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

BackgroundAtherosclerosis 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. MethodsSeveral distinct Leiomodin1 mutant mouse models were generated by clustered regularly interspaced short palindromic repeats (CRISPR). Control (Lmod1WT) and VSMC-restricted Lmod1 knockout (Lmod1SMKO) 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 Lmod1WT and Lmod1SMKO 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 Lmod1WT and Lmod1SMKO mice were subjected to lipid loading with lentiviruses expressing wild-type Lmod1, a nucleation deficient Leiomodin1 (Lmod1ND), or a short hairpin RNA (shRNA) targeting Thrombospondin (Thbs1). ResultsUnder atherogenic conditions, Lmod1SMKO mice displayed unremarkable vessels in several organs but developed diffuse and occlusive coronary atherosclerosis. No such disease was observed in Lmod1WT mice. Time-course studies documented lipid insudation and VSMC foam cell formation in the coronary arteries of Lmod1SMKO 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 Lmod1SMKO mice, with a similar reduction in LMOD1, showed no CAD. Spatial metabolomics uncovered multiple lipid species within coronary atheromata of Lmod1SMKO 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 Lmod1SMKO VSMCs that was rescued by Lmod1WT, Lmod1ND, and Thbs1 shRNA. VSMC-restricted expression of Lmod1ND in mice resulted in negligible coronary atherosclerosis. ConclusionsUnder proatherogenic conditions, Lmod1SMKO 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 PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIVascular 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. C_LIO_LIA 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. C_LIO_LIThe coronary phenotype appears to be independent of LMOD1s actin nucleation activity, but VSMC lipid uptake is thrombospondin-dependent. C_LI What are the clinical implications?O_LILMOD1 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. C_LIO_LIThe rapidly manifesting CAD phenotype in Lmod1 knockout mice enables expedited testing of novel therapeutics to mitigate disease progression. C_LIO_LINew insight into LMOD1 pathobiology will help inform further SNV interrogation of the LMOD1 locus for CAD risk in patients. C_LI