Author ORCID Identifier

https://orcid.org/0000-0002-6456-8004

Date of Graduation

5-2022

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dianna Milewicz

Committee Member

Guangwei Du

Committee Member

Jeffrey A. Frost

Committee Member

Hyun-Eui Kim

Committee Member

Jun-ichi Abe

Abstract

Heterozygous variants in ACTA2 (smooth muscle (SM) α-actin) predispose to thoracic aortic aneurysms and dissections (TAAD) and early-onset coronary artery disease (CAD). The most common ACTA2 mutation is a genetic alteration of arginine 149 to a cysteine, ACTA2 p.Arg149Cys, which accounts for disease in 24% of all ACTA2 mutation carriers.(1) ACTA2 p.Arg149Cys mutation carriers present with either TAAD or CAD but rarely have both diseases. To identify the molecular mechanisms dictating whether an individual with ACTA2 p.Arg149Cys develops TAAD or CAD, CRISPR/Cas9 technology was used to generate the mutant mouse, Acta2R149C/+, in a C57BL6 background. Acta2R149C/+ mice did not develop thoracic aortic disease, despite decreased aortic contraction and disrupted SM α-actin filament formation in Acta2R149C/+ smooth muscle cells (SMCS). Biochemical characterization of the mutant actin revealed increased retention of the mutant SM α-actin in the chaperonin-containing t-complex polypeptide (CCT) folding complex, which was associated with decreased mutant actin in the cytosol. These data suggested that the increased retention of mutant SM α-actin in the CCT complex possibly minimized the effect of the mutation on TAAD and conversely increases the risk for CAD, mirroring the phenotypic divergence observed in mutation carriers.

Single cell RNA sequencing (scRNA-Seq) identified phenotypically modulated SMCs (mSMCs) characterized by upregulation of macrophage, fibroblast, and chrondrocyte markers that appear with atherosclerotic plaque formation in Apoe-/- mice aortas. This transition is dependent on Kruppel-like factor 4 (Klf4). We previously demonstrated that SMCs treated with free cholesterol complexed to methyl-β-cyclodextrin (MBD-Chol) in vitro induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR) pathways upstream of Klf4, and SMCs assume a mSMC phenotype. Blocking the UPR Perk pathway abolishes SMC modulation in vitro and deletion of Perk prevents up to 80% of atherosclerotic plaque burden in hyperlipidemic mice. We sought to determine how ACTA2 p.Arg149Cys causes early-onset CAD. Acta2R149C/+ mice were generated using CRISPR/Cas9. Acta2R149C/+ 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 Acta2R149C/+Apoe-/- aortas developed significantly more plaques than similarly treated Apoe-/- mice (p<0.01) with no difference in plasma lipid levels. 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 Acta2R149C/+Apoe-/- aortas compared to Apoe-/- aortas. Strikingly, Acta2R149C/+ SMCs upregulate modulation markers without exogenous cholesterol due to activation of the UPR Perk-Klf4 signaling pathway by misfolded mutant SM α-actin. The misfolded R149C SM α-actin was also found to activate heat shock factor 1 (HSF1) in Acta2R149C/+ SMCs, which increases endogenous cholesterol biosynthesis through increased levels and activity of HMG CoA reductase. The increased cellular cholesterol levels lead to ER stress, thus engaging an UPR and PERK activation and modulation in the absence of exogenous cholesterol. To confirm a role of Hsf1-induced HMG CoA reductase in the increased plaque burden and SMC modulation, HMG CoA reductase was inhibited with pravastatin and was found to reduce aortic plaque burden in Acta2R149C/+ Apoe-/- mice and block augmented modulation of Acta2R149C/+ SMCs, highlighting a novel mechanism by which a genetic alteration augments SMC modulation and predisposes to increased atherosclerotic burden in the absence of altered lipid levels.

Keywords

atherosclerosis, aorta, genetic, aortic aneurysm, aortic dissection, coronary artery disease, acta2, smooth muscle, vascular biology, cardiovascular

Available for download on Friday, April 28, 2023

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