Role of CEBPb in flow-dependent endothelial dysfunction and atherosclerosis

动脉粥样硬化是一种慢性炎症性疾病，多发生在暴露于 扰乱血流(d-flow)，而处于稳定血流(S-flow)区域的则受到保护。这些机制由 D-血流和S-血流对动脉粥样硬化形成的调节作用尚不清楚。要解决这一关键知识 GAP，我们最近使用小鼠部分颈动脉结扎(PCL)进行了scRNAseq和scATACseq分析 D-血流引起的动脉粥样硬化模型。研究表明，d-flow显著改变了内皮细胞。 转录组和表观基因组图谱，将它们重新编程为炎性、间充质(EndMT)和 免疫细胞样(EndICLT)表型，我们将其定义为“内皮重编程(EndRep)”。而当 炎症和EndMT的促动脉粥样硬化作用是公认的，EndICLT和EndMT的机制和作用 EndRep在动脉粥样硬化中的作用尚不清楚。我们通过重新分析我们的scRNAseq和 ScATACseq数据集作为一个可能调节EndRep的候选转录因子。我们的初步数据进一步 在体内和培养的HAECs中，d-flow均能促进CEBPB的表达。CEBPB生产了三个 不同的蛋白质亚型，LAP1，LAP2，和LIP，通过替代翻译。令人惊讶的是，CEBPB是 主要翻译为HAECs中的LIP蛋白。此外，d-flow刺激LIP的核定位。 HAECs，LIP的过度表达显著诱导EndRep(内皮炎症，EndMT，和 EndICLT)我们的蛋白质组学研究表明，LIP与PSMB9免疫蛋白酶体蛋白结合，而d-Flow- 增加PSMB9活性。此外，先前的研究表明，嘴唇会诱导癌症类型的代谢 重新编程。因此，我们假设d-flow刺激LIP的核表达，LIP结合到 PSMB9，导致EndRep和动脉粥样硬化。我们将通过三个目标来检验这一假设。目标一号将决定 D-flow对唇核表达的影响及其在EndRep中的作用。欧共体特有的五彩纸屑小鼠将用于 谱系追踪以验证EndICLT和EndRep的流动调节。Si-CEBPB或LIP处理人脐静脉内皮细胞 将使用带有EC特异性LIP过度表达(LIPEC-OE)或LIP缺陷(LIPDEF)的质粒和小鼠。一个 采用超声引导下将LIP导入LIPDEF小鼠左侧颈动脉的方法诱导EndRep。目标2 将决定LIP如何以ECs流依赖的方式诱导EndRep。ScRNAseq和scATACseq分析将 使用带有PCL的LIPEC-OE和LIPDEF小鼠进行研究，以识别LIP和血流调节基因/通路。 PSMB9活性的流动调节及其在LIP诱导的EndRep中的作用将在HAECs和小鼠中确定 使用siRNAs和PSMB9抑制剂。目标3将确定LIP在血流中动脉粥样硬化中的作用-以及PSMB9- 依赖的态度。LIPEC-OE和LIPDEF的动脉粥样硬化将在急性PCL和慢性模型中进行研究 应用AAV-PCSK9和西式饮食加或不加PSMB9抑制剂治疗。ScRNAseq和scATACseq 分析将在这些小鼠身上进行。人类冠状动脉CEBPB染色将确定其 病理生理学意义。这些研究可能揭示潜在的抗动脉粥样硬化治疗途径。

Atherosclerosis is a chronic inflammatory disease and preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow) while those in the stable flow (s-flow) regions are protected. The mechanisms by which d-flow and s-flow regulate atherogenesis are still not well-understood. To address this critical knowledge gap, we recently conducted a scRNAseq and a scATACseq assay using the mouse partial carotid ligation (PCL) model of atherosclerosis caused by d-flow. The study revealed that d-flow dramatically alters endothelial transcriptome and epigenomic profiles, reprogramming them into the inflammatory, mesenchymal (EndMT), and immune cell-like (EndICLT) phenotypes, which we defined as “endothelial reprogramming (EndRep)”. While the pro-atherogenic role of inflammation and EndMT is well-established, the mechanisms and role of EndICLT and EndRep in atherosclerosis are unknown. We identified CEBPb through a reanalysis of our scRNAseq and scATACseq datasets as a candidate transcription factor that could regulate EndRep. Our preliminary data further show that the expression of CEBPb increased by d-flow in vivo and cultured HAECs. CEBPb produces three different protein isoforms, LAP1, LAP2, and LIP, through alternative translation. Surprisingly, CEBPb is translated primarily as the LIP protein in HAECs. Further, d-flow stimulates the nuclear localization of LIP in HAECs, and overexpression of LIP dramatically induces EndRep (endothelial inflammation, EndMT, and EndICLT). Our proteomics study shows that LIP binds the PSMB9 immunoproteasome protein, and d-flow- increases the PSMB9 activity. Furthermore, prior studies showed that LIP induces cancer-type metabolic reprogramming. Therefore, we hypothesize that d-flow stimulates nuclear expression of LIP, which binds to PSMB9, leading to EndRep and atherosclerosis. We will test the hypothesis with three aims. Aim 1 will determine the effect of d-flow on LIP nuclear expression and its role in EndRep. EC-specific-Confetti mice will be used for lineage tracing to validate the flow regulation of EndICLT and EndRep. HAECs treated with si-CEBPb or LIP plasmid and mice with EC-specific LIP overexpression (LIPEC-OE) or LIP deficiency (LIPDEF) will be used. An ultrasound-guided method will be used to deliver LIP to the left carotid of LIPDEF mice to induce EndRep. Aim 2 will determine how LIP induces EndRep in ECs flow-dependent manner. scRNAseq & scATACseq assays will be conducted using the LIPEC-OE and LIPDEF mice with the PCL to identify LIP and flow-regulated genes/pathways. Flow-regulation of PSMB9 activity and its role in LIP-induced EndRep will be determined in HAECs and mice using siRNAs and PSMB9 inhibitors. Aim 3 will determine the role of LIP in atherosclerosis in a flow- and PSMB9- dependent manner. Atherosclerosis in LIPEC-OE and LIPDEF will be studied in the acute PCL and chronic model using AAV-PCSK9 and western diet with or without PSMB9 inhibitor treatment. scRNAseq and scATACseq analysis will be conducted in these mice. Human coronary staining for CEBPb will determine its pathophysiological significance. These studies could reveal potential anti-atherogenic therapeutic avenues.