Role of Cu Transporter Proteins in Atherosclerosis

 描述(由申请人提供)： 动脉粥样硬化受炎症、内皮细胞屏障功能障碍和氧化应激的调节，是导致老兵主动脉瘤(AA)和死亡率的主要原因。铜是人体必需的营养物质，参与机体的正常功能，而过量的铜可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，细胞内铜的生物有效性受到铜转运ATPase(ATP7A)的严格控制，ATP7A从胞内铜伴侣Atox1中获得铜，然后将铜输送到包括EcSOD在内的分泌型铜酶，或将铜输出到胞外空间。我们的实验室报道，血管ATP7A-EcSOD轴对高血压和1型糖尿病的内皮功能障碍具有保护作用，当Atox1定位于细胞核时，它作为一种铜依赖的转录因子发挥作用。然而，ATP7A在动脉粥样硬化中的作用及其与核Atox1的关系还完全不清楚。初步数据表明，与ApoE-/-小鼠相比，降低铜输出功能的ATP7A突变(ATP7Amut)小鼠与ApoE-/-小鼠相比，加速了动脉粥样硬化病变和AA的炎性细胞和血管通透性过剩。相比之下，Atox1-/-/ApoE-/-小鼠的动脉粥样硬化病变显著减少，这表明ATP7A具有动脉粥样硬化保护作用，而Atox1具有致动脉粥样硬化作用。值得注意的是，ATP7Amut和Atox1-/-小鼠都显示出通过铜伴侣Atox1介导的铜酶赖氨酸氧化酶活性降低的相似程度，这不能解释ATP7Amut/ApoE-/-小鼠增强动脉粥样硬化或再生障碍性贫血。基于ATP7A缺失的内皮细胞的其他新的初步数据，我们将检验这一新的假设，即炎症下调铜出口蛋白ATP7A会增加细胞内铜，从而刺激“核Atox1”介导的EC屏障功能障碍和ROS依赖的炎症反应。这反过来又促进了过度的炎性细胞募集，从而加速了动脉粥样硬化和再生障碍性贫血。目的1明确ATP7A对炎症诱导的内皮屏障功能障碍和ROS依赖的炎性黏附分子表达的保护作用，这些作用有助于以铜和Atox1依赖的方式促进内皮细胞的白细胞跨内皮细胞迁移。目的2将通过重点研究Atox1的铜依赖转录因子功能，上调抑制VE-钙粘蛋白的miR125b和增加Ros-NFkB的NADPH氧化酶的p47Phox，来确定炎症诱导的细胞内蓄积的铜如何促进内皮屏障功能障碍和ROS依赖的炎症反应。目的3明确ATP7A在体内通过铜和Atox1依赖的方式调节血管通透性和炎症，对动脉粥样硬化和再生障碍性贫血的发展具有保护作用。我们将使用ATP7Amut、ATP7A转基因、Atox1-/-小鼠；或可诱导的EC特异性ATP7A条件缺陷小鼠与高脂饮食的ApoE-/-小鼠杂交，用铜螯合剂或LNA-anti-miR125b处理。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)和同步辐射X射线荧光显微镜(XFM))、活细胞成像、活体显微镜、使用NFkB转基因报告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.