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，并且优先发生在暴露于 受干扰的血流（D-流），而稳定流（S-流）区域中的血流受到保护。的机制 D-flow和S-flow调节动脉粥样硬化形成的机制仍然没有很好的理解。为了解决这个关键的知识 间隙，我们最近进行了scRNAseq和scATACseq测定使用小鼠部分颈动脉结扎（PCL） 由D-流引起的动脉粥样硬化模型。该研究表明，d-血流显著改变了内皮细胞的 转录组和表观基因组谱，将其重编程为炎症，间充质（EndMT）， 免疫细胞样（EndICLT）表型，我们将其定义为“内皮重编程（EndRep）"。而 炎症和EndMT的促动脉粥样硬化作用是公认的，EndICLT和 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鉴定了CEBPb， scATACseq数据集作为一个候选转录因子，可以调节EndRep。我们的初步数据进一步 结果显示，在体内和培养的HAECs中，d-flow增加了CEBPb的表达。CEBPb生产三个 不同的蛋白质亚型，LAP 1，LAP 2和LIP，通过替代翻译。令人惊讶的是，CEBPb 在HAEC中主要翻译为LIP蛋白。此外，d-flow刺激LIP的核定位， HAECs和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结合PSMB 9免疫蛋白酶体蛋白，而d-flow- 增加PSMB 9的活性。此外，先前的研究表明，LIP诱导癌症型代谢 重新编程因此，我们假设d-流刺激LIP的核表达，LIP结合到 PSMB 9，导致EndRep和动脉粥样硬化。我们将以三个目标来检验这个假设。目标1将决定 d-flow对LIP核表达的影响及其在EndRep中的作用EC特异性-Confetti小鼠将用于 谱系追踪以验证EndICLT和EndRep的流动调节。用Si-CEBPb或LIP处理的HAEC 质粒和具有EC特异性LIP过表达（LIPEC-OE）或LIP缺陷（LIPDEF）的小鼠。一个 将使用超声引导的方法将LIP递送至LIPDEF小鼠的左颈动脉以诱导EndRep。目的2 将确定LIP如何以EC流动依赖性方式诱导EndRep。scRNAseq和scATACseq测定将 使用具有PCL的LIPEC-OE和LIPDEF小鼠进行，以鉴定LIP和流动调节基因/途径。 将在HAEC和小鼠中确定PSMB 9活性的流动调节及其在LPS诱导的EndRep中的作用。 使用siRNA和PSMB 9抑制剂。目的3将确定LIP在动脉粥样硬化中的作用， 依赖的方式。将在急性PCL和慢性模型中研究LIPEC-OE和LIPDEF中的动脉粥样硬化 使用AAV-PCSK 9和西方饮食，用或不用PSMB 9抑制剂处理。scRNAseq和scATACseq 将在这些小鼠中进行分析。人冠状动脉CEBPb染色将确定其 病理生理学意义这些研究可以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.