Abstract 3067: Chronic Knockout Of The ETS Transcription Factor ERG Promotes Loss Of Endothelial Cell Identity And Endothelial Cell Dysfunction In An Aortic Endothelial Cell Model Relevant For Atherosclerosis

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Background: The ETS transcription factor ERG is a key endothelial cell (EC) transcription factor. ERG represses vascular inflammation and is downregulated in chronic inflammatory diseases (e.g. liver fibrogenesis, atherosclerosis). Previously, transient ERG knock-down has been associated with endothelial-mesenchymal transition (EndMT) and EC dysfunction; however, the mechanisms underlying EndMT and the EC dysfunction associated with chronic ERG loss have not been elucidated in an atherosclerosis-relevant model. We hypothesize that chronic ERG knockout (KO) in telomerase-immortalized human aortic ECs (teloHAECs) induces EndMT and contributes to EC dysfunction underlying vascular dysregulation in atherosclerosis. Methods: TeloHAECs with CRIPSR/Cas9-mediated deletion of ERG were used. Chromatin accessibility and gene expression in ERG KO and wildtype (WT) teloHAECs were assessed using ATAC-seq (n=2) and RNA-seq (n=5-6). TOBIAS and diffTF were used to identify enriched motifs by footprinting (P<0.05) and to profile TF activity (FDR<0.05), respectively. ERG-mediated EC dysfunction was investigated using immunofluorescence (IF; n=3), western blot (WB; n=3), and a timelapse migration assay (n=3). Results: There were ~ 21,000 differentially accessible chromatin peaks between ERG KO and WT ECs. Integrated analysis of chromatin accessibility and gene expression demonstrated increased activity of TGFβ-SMAD and IL-1β signaling constituents and classical EndMT transcription factors. Loss of ERG downregulated EC genes (e.g., CLDN5 , TIE1 ) and protein expression (e.g., CDH5, vWF) and upregulated mesenchymal genes (e.g., ACTA2 , FSP1 ) and protein distribution (e.g., FN1, COL1A1). Investigation of EC-dysfunction phenotypes revealed increased migration and proliferation in ERG KO teloHAEC - with the percentage of Ki67 positive ECs increasing from 14% to 49% (P<0.05, n=3). Conclusion: ERG KO in cultured aortic ECs markedly impacts chromatin accessibility and gene expression and induces EC identity loss, migration, and proliferation, providing mechanistic insight into the role of ERG in vascular dysfunction in atherosclerosis.