Twist1 Promotes Endothelial Phenotypic Transition and Unstable Plaque Phenotype During Atherosclerosis

Comprehensive investigation of endothelial cell (EC) dysfunction during atherosclerosis with single-cell omics has resulted in the proposal that ECs undergo multiple alternative cell fate decisions during disease, but lack of lineage tracing or spatial localization complicates interpretation of these data. TWIST1, a causal gene for multiple atherosclerotic vascular diseases, is activated with low shear stress in ECs, and EC-Twist1 knockout results in reduced atherosclerosis. However, it remains unclear how Twist1 affects EC phenotype and plaque biology.

We performed EC lineage tracing, in situ analysis, and scRNA-Seq in ApoE^-/- mice, both before disease and after 16 weeks of high-fat diet. We also performed these studies with 2 mouse models of EC Twist1 deletion. We overexpressed TWIST1 in human coronary artery ECs exposed to different flow conditions, followed by bulk RNA-seq. Human scRNA-Seq data were used to validate key findings in the mouse model.

We found that EC phenotypic modulation during atherosclerosis is characterized by both proinflammatory and endothelial-to-mesenchymal transition gene programs, occurring simultaneously along a single-cell fate transition. Human scRNA-Seq data validated a similar endothelial-to-mesenchymal transition during disease. We found that the commonly used Twist1 conditional allele is hypomorphic, leading to reduced Twist1 expression in multiple cell types. Using a mouse model of EC-specific Twist1 deletion, we found reduced EC phenotypic modulation, decreased lesion size, and a more stable lesion phenotype. Integration of TWIST1 overexpression in human coronary artery ECs with the mouse scRNA-seq data identified specific TWIST1-induced targets including CXCL12 and E-selectin during EC phenotypic modulation.

Our study revealed important aspects of EC phenotypic modulation during atherosclerosis, unifying disparate observations in the field. We identified key cellular and molecular mechanisms underlying a top risk locus for multiple vascular diseases, highlighting the promotion of inflammatory endothelial-to-mesenchymal transition by TWIST1 as a key driver of disease risk.