Abstract 12668: Mutant Smooth Muscle α-Actin Predisposes to Atherosclerosis and Augments Cholesterol-Driven Phenotypic Modulation of Vascular Smooth Muscle Cells

Abstract

Heterozygous variants in ACTA2 (smooth muscle α-actin) predispose to thoracic aortic disease and early-onset coronary artery disease (CAD). Single cell RNA sequencing (scRNA-Seq) identified phenotypically modulated SMCs (mSMCs; de-differentiated SMCs with upregulation of macrophage and fibroblast markers) that appear with atherosclerotic plaque formation in the aortas of Apoe -/- mice. Importantly, this transition is dependent on Kruppel-like factor 4 (Klf4). We previously demonstrated that SMCs exposed to free cholesterol complexed to methyl-β-cyclodextrin (MBD-Chol) in vitro activate unfolded protein response (UPR) pathways upstream of Klf4 and assume a mSMC phenotype. Blocking the UPR Perk pathway abolishes modulation. We sought to determine how ACTA2 p.Arg149Cys causes early-onset CAD. Acta2 R149C/+ mice were generated using CRISPR/Cas9. Acta2 R149C/+ mice were crossed into Apoe -/- mice and fed a high fat diet for 3 months. Oil Red O staining and en face aortic analysis showed that Acta2 R149C/+ Apoe -/- aortas developed significantly more plaques than similarly treated Apoe -/- mice (p<0.01) with no difference in plasma lipid levels ( Fig. 1A ). ScRNA-Seq of the root and ascending aorta showed no novel SMC cluster, however mSMC clusters demonstrated increased expression of UPR markers and Klf4 in the Acta2 R149C/+ Apoe -/- aortas compared to Apoe -/- aortas. Strikingly, Acta2 R149C/+ SMCs upregulate modulation markers at much lower levels of cholesterol than WT SMCs due to activation of the UPR Perk-Klf4 by misfolded mutant SM α-actin ( Fig. 1B ). Importantly, blocking Perk abolishes the augmented phenotypic modulation in Acta2 R149C/+ SMCs. These findings identify a novel pathway driving atherosclerosis independent of lipid metabolism and confirm a key role of SMCs in atherosclerotic plaque formation.