The pluripotency factor OCT4 plays an athero-protective role in endothelial cells

摘要 动脉粥样硬化是一种慢性动脉壁疾病，在美国占所有死亡病例的40%。 尽管先前的研究提供了明确的证据表明内皮细胞(EC)激活和 动脉粥样硬化中的表型调节，对EC可塑性的关键调控机制知之甚少 会影响病变的生长和/或稳定性。最近，我们发现胚胎干细胞/iPS因子OCT4 在体细胞中被认为是沉默的，在平滑肌细胞(SMC)中起到动脉粥样硬化的保护作用。 通过调节SMC的表型转变。然后我们问OCT4是否也可能在调节 内皮细胞在动脉粥样硬化发展过程中的可塑性。我们使用一种新的EC特异性Oct4的初步结果 APOE-/-基因敲除小鼠模型显示EC中Oct4的缺失增加了动脉内的总脂质负荷 以及长期喂食高脂饮食的APOE-/-小鼠肺动脉内斑块的出现，表明 Oct4在EC中起着关键的功能作用。进一步了解EC衍生的分子机制 Oct4抑制脂质堆积我们对主动脉和肺进行了单细胞(Sc)rna-seq分析。 EC从EC-Oct4基因敲除和野生型APOE-/-小鼠中分离出来，饲喂西方饮食5周。差异基因 表达分析发现，在Oct4基因敲除EC中，405(主动脉)和250(肺)基因显著异常 与野生型EC相比，包括内皮细胞到间充质细胞相关基因的上调 转化(Endo-MT)、促炎和黏附分子，以及参与 异种生物和血红素代谢。此外，使用体内OCT4芯片-SEQ的结果，我们发现其中一个 在Oct4基因敲除的肺和主动脉EC中显著下调的基因ABCG2是OCT4的假定靶点。 ABCG2是一种三磷酸腺苷结合盒转运体，负责挤出有毒的外来物质，包括来自 细胞。以往的研究表明，EC来源的ABCG2在心脏修复中发挥着关键作用 小鼠心肌梗死及其对氧化应激和神经炎性反应的保护作用 通过抑制核因子-kB信号而影响大脑。然而，目前还没有关于ABCG2的潜在保护作用的数据。 EC在动脉粥样硬化发展过程中的作用。我们的总体假设是，多能因子OCT4发挥着 内皮细胞通过激活血管内皮细胞在动脉粥样硬化形成中的重要保护作用 守门人蛋白ABCG2，一种降低氧化应激和保护EC免受ROS影响的适应性机制。 诱导活化和炎症反应。目标1将检验OCT4起动脉粥样硬化作用的假设- 在早期和晚期通过阻止EC的激活和表型修饰发挥保护作用 动脉硬化。目的2将验证OCT4减弱EC激活和EC炎症的假设 至少部分是通过上调ABCG2来保护EC免受氧化应激的影响 细胞内血红素水平和ROS介导的依赖核因子-kB的EC炎症反应。这些研究将 导致确定治疗或预防动脉粥样硬化的新的细胞特异性治疗方法。

ABSTRACT Atherosclerosis is a chronic disease of the arterial wall which contributes to >40% of all deaths in the USA. Although previous studies provide clear evidence for the importance of endothelial cell (EC) activation and phenotypic modulation during atherosclerosis, little is known about key regulatory mechanisms for EC plasticity that influence lesion growth and/or stability. Recently, we found that the embryonic stem cell/iPS factor OCT4 that was believed to be silenced in somatic cells plays an athero-protective role in smooth muscle cells (SMC) by regulating SMC phenotypic transitions. We then asked if OCT4 might also play a key role in regulating plasticity of EC during atherosclerosis development. Our preliminary results using a novel EC-specific Oct4 knockout Apoe-/- mouse model demonstrated that loss of Oct4 in EC increases the total lipid burden within aortas and occurrence of plaques within pulmonary arteries of Apoe-/- mice fed a long-term high-fat diet, indicating that OCT4 plays a key functional role within EC. To further understand molecular mechanisms whereby EC-derived OCT4 inhibits the accumulation of lipids we performed a single cell (sc)RNA-seq analysis on the aortic and lung EC sorted from EC-Oct4 knockout and wild-type Apoe-/- mice fed a Western diet for five weeks. Differential gene expression analyses identified 405 (aorta), and 250 (lung) significantly dysregulated genes in Oct4 knockout EC as compared to wild type EC, including upregulation of genes involved into Endothelial-to-Mesenchymal transition (Endo-MT), pro-inflammatory and adhesion molecules, and down-regulation of genes involved into xenobiotic and heme metabolism. Also, using results from the in vivo OCT4 ChIP-seq, we found that one of the genes significantly down-regulated in the Oct4 knockout lung and aortic EC, Abcg2, is a putative target of OCT4. ABCG2 is an ATP-binding cassette transporter responsible for extruding toxic xenobiotics, including heme from cells. Previous studies demonstrated that EC-derived ABCG2 plays a pivotal role in cardiac repair after myocardial infarction and protective role against oxidative stress and neuroinflammatory response in mouse brains by inhibiting NF-kB-signaling. However, there is no data about the potential protective role of ABCG2 in EC during atherosclerosis development. Our overall hypothesis is that the pluripotency factor OCT4 plays a critical athero-protective role in EC during the development of atherosclerosis through activation of the gatekeeper protein ABCG2, an adaptive mechanism to decrease oxidative stress and protect EC from ROS- induced activation and inflammatory response. Aim 1 will test the hypothesis that OCT4 plays an athero- protective role by preventing EC from activation and phenotypic modification at the early and late stages of atherosclerosis. Aim 2 will test the hypothesis that OCT4 attenuates EC-activation and EC inflammatory response at least in part through up-regulating Abcg2 that protects EC from oxidative stress by decreasing intracellular heme levels and ROS-mediated NF-kB-dependent EC inflammatory response. These studies will lead to an identification of novel cell-specific therapeutic approaches for treating or preventing atherosclerosis.