. Aim 1 will define the protective role of ATP7A against inflammation-induced endothelial barrier dysfunction and ROS-dependent inflammatory adhesion molecule expression, which contribute to leukocyte transendothelial migration (TEM) in ECs in a Cu- and Atox1-dependent manner. Aim 2 will determine how inflammation-induced accumulated intracellular Cu promotes endothelial barrier dysfunction and ROS-dependent inflammatory responses by focusing on Cu-dependent transcription factor function of Atox1 to upregulate miR125b that represses VE- cadherin as well as p47phox of NADPH oxidase that increases ROS-NFkB. Aim 3 will define the protective role of ATP7A against atherosclerosis and AA development via regulating vascular permeability and inflammation in a Cu- and Atox1-dependent manner in vivo. We will use ATP7Amut, ATP7A transgenic, Atox1-/- mice; or inducible EC-specific ATP7A conditional deficient mice crossed with ApoE-/- mice with high fat diet treated with Cu chelators or LNA-anti-miR125b. Moreover, Cu imaging analysis (64Cu labeling, inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence microscopy (XFM)), live cell imaging, in vivo intravital microscopy, non-invasive Bioluminescence imaging using NFkB transgenic reporter mice will be used. Our study will provide novel insight into Cu transporter ATP7A or nuclear Atox1 as potential therapeutic targets for treatment of vascular inflammatory diseases such as atherosclerosis.