HEG1 in endothelial function and atherosclerosis

总结 动脉粥样硬化是一种慢性炎症性疾病，是心脏病发作和中风的基础。疾病 优先发生在暴露于受干扰血流（d-流）的动脉区域，部分通过改变 流动敏感基因在寻找流动敏感的长非编码RNA时，我们鉴定了流动敏感的 玻璃心（HEG 1）基因作为潜在靶点。先前在斑马鱼和小鼠中使用HEG 1敲除的研究 已经证明了它在心血管发育和血管完整性中的关键作用，但它的作用和 在血管生物学和动脉粥样硬化中的作用机制还远不清楚。最近，我们产生了令人兴奋的 初步数据，包括使用小鼠部分颈动脉结扎（PCL）模型的单细胞RNAseq研究， 表明稳定流（s-流）增加HEG 1表达，扰动流（d-流）降低HEG 1表达。 流动）条件。人主动脉内皮细胞（HAECs）中的HEG 1敲低诱导炎症、屏障 功能障碍和内皮-间质转化（EndMT），关键的促动脉粥样硬化过程。HEG 1具有长 N-末端细胞外结构含有两个高度糖基化（Gly）结构域（电位流传感 结构域），含有高度保守的Cys簇的三个EGF样（EGFL）结构域（潜在的氧化还原敏感性， 流动传感域），跨膜（TM）域，和胞质C-末端（C-端信号转导） 域我们的初步结果表明，HEG 1 1）可以用HEG 1抗体拉动或剪切以诱导HEG 1 1）。 Ca++通量，和2）以Poldip 2依赖的方式对氧化还原敏感。基于这些令人兴奋的数据，我们 假设HEG 1蛋白是一种氧化还原敏感性机械传感器，介导了 稳定的血流，而由d-血流引起的HEG 1损失和功能障碍诱导内皮功能障碍，导致 动脉粥样硬化我们将在三个目标中测试这一假设：目标1将确定HEG 1在血流中的作用， 使用siRNA介导的敲低依赖性EC功能（炎症、EndMT和通透性），或 HEG 1的过表达（使用表达WT或3种截短突变体ΔGly、ΔEGFL或ΔTM+C-的AAV-HEG 1） 在HAEC和永生化小鼠主动脉EC（iMAEC）中。EC靶向的HEG 1缺失小鼠（HEG 1-EC-/-）将 不与AAV-HEG 1构建体一起使用或与AAV-HEG 1构建体一起使用，用于EC功能的体内验证。目标2将测试HEG 1是否 一种氧化还原敏感的机械传感器，通过关注细胞内钙的即时变化（秒），急性 信号蛋白的激活（秒-分钟），以及响应剪切应力的缓慢（>小时）细胞变化 或使用包被有HEG 1 mAb的磁珠的张力。对于这些研究，HAEC和iMAEC处理 与siHEG 1或目标1中所述的相同AAV 9-HEG 1构建体。目标3将决定作用 使用注射了AAV-PCSK 9的HEG 1-EC-/-小鼠的动脉粥样硬化中的HEG 1。我们将进一步测试是否转导 与AAV-HEG 1构建体联合使用可以预防HEG 1-EC-/-小鼠中的动脉粥样硬化。这些研究将确定， HEG 1通过充当氧化还原敏感性机械传感器介导稳定血流的动脉粥样硬化保护作用， 可能揭示动脉粥样硬化的新治疗策略。

SUMMARY Atherosclerosis is a chronic inflammatory disease that underlies heart attacks and stroke. The disease preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), in part by altering expression of flow-sensitive genes. While looking for flow-sensitive long non-coding RNAs, we identified the flow-sensitive heart of glass (HEG1) gene as a potential target. Previous studies using HEG1 knockouts in zebra fish and mice have demonstrated its critical role in cardiovascular development and vascular integrity, but its role and mechanisms of action in vascular biology and atherosclerosis are far from clear. Recently, we generated exciting preliminary data, including a single-cell RNAseq study using the mouse partial carotid ligation (PCL) model, demonstrating that HEG1 expression is increased by stable flow (s-flow) and decreased under disturbed flow (d- flow) conditions. HEG1 knockdown in human aortic endothelial cells (HAECs) induces inflammation, barrier dysfunction, and endothelial-mesenchymal transition (EndMT), key pro-atherogenic processes. HEG1 has a long N-terminal extracellular structure containing two highly glycosylated (Gly) domains (potential flow sensing domain), three EGF-like (EGFL) domains containing highly conserved Cys clusters (potential redox-sensitive, flow-sensing domain),a transmembrane (TM) domain, and the cytosolic C-terminal (C-term signal transduction) domain. Our preliminary results show that HEG1 1) can be pulled with a HEG1 antibody or sheared to induce Ca++ flux, and 2) is redox-sensitive in a Poldip2-dependent manner. Based on these exciting data, we hypothesize that HEG1 protein is a redox-sensitive mechanosensor, mediating the atheroprotective effects of stable flow, while HEG1 loss and malfunction by d-flow induces endothelial dysfunction leading to atherosclerosis. We will test this hypothesis in three aims: Aim 1 will determine the role of HEG1 in flow- dependent EC function (inflammation, EndMT, and permeability) using siRNA-mediated knockdown or overexpression of HEG1 (using AAV-HEG1 expressing WT or 3 truncation mutants ΔGly, ΔEGFL or ΔTM+C- term) in HAECs and immortalized mouse aortic ECs (iMAECs). EC-targeted HEG1-null mice (HEG1-EC-/-) will be used without or with the AAV-HEG1 constructs for in vivo validation of EC function. Aim 2 will test if HEG1 is a redox-sensitive mechanosensor by focusing on immediate changes (seconds) in intracellular calcium, acute activation (seconds-minutes) of signaling proteins, and slow (>hours) cell changes in response to shear stress or tensional force using magnetic beads coated with HEG1 mAb. For these studies, HAECs and iMAECs treated with siHEG1 or the same AAV9-HEG1 constructs described in Aim 1 will be used. Aim 3 will determine the role of HEG1 in atherosclerosis using HEG1-EC-/- mice injected with AAV-PCSK9. We will further test if transduction with the AAV-HEG1 constructs can prevent atherosclerosis in HEG1-EC-/- mice. These studies will define if HEG1 mediates the atheroprotective effects of stable flow by serving as a redox-sensitive mechanosensor and may reveal novel therapeutic strategies for atherosclerosis.