Sunday, June 1, 2025
6/1/2025
Role of CEBPb in flow-dependent endothelial dysfunction and atherosclerosis - 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.
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