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率和死亡率的重要原因，导致以下疾病 冠状动脉疾病、外周动脉疾病和中风。众所周知，动脉粥样硬化有一个局部 倾向于扰动流区(d流)，而稳定层流区(S流)相对受到保护。 血管内皮细胞(EC)由d-flow激活是动脉粥样硬化的早期步骤，已知暴露于 D-Flow显示更高水平的炎症、增殖、细胞凋亡和衰老。我们最近展示了 ZBTB46是内皮细胞中一种流动调节的转录因子，受d-Flow下调，其 内皮细胞的过度表达抑制了细胞的增殖。虽然ZBTB46在动脉粥样硬化中的作用尚不清楚，但我们的 初步研究表明，ZBTB46调控内皮细胞中的一些基因，可能参与 动脉粥样硬化，包括P21和SFRP2。我们建议检验降低ZBTB46的总体假设 内皮细胞对d-flow的反应导致EC基因表达的下游变化，包括 降低P21和SFRP2基因表达，促进EC活化、凋亡和衰老； 因此，局灶性易患动脉粥样硬化。我们解决了该项目的三个具体目标：1) 确定ZBTB46参与流量调节EC增殖的下游靶点。首先通过合并损失 根据函数方法和各种流动条件的函数和增益，我们将确定P21和/或SFRP2是否 在S-Flow下观察到ZBTB46对EC增殖的抑制作用。我们还将测试P21和 SFRP2是ZBTB46的直接或间接靶点。最后，确定ZBTB46的其他潜在目标作为 转录因子，我们将使用CHIP-SEQ在ECs中表达ZBTB46，并用该数据来检测其他基因的作用 未来可能参与动脉粥样硬化的ZBTB46的潜在下游靶点。2)确定 血流调节的ZBTB46在内皮细胞凋亡、衰老和炎症中的作用我们将测试EC-ZBTB46 表达影响暴露于d-血流的动脉粥样硬化区域的EC功能的这些方面，通过使用 小干扰RNA和腺病毒介导的ZBTB46在S-Flow和D-Flow联合条件下的过表达 用过氧化氢和肿瘤坏死因子等相关刺激。3)确定EC-ZBTB46对 活体动脉粥样硬化。由于ZBTB46也在经典的树突状细胞中表达，已知这些树突状细胞会影响 在动脉粥样硬化方面，我们的团队已经培育出高脂血症EC靶向ZBTB46-KO小鼠。使用这些小鼠 在动脉粥样硬化的实验模型中，我们将确定ZBTB46在EC中的特异性丢失是否有助于 Flow通过促进内皮细胞的凋亡、衰老和/或炎症而介导动脉粥样硬化的易感性。 成功完成我们的目标可能会找到一个新的EC职能监管机构，有助于 并可能确定治疗动脉粥样硬化的新的潜在靶点。

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.