The role of shear induced endothelial ZBTB46 in atherosclerosis

项目摘要 动脉粥样硬化是世界上发病和死亡的重要原因，导致以下疾病 冠状动脉疾病、外周动脉疾病和中风。已知动脉粥样硬化具有局灶性 倾向于扰动流（d-流）区域，而稳定层流（s-流）区域相对受到保护。 内皮细胞（EC）被d-流激活是动脉粥样硬化的早期步骤，并且已知暴露于 D-Flow显示较高水平的炎症、增殖、凋亡和衰老。我们最近的研究表明 ZBTB 46是内皮细胞中的一个流调节转录因子，被d-flow下调， EC中的过表达抑制增殖。虽然ZBTB 46在动脉粥样硬化中的作用尚不清楚，但我们的研究表明， 初步研究表明，ZBTB 46调节了EC中的许多基因，这些基因可能参与了 动脉粥样硬化，包括P21和SFRP 2。我们建议检验ZBTB 46降低的总体假设， EC中响应于d-流的表达导致EC基因表达的下游变化，包括 降低P21和SFRP 2基因表达，促进EC活化、凋亡和衰老，以及 从而导致动脉粥样硬化的局灶性倾向。本项目有三个具体目标：1） 确定ZBTB 46参与流量调节EC增殖的下游靶标。首先，结合损失 函数和函数增益方法以及各种流动条件，我们将确定P21和/或SFRP 2是否 负责ZBTB 46对S流下观察到的EC增殖的抑制作用。我们还将测试P21和 SFRP 2是ZBTB 46的直接或间接靶点。最后，为了确定ZBTB 46的其他潜在靶点， 我们将在表达ZBTB 46的EC中使用ChIP-seq，并使用数据来测试其他转录因子的作用。 ZBTB 46的潜在下游靶点可能在未来参与动脉粥样硬化。2)确定 流动调节ZBTB 46对EC凋亡、衰老和炎症的作用。我们将测试EC-ZBTB 46是否 表达影响EC功能的这些方面，这些方面在暴露于d-flow的atheroprone区域中可见，通过使用 siRNA和腺病毒介导的ZBTB 46在s-flow和d-flow条件下的联合过表达 与相关刺激如过氧化氢和TNF α。3)测定EC-ZBTB 46对 体内动脉粥样硬化。由于ZBTB 46也在已知影响细胞增殖的典型树突状细胞中表达， 为了治疗动脉粥样硬化，我们的团队已经开发了高脂血症EC靶向ZBTB 46-KO小鼠。用这些老鼠 在动脉粥样硬化的实验模型中，我们将确定EC中特异性ZBTB 46的丢失是否有助于动脉粥样硬化的发生。 血流通过促进EC凋亡、衰老和/或炎症介导对动脉粥样硬化的易感性。 成功完成我们的目标将有可能找到一种新的EC功能调节剂， 动脉粥样硬化，并可能确定动脉粥样硬化的新的潜在治疗靶点。

Project Summary Atherosclerosis is a significant cause of morbidity and mortality in the world, contributing to diseases such as coronary artery disease, peripheral arterial disease, and stroke. It is known that atherosclerosis has a focal propensity to areas of disturbed flow (d-flow) while areas of steady laminar flow (s-flow) are relatively protected. Endothelial cell (EC) activation by d-flow is an early step in atherosclerosis, and it is known that ECs exposed to d-flow show higher levels of inflammation, proliferation, apoptosis, and senescence. We have recently shown that ZBTB46 is a flow regulated transcription factor in ECs, is down-regulated by d-flow, and that its overexpression in ECs inhibits proliferation. While the role of ZBTB46 in atherosclerosis is not known, our preliminary studies suggest that ZBTB46 regulates a number of genes in ECs potentially involved in atherosclerosis, including P21 and SFRP2. We propose to test the overall hypothesis that decreased ZBTB46 expression in ECs in response to d-flow leads to downstream changes in EC gene expression including decreased P21 and SFRP2 gene expression, promoting EC activation, apoptosis, and senescence, and consequently a focal predisposition to atherosclerosis. We address three specific aims for this project: 1) Determine the downstream targets of ZBTB46 involved in flow regulated EC proliferation. First by combining loss of function and gain of function methods and various flow conditions, we will determine if P21 and/or SFRP2 are responsible for the inhibitory effect of ZBTB46 on EC proliferation seen under s-flow. We will also test if P21 and SFRP2 are direct or indirect targets of ZBTB46. Finally, to identify other potential targets of ZBTB46 as a transcription factor, we will use ChIP-seq in ECs expressing ZBTB46, and use the data to test the role of other potential downstream targets of ZBTB46 that could be involved in atherosclerosis in the future. 2) Determine the role of flow regulated ZBTB46 on EC apoptosis, senescence, and inflammation. We will test if EC-ZBTB46 expression affects these aspects of EC function which are seen in atheroprone areas exposed to d-flow, by using siRNA and adenovirus mediated overexpression of ZBTB46 under s-flow and d-flow conditions in conjunction with relevant stimuli such as hydrogen peroxide and TNFa. 3) Determine the effect of EC-ZBTB46 on atherosclerosis in vivo. As ZBTB46 is also expressed in classical dendritic cells which are known to affect atherosclerosis, our team has developed hyperlipidemic EC-targeted ZBTB46-KO mice. Using these mice in experimental models of atherosclerosis, we will determine if loss of ZBTB46 specifically in EC contributes to the flow mediated susceptibility to atherosclerosis by promoting EC apoptosis, senescence, and/or inflammation. Successful completion of our aims will potentially find a novel regulator of EC functions contributing to atherosclerosis and may identify novel potential therapeutic targets for atherosclerosis.