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 pr...