HEG1 in endothelial function and atherosclerosis

HEG1在内皮功能和动脉粥样硬化中的作用

• 批准号: 10272942
• 负责人: Hanjoong Jo
• 金额: $ 67.8万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272942
• 关键词: ATAC-seq; Acute; Antibodies; Arteries; Atherosclerosis; Biology; Blood Vessels; Blood flow; C-terminal; CCM1 gene; Calcium; Cardiomegaly; Cardiovascular system; Carotid Arteries; Cause of Death; Cell physiology; Cells; Chromatin; Chronic; Coronary artery; Cytoplasmic Tail; Data; Dependence; Development; Disease; EGF gene; EGF-Like Domain; Endothelial Cells; Endothelium; Exposure to; Extracellular Structure; Functional disorder; Genes; Glass; Glycocalyx; Heart; Hour; Human; In Vitro; Inflammation; Inflammatory; Integral Membrane Protein; Intercellular Junctions; Knock-out; Knockout Mice; Ligation; Mediating; Mesenchymal; Modeling; Modification; Monoclonal Antibodies; Mus; Mutagenesis; Myocardial Infarction; N-terminal; Oxidation-Reduction; Permeability; Phenotype; Play; Prevention; Process; Proteins; Regulation; Role; Signal Transduction; Signaling Protein; Small Interfering RNA; Stains; Stroke; Structure; Testing; Transmembrane Domain; Ultrasonography; Untranslated RNA; Validation; Zebrafish; atheroprotective; base; cardiogenesis; cell immortalization; chronic inflammatory disease; clinically significant; endothelial dysfunction; in vivo; inhibitor/antagonist; knock-down; magnetic beads; mutant; new therapeutic target; novel; novel therapeutic intervention; overexpression; prevent; release of sequestered calcium ion into cytoplasm; response; sensor; shear stress; single-cell RNA sequencing; transcriptome sequencing; vector; western diet

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.

概括 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 优先发生在暴露于分布的血流（D-Flow）的动脉区域中，部分原因是 流动基因。在寻找流敏感长的非编码RNA时，我们确定了流敏感 玻璃（HEG1）基因的心脏作为潜在靶标。先前使用斑马鱼和小鼠中HEG1敲除的研究 已经在心血管发展和血管完整性中表现出了关键作用，但其作用和 血管生物学和动脉粥样硬化中的作用机制远非清晰。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连接（PCL）模型的单细胞RNASEQ研究 证明HEG1表达通过稳定的流量（S-Flow）增加并在受干扰的流动下减少（D- 流）条件。人主动脉内皮细胞（HAEC）中的HEG1敲低诱导感染，屏障 功能障碍和内皮 - 间质转变（ENDMT），关键的亲动力学过程。 HEG1有很长的 N末端的细胞外结构，其中包含两个高糖基化（gly）结构域（电势流传感器 域），三个EGF样（EGFL）域，其中包含高度组成的CYS簇（潜在的氧化还原敏感， 流动域），跨膜（TM）结构域和胞质C末端（C-Term信号转导） 领域。我们的初步结果表明HEG1 1）可以用HEG1抗体拉或剪切以诱导 Ca ++通量和2）以Poldip2依赖性方式对氧化还原敏感。基于这些令人兴奋的数据，我们 假设HEG1蛋白是一种对氧化还原敏感的机制，介导 稳定的流动，而D-Flow的HEG1损失和故障会诱导内皮功能障碍，导致 动脉粥样硬化。我们将在三个目标中检验这一假设：AIM 1将确定HEG1在流动中的作用 - 使用siRNA介导的敲低或 HEG1的过表达（使用表达WT或3截断突变体ΔGly，ΔEGFL或ΔTM+C-的AAV-HEG1的过表达 术语）在HAEC和永生的小鼠主动脉EC（IMAEC）中。 EC靶向的HEG1-NULL小鼠（HEG1-EC - / - ）将 在没有AAV-HEG1构建体的情况下用于体内验证EC功能。 AIM 2将测试HEG1是否 通过关注细胞内钙的立即变化（秒），一种对氧化还原敏感的机理使用者 信号蛋白的激活（秒）激活（几分钟），以及缓慢（>小时）的细胞变化，响应剪切应力 或使用涂有HEG1 mAb的磁珠的拉伸力。对于这些研究，HAEC和IMAEC治疗了 使用SIHEG1或AIM 1中描述的相同的AAV9-HEG1构建体。 AIM 3将决定角色 使用AAV-PCSK9注入的HEG1-EC - / - 小鼠在动脉粥样硬化中的HEG1的of。我们将进一步测试是否翻译 使用AAV-HEG1构建体可以防止HEG1-EC - / - 小鼠的动脉粥样硬化。这些研究将定义是否 HEG1通过用作氧化还原敏感的机械传感器和 可能揭示了动脉粥样硬化的新型治疗策略。