Role of Cu Transporter Proteins in Atherosclerosis

 描述（由申请人提供）： 由炎症、内皮细胞（EC）屏障功能障碍和氧化应激调节的动脉粥样硬化是退伍军人人群中主动脉瘤（AA）和死亡率的主要原因。铜（Cu）是一种重要的营养物质，参与机体的正常功能，而过量的Cu会导致包括动脉粥样硬化在内的炎症性疾病，其机制尚不清楚。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（ATP 7A）的严格控制，该ATP酶从细胞溶质铜伴侣Atox 1获得铜，然后将铜递送到分泌型铜酶（包括ecSOD），或将铜输出到细胞外空间。我们的实验室报道，血管ATP 7A-ecSOD轴保护高血压和1型糖尿病的内皮功能障碍，Atox 1作为一个铜依赖性转录因子，当它定位在细胞核。然而，ATP 7A在动脉粥样硬化中的作用及其与核Atox 1的关系完全未知。初步数据表明，与Apoe-/-小鼠相比，铜输出功能降低的ATP 7A突变小鼠（ATP 7Amut）与Apoe-/-小鼠杂交会加速动脉粥样硬化病变和AA，并伴有过多的炎症细胞和血管渗透性。相比之下，Atox 1-/-/ApoE-/-小鼠表现出动脉粥样硬化病变的显著减少，表明ATP 7A是动脉粥样硬化保护的，而Atox 1是促动脉粥样硬化的。值得注意的是，ATP 7Amut和Atox 1-/-小鼠均显示出通过Cu伴侣Atox 1介导的Cu酶赖氨酰氧化酶活性降低的相似程度，这不能解释ATP 7Amut/ApoE-/-小鼠中动脉粥样硬化或AA的增强。基于ATP 7A缺失的EC的额外的新的初步数据，我们将测试新的假设，即Cu出口商ATP 7A通过炎症下调增加细胞内Cu，刺激“核Atox 1”介导的EC屏障功能障碍和ROS依赖性炎症反应。这反过来又促进了过量的炎症细胞募集，从而加速动脉粥样硬化和AA。目的1将确定ATP 7A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，这些炎症粘附分子表达以Cu和Atox 1依赖的方式促进EC中的白细胞跨内皮迁移（TEM）。目的2将通过关注Atox 1的Cu依赖性转录因子功能以上调抑制VE-钙粘蛋白的miR 125 b以及增加ROS-NFkB的NADPH氧化酶的p47 phox来确定炎症诱导的细胞内累积的Cu如何促进内皮屏障功能障碍和ROS依赖性炎症反应。目的3将确定ATP 7A通过在体内以Cu和Atox 1依赖的方式调节血管通透性和炎症来对抗动脉粥样硬化和AA发展的保护作用。我们将使用ATP 7Amut、ATP 7A转基因Atox 1-/-小鼠;或与ApoE-/-小鼠杂交的诱导型EC特异性ATP 7A条件性缺陷小鼠，所述ApoE-/-小鼠具有用Cu螯合剂或LNA-抗-miR 125 b处理的高脂肪饮食。此外，将使用Cu成像分析（64 Cu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、体内活体显微镜检查、使用NFkB转基因报告小鼠的非侵入性生物发光成像。我们的研究将提供新的见解铜转运蛋白ATP 7A或核Atox 1作为治疗血管炎症性疾病，如动脉粥样硬化的潜在治疗靶点。

 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.