Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

ABSTRACT Background Atherosclerosis is the primary underlying cause of coronary artery disease (CAD). Leiomodin1 is a vascular smooth muscle cell (VSMC)-restricted CAD risk gene whose role in coronary artery pathophysiology is unknown. Global loss of Leiomodin1 causes lethal neonatal visceral myopathy, requiring unique approaches for study in VSMCs. Methods Several distinct Leiomodin1 mutant mouse models were generated by clustered regularly interspaced short palindromic repeats (CRISPR). Control ( Lmod1 WT ) and VSMC-restricted Lmod1 knockout ( Lmod1 SMKO ) mice were subjected to various atherogenic regimens. Atherosclerosis and LMOD1 expression in mouse and human coronary arteries were assessed by histopathology and confocal immunofluorescence microscopy. Coronary arteries from Lmod1 WT and Lmod1 SMKO mice were analyzed with assorted stains and antibodies, immunogold lineage tracing, spatial metabolomics/transcriptomics, and single-cell RNA sequencing (scRNA-seq). Mouse aortic SMCs from Lmod1 WT and Lmod1 SMKO mice were subjected to lipid loading with lentiviruses expressing wild-type Lmod1 , a nucleation deficient Leiomodin1 ( Lmod1 ND ), or a short hairpin RNA (shRNA) targeting Thrombospondin ( Thbs1 ). Results Under atherogenic conditions, Lmod1 SMKO mice displayed unremarkable vessels in several organs but developed diffuse and occlusive coronary atherosclerosis. No such disease was observed in Lmod1 WT mice. Time-course studies documented lipid insudation and VSMC foam cell formation in the coronary arteries of Lmod1 SMKO mice as early as six days post-regimen. Immunogold lineage tracing demonstrated 46% of coronary plaque cells being of VSMC origin, with most showing evidence of lipid uptake. An intronic deletion of Lmod1 , containing a conserved region where the single nucleotide variant associated with CAD exists, showed attenuated LMOD1 expression; heterozygous Lmod1 SMKO mice, with a similar reduction in LMOD1, showed no CAD. Spatial metabolomics uncovered multiple lipid species within coronary atheromata of Lmod1 SMKO mice, and spatial/scRNA-seq of similar coronary lesions disclosed altered lipid pathways with a consistent elevation in Thbs1 . In vitro mechanistic studies revealed lipid accumulation in Lmod1 SMKO VSMCs that was rescued by Lmod1 WT , Lmod1 ND , and Thbs1 shRNA. VSMC-restricted expression of Lmod1 ND in mice resulted in negligible coronary atherosclerosis. Conclusions Under proatherogenic conditions, Lmod1 SMKO mice present with rapidly manifesting coronary atherosclerosis that appears to be independent of the actin nucleation function of LMOD1. Targeting Thbs1 represents a viable strategy to mitigate VSMC foam cell formation. Clinical Perspective What is new? Vascular smooth muscle cell (VSMC) loss of Leiomodin1 ( Lmod1 ) causes diffuse and occlusive coronary atherosclerosis in mice, with little or no such disease in other vascular beds. A novel immunogold lineage tracing assay shows VSMC migration to the intima as early as six days following an atherogenic regimen, and quantitative studies demonstrate that 46% of coronary plaque cells are of SMC origin. The coronary phenotype appears to be independent of LMOD1’s actin nucleation activity, but VSMC lipid uptake is thrombospondin-dependent. What are the clinical implications? LMOD1 is an annotated smooth muscle cell-restricted risk allele for human coronary artery disease (CAD), offering new insight into the role of smooth muscle cells in atherogenesis. The rapidly manifesting CAD phenotype in Lmod1 knockout mice enables expedited testing of novel therapeutics to mitigate disease progression. New insight into LMOD1 pathobiology will help inform further SNV interrogation of the LMOD1 locus for CAD risk in patients.