Abstract Body: Aim: The ETS transcription factor ERG supports endothelial function by repressing inflammation and maintaining endothelial cell (EC) identity in healthy vasculature. Loss of ERG induces endothelial to mesenchymal transition (EndMT) and exacerbates chronic inflammatory diseases. To investigate ERG function in atherosclerosis, we leveraged an EC-specific Erg knockout (Erg^EC-KO) in a PCSK9-overexpression murine atherosclerosis model.

Methods: Atherosclerosis was induced in 10-week-old Erg^EC-KO or control mice that had a Cre-inducible EC lineage tag (Cdh5^CreERT2; R26-CAG-LSL-Sun1-sfGFP) with AAV8-PCSK9 and 12 weeks of high-cholesterol diet (HCD, 1.25%). Plaque burden and morphology were characterized with Oil Red O, H&E, Movat, and immunofluorescence imaging. Aortic intimal cells were isolated with gentle digestion and profiled with single-cell RNA sequencing (scRNA-seq) in early and late plaque (4 and 12 weeks of HCD, n=6 mice/sample). Endothelial permeability was assessed with accumulation of IV TRITC-dextran (70 kDa) or DiI-LDL.

Results: Loss of ERG led to a 1.6-fold increase in aortic plaque burden and plaque formation at atheroprotective sites (greater curvature; p≤0.03; n=9-10). A 5.3-fold increase in EC lineage cells was observed in brachiocephalic artery plaques (p=0.0001; n=6-15), which underwent EndMT (31.5-fold increase in GFP⁺ACTA2⁺ cells, p=0.02, n=3-5). Aortic intimal scRNA-seq in early plaque revealed increased proportions of inflammatory macrophages and foam cells in Erg^EC-KO mice (6.9 and 2.7-fold). Erg^EC-KO ECs showed a marked loss of endothelial identity (67 transcripts including Nos3, Tek, Vwf, p=1.42e^-91) that preceded the gain of mesenchymal traits in late plaque (66 transcripts including Col8a1, Fbln5, Vim, p=1.19e^-96). Pathway and ligand-receptor analyses highlighted alterations in cell adhesion (Cadm1, Gja5) and migration programs (Sdc4), which were confirmed with increased endothelial permeability prior to atherosclerosis (TRITC-dextran, p=0.0007, n=8) and increased monocyte LDL uptake in early plaque (p=0.04, n=7-9).

Conclusion: Endothelial Erg KO increases plaque burden in murine atherosclerosis. Loss of ERG promotes EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior, which is associated with increased permeability and lipid accumulation. These findings implicate ERG dysfunction as a novel mechanism by which EC identity and resilience are compromised in atherosclerotic cardiovascular disease.