LMO7 regulates smooth muscle cell cholesterol esterification to influence plaque stability

LMO7 regulates smooth muscle cell cholesterol esterification to influence plaque stability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LMO7 regulates smooth muscle cell cholesterol esterification to influence plaque stability Yi Xie, Hanna Winter, Noemi Rotlan, Allison Ostriker, Raja Chakraborty, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1359588/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Smooth muscle cells (SMC) can undergo distinct fate transitions that influence atherosclerotic plaque phenotype: SMC make up the protective fibrous cap that prevents plaque rupture, but can also transdifferentiate to a destabilizing pro-inflammatory phenotype. We found that LMO7, a scaffolding protein that modulates SMC phenotype, is induced in atherosclerosis. SMC LMO7 deletion produced a paradoxical phenotype with enhanced indices of plaque stability despite greater lipid content. We determined that LMO7 promotes SOAT1 ubiquitination and degradation. LMO7 depletion thus favored cholesterol esterification and “safe” storage, relieving ER stress and subsequent induction of KLF4 and SMC transdifferentiation. Immunostaining and single cell sequencing confirmed that SMC LMO7 depletion enhanced cap stabilizing fate transitions while substantially reducing inflammatory transdifferentiation. Similarly, LMO7 was induced in human atherosclerosis, but at lower levels in stable versus ruptured plaques. These findings reveal that SMC cholesterol esterification, rather than total cholesterol content, influences plaque composition and stability. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Atherosclerosis is the main cause of morbidity and mortality in the US and Europe. Despite widespread use of lipid lowering agents that help slow the progression, complications of atherosclerotic plaque rupture, including myocardial infarction and stroke, remain major challenges 1 . Accordingly, plaque stability is a key factor in prognosis 2, 3 . New approaches to reduce the incidence of thrombotic events would significantly improve morbidity and mortality consequent to atherosclerosis. Cholesterol accumulation in the vessel wall leads to plaque formation. Macrophages that engulf the excess cholesterol become foam cells and contribute to a pro-inflammatory milleu 4 . Excessive intracellular cholesterol promotes apoptosis and formation of a necrotic core in the lesions. Smooth muscle cells (SMC) form a protective fibrous cap, providing an essential stabilizing function to separate the pro-thrombotic necrotic core from the lumen 5 . Medial SMC migrate from the plaque shoulder to cover the plaque core at early stages 6 . These SMC secrete copious extracellular matrix (ECM) to form the fibrous cap. Because the thickness and stability of the fibrous cap predicts the vulnerability to rupture 7 , SMC are thought to play a protective role in plaques. Recent fate mapping studies have expanded our understanding of the heterogeneity and roles of SMC-derived populations in atherosclerosis. Notably, SMC transdifferentiation to a macrophage-like state is now appreciated to be a major source of foam cells in mouse and human lesions, with 50% of foam cells derived from SMC in human coronary lesions in one analysis 8-10 . The finding that SMC undergo phenotypic modulation to macrophage-like cells in hyperlipidemic conditions 11 suggests a new paradigm where SMC-derived cells can play distinct roles within lesions: the macrophage-like SMC can contribute to the inflammatory lesion core, whereas matrix synthesizing SMC-derived cells act as a protective component in the fibrous cap. Single cell RNA sequencing (scRNAseq) analyses have demonstrated that SMC can give rise to distinct populations 12 including inflammatory, matrix-producing and osteogenic subtypes 13 . Several studies have recently shown that Kruppel Like Factor 4 (KLF4), a transcription factor implicated in pluripotency, is a key factor promoting SMC dedifferentiation 9, 13, 14 . KLF4 has been shown to play a fundamental role in the SMC phenotypic switching, including in the transition to the pro-inflammatory macrophage-like state 9 . Although expressed in many hematopoietic cells including macrophages, LGALS3 has been identified as a KLF4-regulated marker of phenotypically modulated SMC 13 . LGALS3 expression may represent an early-stage transition that subsequently gives rise to multiple distinct phenotypes during atherosclerosis progression 13 . Early signals through which hypercholesterolemia influences SMC transcription factors and phenotypic switching in atherosclerosis are still poorly understood. We have recently identified LIM domain only protein 7 (LMO7) as a potent modulator of SMC phenotype 15 . LMO7 is a scaffolding protein containing multiple protein-protein interaction domains. LMO7 loss of function in SMC enhances cell proliferation and ECM deposition following vascular injury 15 . Given the roles for SMC phenotypic modulation, proliferation, and ECM synthesis in atherosclerosis, we hypothesized that LMO7 loss of function may lead to more stable lesions with a thicker fibrous cap. Methods Mice. Lmo7 fl/fl mice on a C57BL/Swiss mixed background were a gift from Dr. Ju Chen (UCSD). Myh11 -CreER T 2 and Apoe −/− mice were purchased from Jackson Laboratory. Mouse strain with Gt(ROSA26)-ZsGreen reporter was a generous gift of Dr. Daniel Greif (Yale). For lineage tracing studies, the Gt(ROSA26)-ZsGreen reporter strain was crossed with Myh11-CreER T 2 mice on the Apoe −/− background. Recombination was induced by injecting 6 week old mice with 1 mg/kg tamoxifen for 5 days, followed by 5 day washout. Inducible, smooth muscle-specific Lmo7 knockout mice were generated by crossing the Lmo7 fl/fl line with the above-mentioned ROSA26-ZsGreen / Myh11-CreER T2 / Apoe −/− reporter line. Genotyping of all the mice was performed by PCR. Mice were housed in pathogen-free conditions at Yale University. All experiments were approved by the institutional animal care and use committee of Yale University. Human tissue studies. Human carotid arteries of the Munich Vascular Biobank 16 were taken during carotid endarterectomy (CEA) followed by fixation for 48 hours in 2% zinc-formalin and paraffin embedding. Per specimen, four 5µm thick sections were stained with hematoxylin and eosin (HE) and Elastin van Gieson’s staining. Based on the histochemical stains, plaques were characterized according to their features as stable or unstable/ruptured following the American Heart Association (AHA) classification after Stary et al. 17 and Redgrave et al. 18 to determine critical fibrous thickness. Plaque and the non-diseased control portion of the carotid biopsy (as illustrated in Fig. 6 a) were separated and lysed for RNA analysis. Animal experiments. After tamoxifen injection and washout, mice were fed with high fat diet (HFD, 40% kcal%, 1.25% cholesterol, Research Diets D12108C) starting at 8 weeks of age for 0–12 weeks as indicated. For blood lipid assays, mice were starved overnight and anesthetized with 100mg/kg ketamine and 10mg/kg xylazine to collect blood by retro-orbital bleeding before and after HFD feeding. The blood samples were centrifuged at 10,000 rpm for 10 min at R.T. and the plasma (upper phase) was collected for total cholesterol and triglyceride measurement using Cholesterol Assay Kit (Abcam, ab65390) and L-Type Triglyceride M Enzyme Kit (Fujifilm), respectively. Mice were sacrificed at indicated time points post HFD feeding and were immediately perfused first with sodium nitroprusside (SNP) in PBS then 4% paraformaldehyde (PFA). The aortic root (sinus) and the whole aorta as well as brachiocephalic artery (BCA), left common carotid artery, and left subclavian artery branches were dissected and immersed in cold 4% PFA for overnight followed by dehydration in 30% sucrose for another 24 hours. Then the vessels were embedded in OCT compound (Tissue Tek, Elkhart, IN) for cryosectioning. Mouse single cell RNA sequencing sample preparation and sequencing. After 12 weeks HFD, mice were sacrificed and perfused with PBS. The aortic root, aortic arch and the three major branches, including brachiocephalic artery, left common carotid artery and left subclavian artery were dissected. Tissues were briefly digested in adventitia stripping mix (175U/ml Collagenase II and 1.25U/ml elastase in HBSS) at 37°C for 10 min, and adventitia was peeled off. Then the vessels were cut into 1-2mm long rings and digested in digestion mix (600U/ml collagenase II and 2.5U/ml elastase in HBSS) for 1 h at 37°C with shaking at 700 rpm. Cell suspensions were filtered through 70 µm cell strainer and pelleted at 500 x g for 5 min at 4°C. Cells were then stained in viability staining buffer (0.01 µg/ml DAPI in PBS + 2% FBS) for 5 min at 4°C and maintained in this buffer for cell sorting. DAPI negative Cells were sorted using FACSAria II (BD Biosciences) into DMEM + 10% FBS. Cell numbers were counted and ~ 10,000 cells per sample were used immediately for single-cell library preparation at Yale Keck Biotechnology Resource Laboratory. Cells were loaded into a 10x Genomics microfluidics chip and encapsulated with barcoded oligo-dT-containing gel beads using the 10x Genomics Chromium controller according to the manufacturer’s instructions. Single-cell 3’ V3.1 libraries were then constructed according to the manufacturer’s instructions. Libraries from control and Lmo7 iΔSM samples were multiplexed into one lane and sequenced with setting of paired-end 150bp on an Illumina HiSeq 4000 instrument. Analysis of scRNASeq data. Fastq files from each mouse genotype were aligned to the reference genome containing ZsGreen1 gene individually using CellRanger Software (10x Genomics). The dataset was then analyzed using the Partek Flow platform. The dataset was trimmed of cells with read count fewer than 600 or more than 15,000, cells expressing fewer than 500 genes or more than 4,000 genes, and cells with more than 10% mitochondrial counts. The gene expression values then underwent library-size normalization, where raw gene counts from each cell were normalized relative to the total number of read counts present in that cell. Principal component analysis (PCA) was used for dimensionality reduction, followed by cell clustering using a graph-based clustering approach. t-distributed stochastic neighbor embedding (t-SNE) was then used for two-dimensional visualization of the resulting clusters. Contractile SMC, modulated SMC, fibromyocyte, macrophage, endothelial cell, T cell, neutrophil and fibroblast populations were classified based on the gene expression profile. Cells within the specific classification were combined for further analysis for differential gene expression analysis between control and Lmo7 iΔSM groups. Histological staining. Haemotoxylin & Eosin (H&E) staining was performed at Yale Pathology Tissue Service using standard protocols. For Picro-Sirius Red (PSR) staining, tissue sections were washed with water to remove OCT and immersed in staining solution (0.1% Direct Red 80 (Sigma, #365548) in a saturated aqueous solution of picric acid) for 1h. The sections were then washed with two changes of 0.5% acetic acid solution, dehydrated with three changes of 100% ethanol, cleared with Histoclear (National Diagnostics, HS-200) and mounted with DPX mounting media (Electron Microscopy Sciences). For en face Oil Red O (ORO) staining of aortas, the dissected aorta was cut longitudinally and pinned on a silicon plate. The aorta was fixed with 4% PFA for 1h at RT, briefly rinsed with 78% methanol and immersed in the ORO staining solution (0.2% w/v ORO solution in methanol mixed with 1M NaOH at 7:2 ratio) for 1h. Then the aorta was washed with 78% methanol twice and mounted with OCT medium between two microscope slides. Tissue sections and cell ORO staining follows the same protocol as above. Micrographs of ORO staining was taken with a Nikon Eclipse 80i microscope using NIS Elements software. For filipin staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at home temperature, followed by incubation with 1.5 mg/ml glycine in PBS for 10 min at room temperature (R.T) to quench the paraformaldehyde. Cells were then stained with filipin working solution (0.05 mg/ml filipin (Sigma, F-9765) in PBS + 10% FBS) for 2 hrs at R.T. Cells were rinsed with PBS twice and were imaged on a Nikon Eclipse Ti Confocal Microscope. Immunohistochemistry and immunofluorescence. For Immunohistochemistry, cryosections were air-dried completely and fixed/permeabilized in cold acetone for 10 min. OCT was removed by distilled H 2 O washing. For TGF-β (MAB1835, clone #1D11, R&D Systems, 1: 25), staining was performed using Mouse-on-Mouse (M.O.M.™, BMK-2202, Vector Laboratories) kit and developed using DAB kit (SK-4100, Vector Laboratories) per manufacturer’s protocol. For Ter-119 (14-5921-81, Invitrogen, 1: 100), staining was performed using goat anti-rat HRP (31470, Invitrogen, 1: 500) and developed using DAB kit, followed by Methyl Green counterstain. The sections were mounted in DPX mounting media (Electron Microscopy Sciences) and imaged on a Nikon Eclipse 80i microscope using NIS Elements software. For Immunofluorescence, cryosections were air-dried completely and washed with distilled H 2 O to remove OCT. The sections were then permeabilized/blocked in blocking buffer (4% goat normal serum, 1% BSA and 0.5% Triton X-100 in PBS) at room temperature (R.T.) for 1 hr, incubated with primary antibodies at 4°C for 20 hrs, and secondary antibodies at R.T. for 1 hr in blocking buffer. Finally, the sections were stained with DAPI for nuclei at R.T. for 5 min and mounted with Vectashield Mounting Media (Vector Laboratories). The following primary antibodies were used: LMO7 (sc-98422x, Santa Cruz, 1: 100), LGALS3 (CL8942AP, Cedarlane, 1: 100), ACTA2 (sc-32251 AF546, Alexa 546-conjucated, Santa Cruz, 1: 300), CD68 (MCA1957, Bio-Rad, 1: 100), Ki67 (MA5-14520, Thermo, 1: 200), KLF4 (#4038, Cell Signaling, 1: 100), p-SMAD3 (S423/425) (ab52903, Abcam, 1: 100), ATF4 (#11815, Cell Signaling, 1: 100), SOAT1 (ab39327, Abcam, 1: 100). Appropriate fluorescent secondary antibodies were purchased from Molecular Probes and diluted 1: 300 in blocking buffer. For cleaved Caspase 3 (cCasp3) staining (#9664, Cell Signaling, 1: 150), VECTASTAIN® Elite ABC kit (PK-6101, Vector Laboratories) was used and developed by Tyramide signal amplification (NEL705A001KT, Akoya Biosciences) steps. Micrographs were captured on a Nikon Eclipse Ti Confocal Microscope using Volocity software. Primary tissue or cell isolation and cell culture. To isolate primary SMC for cell culture mice were sacrificed, perfused with PBS, and the descending aortas were isolated by microdissection. The vessels were briefly digested in adventitia stripping mix (175U/ml Collagenase II and 1.25U/ml elastase in HBSS) for 10 min, and the adventitia was peeled off. Then the vessels were cut into 1-2mm long rings and digested in digestion mix (400U/ml collagenase II, 2.5U/ml elastase and 0.2mg/ml soybean trypsin inhibitor in HBSS) for 1hr at 37°C with shaking at 700rpm. The cell suspension was centrifuged at 20 X g for 5 min, washed once with DMEM + 20% FBS + Penicillin/Streptomycin (Pen/Strep) and plated in culture dish in the same media. Cells were subcultured 1: 3 ~ 4 upon becoming 90% confluent. Primary human aortic SMCs were purchased from Lonza at passage 3 and propagated in M199 media with 10% FBS, 2.7 ng/ml EGF, 2 ng/ml FGF and Pen/Strep (complete media). Cells were used for experiments between passages 4–7. Drugs used in the experiments: Cycloheximide (Sigma, C7698-1G), MG132 (Selleckchem, S2619), methyl-β-cyclodextrin cholesterol (Sigma, C4951), K-604 (Biovision, B-2926). Transient transfection of siRNA. Transient transfection of small interfering RNA (siRNA) in hCASMC was performed using Lipofectamine® RNAiMAX (Life Technologies). Cells were plated the day before transfection to reach a confluency of 70%. Transfection reagent and siRNA were mixed in OPTI-MEM (Gibco) and added to the cells as per the Lipofectamine instructions. M199 (human ASMC) or DMEM (mouse ASMC) media with 20% FBS was added to the cells after 6 hours. After another 18 hours, the media was changed to M199 complete media (human ASMC) or DMEM + 20% FBS (mouse ASMC). siRNA for LMO7 or SOAT1 and nonsilencing siRNAs (siControl) were purchased from Invitrogen. Cholesterol biochemical assay. Mouse ASMCs were starved for 16 hrs and treated with 10 ug/ml cholesterol for 72 hrs. Cells were washed with PBS and scraped in 1ml PBS. Cells were spun down at 200 X g for 5 min. 1/10 of the cells were lysed in RIPA buffer and used for protein quantification and the remainder were used for cholesterol assay. Cholesterol was extracted from these cells with 200 ul of chloroform: isopropanol: NP40 (7:11:0.1). The pellet was spun at 15,000xg for 10 min and the supernatant containing lipid was transferred to a clean glass bottle and dried under nitrogen gas. Dried lipid was dissolved with 200 ul 1X Reaction Buffer provided in the Amplex® Red Cholesterol Assay Kit (Invitrogen, A12216). Total cholesterol (TC) was measured using the kit with esterase in the reaction, while free cholesterol (FC) was measured without esterase. Cholesterol ester (CE) level was calculated by subtracting FC from TC. The mass of each cholesterol fraction was normalized to the mass of protein of the corresponding sample. Cell lysis and western blotting. Cells were briefly washed with PBS and scraped in cold 1X RIPA buffer supplemented with Protease inhibitor cocktail and PhosSTOP (Roche) on ice. Cell lysates were centrifuged at 20,000 x g for 3 min at 4°C and the supernatant was mixed with loading buffer and heated at 95°C for 5 min. Equal amounts of protein from different samples were separated on SDS-PAGE gel, transferred onto PVDF membrane and immunoblotted with primary antibodies at 4°C for 16 hrs and secondary antibodies (Pierce) at R.T. for 1 h. Blots were developed using SuperSignal® Chemiluminescence Substrate (Pierce). Digital images were taken with Gel Doc™ XR + System using ImageLab software. Primary antibodies used: LMO7 for human samples (ab224113, Abcam, 1: 1000), LMO7 for mouse samples (sc-98422x, Santa Cruz, 1: 2000), LGALS3 (CL8942AP, Cedarlane, 1: 1000), MYH11 (M7786, Sigma, 1:1000), KLF4 (ab129473, Abcam, 1: 1000), ATF4 (#11815, Cell Signaling, 1: 1000), CHOP (#2895, Cell Signaling, 1: 500), SOAT1 (ab39327, Abcam, 1: 1000), Ubiquitin (13724, Cayman, 1: 1000), β-Tubulin (sc-9104, Santa Cruz, 1: 500), GAPDH (#2118, Cell Signaling, 1: 2000), β-actin (#3700, Cell Signaling, 1: 1000). Secondary antibodies conjugated with HRP were purchased from Thermo Fisher and used at 1: 3000 dilution. Densitometry of bands was quantitated using ImageJ. Immunoprecipitation. Cells were washed and scraped as above except using 1X Cell Lysis Buffer (Cell Signaling). After centrifugation, equal amount of cell lysate from each sample was mixed with primary antibodies with gentle rotation at 4°C for 16 hrs. Then magnetic beads (Bio-Rad) were added and rotated for another 2 hrs to pull down antibody-protein complexes. Beads were washed with Cell Lysis Buffer twice and heated in the presence of loading buffer at 95°C for 5 min. Primary antibodies used for IP: SOAT1 (ab39327, Abcam, 1: 100), normal Rabbit IgG (#2729, Cell Signaling). Western analysis was the same as above. qRT-PCR. Cells were briefly washed with PBS and scraped in TRIzol™ (Invitrogen) and RNA was extracted and purified using Direct-zol™ RNA Miniprep Plus kit (ZYMO Research). cDNA was reverse transcribed using 5X All-In-One RT MasterMix (G486, ABM) with equal amount of RNA from each sample. cDNA was then diluted 1: 10 and used for quantitative real-time PCR analysis (Bio-Rad CFX96 Real-Time System) using BrightGreen qPCR MasterMix (MasterMix-S, ABM). For miRNA analysis, miRNAs were purified using miRNeasy Kit (217004, Qiagen) and reverse transcribed using miRNA cDNA Synthesis Kit (G902, ABM), followed by real-time qPCR analysis using BrightGreen miRNA qPCR MasterMix (MasterMix-mS, ABM). qPCR was analyzed using the delta delta CT method. All samples were run in duplicate, and signals were normalized to the indicated housekeeping genes/microRNAs. Statistical analysis. Values are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using Graph-Pad (Prism7). Comparisons between two samples were performed using the nonparametric Mann-Whitney U test. Data sets with three or more groups were analyzed by 2-way ANOVA followed by Holm-Sidak multiple comparisons testing. Comparisons between two curves were performed by Nonlinear Regression with Extra sum-of-squares F test. P-value smaller than 0.05 was considered significant. Results LMO7 is induced in mouse atherosclerosis We examined LMO7 expression in SMC and other cells during murine atherosclerosis development. We generated a smooth muscle-specific, tamoxifen-inducible, ZsGreen reporter mouse in the Apoe -/- background ( Myh11_CreER T2 ; Gt(ROSA26)-ZsGreen; Apoe -/- ). Tamoxifen treatment results in fluorescent labeling of SMCs and their daughter cells (Suppl Fig. 1a). We subjected these mice to high fat diet (HFD) feeding, harvested the brachiocephalic artery (BCA) at multiple time points, and immunostained for LMO7 and LGALS3 13 . LMO7 expression was relatively low prior to HFD feeding but was greatly induced in the plaque by HFD, although not in medial layer (Suppl Fig. 1a). We found that cholesterol loading of cultured SMC was sufficient to induce robust expression of LMO7 in vitro (Suppl Fig. 1b). LMO7 staining was found in ZsGreen + and ZsGreen - cells and was most prominent after 4-weeks HFD feeding in ZsGreen - /LGALS3 + cells, which are likely macrophages. However, from 8 weeks onward, there was extensive LMO7 staining in ZsGreen + cells, suggesting potential roles in SMC as well as in macrophages. Intriguingly, LGALS3 staining directly correlated with the LMO7 staining pattern in ZsGreen + cells, suggesting a potential role for LMO7 in transdifferentiating SMC (Suppl Fig. 1a, arrows). Global Knockout of LMO7 induces larger plaque but with a stabilized structure To investigate the function of LMO7 during atherosclerosis progression, we compared Lmo7 global knockout mice ( Lmo7 -/- ) crossed with Apoe -/- mice to Apoe -/- control after 12 weeks HFD. There was no significant difference in total plasma cholesterol or triglycerides before and after HFD feeding between the two groups (Suppl Fig. 2a). We observed a significantly larger Oil Red-O (ORO) positive area in the whole aorta en face , in segments of the aortic arch, and in the brachiocephalic artery (BCA) in the Lmo7 -/- mice compared to the controls (Suppl Fig. 2b-c). We further examined the histological structure of BCA plaques. Lesion size was larger in the Lmo7 -/- mice, but the necrotic core was smaller, and fibrous cap was thicker (Suppl Fig. 2d), suggesting that LMO7 deletion enhanced plaque stability. We also note a reduced fraction of Lmo7 -/- mice with erythrocyte infiltration in the plaque (Ter-119 staining), a surrogate for spontaneous plaque rupture (Suppl Fig. 2e). Consistent with this, we observed a larger collagen + area in the Lmo7 -/- BCA plaques using polarized microscopy of Picro-Sirius Red (PSR) stained sections (Suppl Fig. 2f). These data reveal that global LMO7 knockout yielded a larger but more stabilized plaque. Knockout of LMO7 in SMC alters SMC phenotype and promotes features of plaque stability To investigate the function of LMO7 specifically in SMC during atherosclerosis progression, we generated Gt(ROSA26)-ZsGreen , Lmo7 fl/fl , Myh11_creER T2 , Apoe -/- (Lmo7 iΔSM ) mice. These mice, along with Gt(ROSA26)-ZsGreen, Lmo7 +/+ , Myh11_creER T2 , Apoe -/- (control) mice, were injected with tamoxifen and fed with HFD for 12 weeks. There was no significant difference in total cholesterol or triglycerides between the two groups (Fig. 1a). There was no difference in ORO staining in the whole aorta en face between the Lmo7 iΔSM and control mice, but we observed a significantly larger ORO positive area in the aortic arch and BCA plaques in the Lmo7 iΔSM mice (Fig. 1b-c). H&E staining in BCA plaques showed no difference in lesion size between the Lmo7 iΔSM and control mice; however, we noted multiple features of enhanced stability in the Lmo7 iΔSM mice, including a smaller necrotic core and thicker fibrous cap (Fig. 1d), lower fraction of mice with Ter-119 + staining (Fig. 1e), and a larger collagen + area (Fig. 1f). Cell composition is an important determinant of plaque stability 19, 20 , with a greater SMC to macrophage ratio in the lesion, and especially in the fibrous cap area, correlating with plaque stability 21 . We observed a larger ACTA2 + (SMC) area and smaller CD68 + (macrophage) area in the plaques of Lmo7 iΔSM mice compared to controls, and a higher percentage of ACTA2 + cells and a lower percentage of CD68 + cells in the fibrous cap area of Lmo7 iΔSM mice (Fig. 1g), indicating a more favorable cellular composition. We noticed a marked increase in SMC-derived (ZsGreen + ) cells in the BCA lesions of Lmo7 iΔSM mice compared to controls (Fig. 1g, 2a). To quantitatively assess the phenotypes of these SMC-derived cells, we immunostained for ACTA2 and LGALS3. Similar to CD68 (Fig. 1g), there was a reduction in LGALS3 + cells in the Lmo7 iΔSM compared to controls (Fig. 2a). In the fibrous cap, we noted more ZsGreen + cells in Lmo7 iΔSM versus control lesions, and a higher percentage of these cells were ACTA2 + (Fig. 2a). There was also a trend toward a lower percentage of ZsGreen + LGALS3 + cells relative to total DAPI + nuclei in the cap (Fig. 2a). In the ZsGreen + population, there were more ACTA2 + and fewer LGALS3 + cells, indicating that the phenotypic modulation of SMC was skewed toward more protective and less inflammatory states in the Lmo7 iΔSM mice. Moreover, the percentage of ZsGreen - LGALS3 + cells relative to DAPI + nuclei was also reduced (Fig. 2a), indicating that the recruitment, or retention of macrophages in the lesion was also suppressed in the Lmo7 iΔSM mice. To determine whether the increased number of SMC-derived (ZsGreen + ) cells in the Lmo7 iΔSM lesions was due to altered SMC proliferation and/or apoptosis, we immunostained for Ki67, a proliferation marker, and cleaved caspase 3 (cCasp3), an apoptosis marker, in the BCAs. There were more ZsGreen + Ki67 + cells in the fibrous cap of the Lmo7 iΔSM mice (Suppl Fig. 3), indicating enhanced SMC proliferation. This is consistent with our prior report of enhanced SMC proliferation in Lmo7 iΔSM mice following vascular injury 15 . We found that there were fewer ZsGreen + cCasp3 + cells within fibrous cap cells (DAPI + ) and in the ZsGreen + SMC (Fig. 2b), revealing that LMO7 limits proliferation and promotes apoptosis in plaque SMCs. Phenotypic modulation by cholesterol treatment is suppressed in LMO7-deficient SMC We next determined whether LMO7 could regulate cholesterol-induced SMC transdifferentiation in vitro by treating primary mouse aortic SMC isolated from WT or Lmo7 -/- mice with cyclodextrin-balanced cholesterol (as in 11 ). As expected, qPCR analysis indicated that cholesterol treatment inhibited Myh11 and induced Lgals3 RNA in WT SMC (Fig. 2c). Cholesterol-induced changes in these lineage markers were significantly reduced in Lmo7 -/- SMC (Fig. 2c), consistent with an inhibited transdifferentiation in the Lmo7 iΔSM mice (Fig. 2a). Cholesterol treatment reduces SMC collagen expression in vitro 22 . We noted higher RNA levels of several collagen genes in Lmo7 -/- cells compared to WT both at baseline and after cholesterol treatment (Suppl Fig. 4), consistent with the higher ECM content in the Lmo7 iΔSM mice (Fig. 1f). We further evaluated KLF4, a master regulator of SMC phenotypic modulation 9 . Similar to Lgals3 , cholesterol treatment induced Klf4 mRNA in both WT and Lmo7 -/- SMC, but to a lesser extent in the Lmo7 -/- cells (Fig. 2d). These differences were also observed in the protein levels of KLF4, LGALS3 and MYH11 in WT and Lmo7 -/- SMC lysates (Fig. 2f). Immunostaining for KLF4 was also reduced in BCA lesions in the Lmo7 iΔSM mice (Fig. 2e). Collectively, these data indicate that LMO7 is required for optimal KLF4 expression and SMC phenotypic modulation in response to cholesterol. LMO7 regulates lineage markers and KLF4 expression via TGF-β-miR-145 axis Our previous findings demonstrated that loss of LMO7 in SMC induces stronger TGF-β signaling in vascular injury models 15 . Here, in atherosclerosis, we also observed stronger immunostaining for TGF-β as well as p-SMAD3, the major signal-transducer of this pathway, in BCAs of Lmo7 iΔSM mice compared to WT lesions (Suppl Fig. 5a, b). Considering the protective role of the TGF-β pathway in SMC in atherosclerosis 23 , we tested whether TGF-β contributes to the anti-transdifferentiation effect of LMO7 depletion. Co-treatment of cultured SMC with cholesterol and SB431542, an inhibitor of TGFβRI, largely eliminated the difference between WT and Lmo7 -/- in expression of Myh11, Lgals3, and Klf4 (Suppl Fig. 5c). Previous studies have demonstrated that TGF-β suppresses KLF4 expression by inducing the miR-143/145 microRNA cluster 24 . Moreover, cholesterol treatment induces KLF4 by inhibiting miR-145 in SMC 25 . Therefore, we assessed miR-145 expression in the Lmo7 -/- SMC after cholesterol and SB431542 treatment. We noticed higher levels of miR-145 at baseline in the Lmo7 -/- SMC (Suppl Fig. 5d), consistent with increased TGF-β signaling 15 . Cholesterol treatment inhibited miR-145 expression in both WT and Lmo7 -/- SMC, likely due to the suppression of TGF-β signaling, however, miR-145 expression remained significantly higher in Lmo7 -/- SMC compared to WT SMC (Suppl Fig. 5d). Lastly, co-treatment of SMC with TGFβRI inhibitor and cholesterol further suppressed miR-145 levels in Lmo7 -/- SMC down to the expression observed in WT cells (Suppl Fig. 5d). The regulation of miR-145 is consistent with that of KLF4 by LMO7. These data suggest that the TGF-β-miR-145-KLF4 axis could, at least in part, mediate the LMO7-regulated SMC phenotypic modulation. LMO7 regulates ER stress via cholesterol metabolism Endoplasmic reticulum (ER) stress is elevated in SMC in hyperlipidemic conditions 26 , and has also been implicated in KLF4 regulation: ER stress activates ATF4, which in turn transactivates the KLF4 gene and inhibits KLF4 protein degradation 27 . We therefore hypothesized that LMO7 may influence KLF4 expression by modifying cholesterol-induced ER stress. Cholesterol treatment induced a significant upregulation of ATF4 and its downstream pro-apoptotic gene CHOP in WT SMC, but not in the Lmo7 -/- SMC (Fig. 3a). Additionally, immunostaining showed less ATF4 expression in the Lmo7 iΔSM mice compared to control (Fig. 3b), indicating attenuated ER stress. Excess of free cholesterol (FC) is a major inducer of ER stress 26, 28 . We determined that FC levels as measured by filipin staining were lower in the Lmo7 -/- SMCs compared to WT cells following cholesterol treatment (Fig. 3c). Differential cholesterol uptake does not likely explain this difference in FC as our cell-based transdifferentiation assays employ a water-soluble cholesterol formulation. Furthermore, there was no difference in Lrp1 mRNA, the major receptor for LDL uptake in SMC 29 between WT and Lmo7 -/- SMC (Suppl Fig. 6a). We also found no differences in mRNA for critical genes in the cholesterol biosynthesis pathway, including Sqle, Cyp51 , Dhcr24 and Hmgcr 30 between WT and Lmo7 -/- SMC either at baseline or with cholesterol (Suppl Fig. 6b). There was also no change in ABCA1, the main membrane transporter which mediates cholesterol efflux 31 (Suppl Fig. 6c). We hypothesized that FC was reduced in Lmo7 -/- SMC through enhanced conversion of FC to cholesterol ester (CE), an important mechanism shown to reduce cholesterol cytotoxicity 26, 32 . This could potentially explain the in vivo data showing higher ORO positive area in the aortic arch (Fig. 1b) and BCA (Fig. 1c) of Lmo7 iΔSM mice, as ORO stains specifically neutral lipids including CE but not FC. We performed ORO staining on cholesterol-treated mouse SMC in culture and observed more ORO + lipid droplets in the Lmo7 -/- SMC (Fig. 3d). This observation correlated with increased cholesterol esters found in LMO7-deficient SMC (Fig. 3e). Total cholesterol (TC) was similar in WT and Lmo7 -/- SMC but FC reduced in cells lacking Lmo7 (Fig. 3e). Together, these data suggested that the reduced level of FC in Lmo7 -/- SMC is likely due to its accelerated conversion to CE. LMO7 regulates SOAT1 expression by mediating its ubiquitination and proteasomal degradation Sterol o-acyltransferase (SOAT; also called ACAT) catalyzes the conversion of FC to CE. Notably, we observed an increase expression of SOAT1, the major SOAT isoform in SMC, in Lmo7 -/- SMC compared to WT SMC (Fig. 4a). Concordantly, in vivo analysis showed stronger SOAT1 immunofluorescence in ZsGreen + cells in the BCA of the Lmo7 iΔSM mice (Fig. 4b), suggesting that LMO7 may attenuate SOAT1 expression. We did not see a significant difference in Soat1 mRNA between WT and Lmo7 -/- SMC (Suppl Fig. 6d), suggesting that LMO7 might control SOAT1 expression at the post-transcriptional level. Thus, we analyzed whether LMO7, an F-box protein that mediates proteasomal degradation of target proteins 15 , interacts directly with SOAT1. Our results show that SOAT1 and LMO7 co-immunoprecipitate (Fig. 4c) and suppression of protein synthesis using cycloheximide (CHX) revealed a slower rate of SOAT1 protein decay (enhanced protein half-life) in the Lmo7 -/- SMC compared to WT cells (Fig. 4d). These results suggest that LMO7 might regulate SOAT1 protein stability and degradation. Indeed, we observed that the proteasome inhibitor MG-132 enhanced the SOAT1 ubiquitin signal in control but not LMO7 depleted cells (Fig. 4e), indicating that LMO7 can mediate the ubiquitination of SOAT1 protein. To verify the function of SOAT1 in LMO7-mediated SMC phenotypic modulation, we next assessed cholesterol-induced SMC transdifferentiation in the presence of the SOAT1 inhibitor K-604. In both WT and Lmo7 -/- SMC, SOAT1 inhibition enhanced the induction of KLF4, LGALS3, and the ER stress markers ATF4 and CHOP (Fig. 4f). Importantly, SOAT1 inhibition abrogated the effect of Lmo7 -/- , such that levels of KLF4 and LGALS3 were comparable to WT controls (Fig. 4f). These data suggest that modulation of cholesterol-induced SMC transdifferentiation, by LMO7, requires SOAT1-dependent regulation of cholesterol metabolism. Single cell RNA sequencing revealed a distinct cellular composition of SMC-derived cells in the Lmo7 iΔSM mice To comprehensively investigate the changes in plaque composition and SMC phenotype with loss of LMO7, we performed single cell RNA sequencing (scRNAseq) analysis of cells isolated from aortic root, aortic arch and its three major branches which were harvested from control and Lmo7 iΔSM mice (both on Apoe -/- background) fed a HFD for 12 weeks. We combined cell data points from WT and Lmo7 iΔSM groups for analysis and identified 11 populations by graph-based clustering (Fig. 5a). Based on the gene expression profile, we identified all the major cell types in the atherosclerotic lesion, including SMCs, fibroblasts, macrophages (including classically activated- “Macrophage 1”, and alternatively activated- “Macrophage 2” clusters), endothelial cells, neutrophils, and T lymphocytes (Fig. 5b). Five SMC-derived ZsGreen+ clusters were identified (Fig. 5b). Based on the published defining markers for various subpopulations 12, 13 , they were classified as “SMC” (classical contractile phenotype SMC), cap-stabilizing “Phactr1 + SMC” 13 , “Transitioning SMC” ( Klf4 + Lgals3 + Myh11 low ), inflammatory “Macrophage-like cell” (expressing both SMC and macrophage markers) or rupture-protective “Fibromyocyte” 12 (Fig. 5a, defining genes shown in Fig. 5b). Interestingly, WT and Lmo7 iΔSM mice displayed distinct profiles of SMC-derived populations: WT mice contained 52.2% transitioning SMC, while this population was dramatically reduced (8.0%) in Lmo7 iΔSM mice (Fig. 5c). Conversely, there were many more contractile SMC and slightly increased fibromyocytes in Lmo7 iΔSM mice (Fig. 5c). Interestingly, the macrophage1 cluster was also reduced in Lmo7 iΔSM plaques, suggesting reduced inflammation (Fig. 5c). These data were consistent with the plaque characterization based on immunostaining and lineage tracing in Fig. 1-2. LMO7 is induced in human atherosclerosis and enriched in ruptured lesions To determine whether these roles of LMO7 may translate to human atherosclerosis, we evaluated LMO7 mRNA levels in human atherosclerotic arteries from patients undergoing carotid endarterectomy. These samples included normal control regions (Fig. 6a). There was an increase in LMO7 mRNA in all atherosclerotic regions (average of 3-fold induction) compared to a non-diseased control segment of the same artery in each patient (Fig. 6b). Additionally, there was 5.7-fold more LMO7 mRNA in ruptured human plaque samples compared to stable plaques (Fig. 6b). These data reveal a positive correlation between SMC LMO7 expression and atherosclerotic plaque vulnerability in patients. Discussion The most salient finding of our study is the identification of LMO7 as a key molecule that integrates cellular cholesterol metabolism and SMC transdifferentiation affecting atherosclerotic plaque composition and stability. We demonstrate that cholesterol-induced LMO7 promotes SOAT1 ubiquitination and degradation leading to accumulation of FC. LMO7 depletion stabilizes SOAT1, promoting cholesterol esterification in SMC. This alleviates ER stress-induced induction of KLF4 and inflammatory SMC fate transitions (Fig. 6 c). Finally, our study also reveals that enhancing cholesterol esterification dramatically alters SMC phenotypic modulation and promotes features of plaque stability. The pathogenesis of atherosclerosis involves the actions of multiple cell types. The complex role of SMCs in this disease was underestimated until recently. Previously, SMCs were thought to de-differentiate to a synthetic phenotype and participate in fibrous cap formation. While SMC transitions are indeed necessary for fibrous cap formation 6 , 33 , recent studies showed that SMC can also transform into a range of diverse phenotypes in lesions, including macrophage-like cells 9 , 11 , fibromyocytes 12 , osteoblasts and chondrocytes 23 . Since these different phenotypic transitions likely exert distinct or even opposite effects with respect to lesion growth and stability, it is crucial to understand the mechanisms controlling cell fate. In the current study, we demonstrated that loss of LMO7 in SMC led to alteration of intracellular cholesterol processing and phenotypic modulation. A comprehensive scRNAseq analysis showed that the “transitioning” SMC state ( Klf4 + Lgals3 + Myh11 low ) was substantially reduced in the Lmo7 iΔSM mice, consistent with analysis of lesion cell composition by immunostaining. Moreover, the LMO7-deficient SMC-derived cells presented enhanced proliferation and viability and deposited larger amounts of ECM, leading to features of stable plaque. We demonstrated that LMO7 modulates cholesterol-induced SMC phenotypic switching in vitro via regulation of KLF4 and LGALS3 expression, the two key players in this process 9 , 13 . We further showed that suppression of KLF4 and LGALS3 expression in Lmo7 −/− SMC was due to the attenuated ER stress and ATF4 activity. Previous studies showed that unresolved ER stress leads to apoptosis in macrophages and SMCs 26 , 32 . ER stress could also induce dramatic phenotypic modulation in SMCs by activating ATF4, thus inducing KLF4 expression 27 . The Milewicz group recently published a study demonstrating that inhibiting the PERK-eIF2α-ATF4 pathway suppresses SMC transition to macrophage-like cells by cholesterol treatment 34 . This study supports our finding that cholesterol-induced ER stress is a critical mediator of adverse SMC phenotypic modulation in atherosclerosis, and approaches to suppress SMC ER stress may have therapeutic utility. In addition to cholesterol metabolism and ER stress, our data suggest that LMO7 may also regulate KLF4 and LGALS3 expression via TGF-β1 signaling. Our previous study showed that knockout of LMO7 in SMC induced elevated TGF-β1 expression and signaling, contributing to enhanced collagen deposition in injury-induced vascular remodeling 15 . Interestingly, cholesterol treatment could suppress TGF-β1 signaling and downstream effectors 35 . We now report that SMC LMO7 depletion enhances TGF-β1 expression and signaling in atherosclerotic plaques, and that TGF-β1 inhibition blunts the protective effect of LMO7 depletion on cholesterol-induced SMC transdifferentiation in vitro . These data indicate that LMO7 can influence KLF4 expression through a TGF-β1-dependent miR-145 axis. It is likely that the ability of LMO7 to regulate both TGF-β1 signaling and cholesterol esterification accounts for the potent effects of this protein on SMC fate transitions in atherosclerosis. Lmo7 global knockout or only in SMC-derived cells elicited a similar phenotype of smaller necrotic core and thicker fibrous cap, although Lmo7 −/− mice had larger lesions compared to control, while the SMC Lmo7 deficient mice did not, indicating that LMO7 may also play roles in other relevant cell types. For example, TGF-β1-induced endothelial-mesenchymal transitions can contribute to atherosclerosis 36 . Macrophages may also play a role in the phenotype of Lmo7 −/− mice. Intriguingly, in our scRNAseq analysis with Lmo7 iΔSM mice, we observed a dramatic alteration of macrophage subpopulations, indicating that SMC exert a paracrine effect on macrophages, potentially through TGF-β1 signaling, which has been shown to influence macrophage classical versus alternative activation patterns 37 , 38 . Our findings suggest that targeting LMO7 specifically in SMC has beneficial effects on lesion composition without enhancing lesion size. In atherosclerosis, monocyte-derived macrophages were thought to be the major cell type to process excessive cholesterol, which internalize modified lipoproteins and become foam cells. However, recent studies in human and mouse lesions revealed that majority of the foam cells are derived from SMCs 8 , 39 . This finding highlights the role of SMC in cholesterol storage and its contribution to the control of lipid content in the plaque. In our study, loss of LMO7 in SMC enhanced the conversion of FC to CE, a critical step of lipid droplet formation in foam cells, and increase in foam cell number is associated with reduced SMC transition to macrophage-like cells, as well as with a more stable plaque. Interestingly, previous studies showed that foamy cells derived from macrophages are less inflammatory than non-foamy cells 40 , 41 , suggesting that foam cell formation for lipid storage is a protective mechanism in hyperlipidemic disease. Current treatments targeting atherosclerotic vascular diseases, including statins and anti-PCSK9 antibodies, aim to lower circulating lipid levels. These therapies have proven to be effective, but there are still an estimated 805,000 myocardial infarctions per year, and stroke accounts for > 5% of all deaths in the US 1 , indicating that additional approaches are required to prevent the occurrence of atherothrombotic events. Enhancing plaque stability may be a beneficial strategy. In our study, when cholesterol esterification was enhanced via elevated SOAT1 by manipulating LMO7 expression globally, or in a SMC-specific manner, it induced features of stabilized plaque, with smaller necrotic cores and thicker fibrous caps. This change in cholesterol storage did not affect circulating cholesterol and triglyceride or cellular TC levels, indicating that promoting cholesterol storage in the esterified form can by itself enhance lesion stability, or potentially combine with the existing lipid-lowering therapies to further reduce incidence of myocardial infarction and stroke. Cholesterol esterification is catalyzed by SOAT, which may therefore serve as a potential drug target. SOAT inhibition has been evaluated in mouse atherosclerosis with conflicting results 42 – 46 . Studies from the Chang lab showed that myeloid-specific knockout of ACAT1/SOAT1 in Apoe −/− mice reduced lesional macrophage content and suppressed atherosclerosis progression 45 , 46 . Additionally, administration of an ACAT/SOAT inhibitor in Apoe −/− mice reduced atherosclerosis 44 . However, Accad et al. reported that global knockout of SOAT1/ACAT1 reduced neutral lipids but resulted in more macrophages in the lesions 42 . Further, Fazio et al. demonstrated that transplantation of ACTA1/SAOT1-deficient bone marrow to the LDLR −/− mice exacerbated atherosclerosis 43 , suggesting a beneficial role of macrophage SOAT1. Importantly, in patients receiving standard care for secondary prevention, administration of ACAT/SOAT inhibitors elicited less normalized atheroma volume regression compared to placebo group (ATIVATE trial 47 ), and increased the incidence of major cardiovascular events in patients with familial hypercholesterolemia (CAPTIVATE trial 48 ), indicating a protective role of SOAT activity in human cardiovascular disease. Our results that loss of LMO7 in SMC promoted plaque stability by induction of SOAT1 expression provide additional insights into the beneficial role of SOAT1. This assertion is strengthened by the finding that inhibition of SOAT activity in mouse ASMC enhanced their transition to macrophage-like cells by cholesterol treatment 11 and abolished the protective effect of LMO7 depletion on this process. These findings suggest that SOAT1 in SMCs attenuates the cholesterol-induced SMC phenotypic transitions that destabilize atherosclerotic plaques, such that enhancing SMC SOAT1 activation and cholesterol esterification may be a potential therapeutic strategy. We report that LMO7 is induced by cholesterol in SMC in plaques, and regulates cholesterol processing, which, in turn, influences SMC phenotypic transitions, plaque composition, and features of stability. Notably, LMO7 is also induced in human atherosclerosis compared to healthy vessel and is expressed at higher levels in ruptured or unstable compared to stable lesions. Interestingly, an LMO7 SNP (rs2273996) has been associated with human coronary artery disease 49 . These studies suggest that LMO7 expression and binding partners and cholesterol esterification specifically in SMC could be of interest for targeting lesion stability. Declarations Acknowledgements This work was supported by grants from the NIH to K.A.M. (R01HL151222, R01HL142090, R01 HL146101), to J.H. (R01HL115247A, R01HL122815), and to CF-H (R35HL135820). Research in L.M.’s laboratories is funded by the Swedish Heart-Lung-Foundation (20210519), the Swedish Research Council (Vetenkapsrådet, 2019-01577), the German Center for Cardiovascular Research (DZHK), the SFB1123 and TRR267 of the German Research Council (DFG), the National Institutes of Health (NIH; 1R011HL150359-01), the Bavarian State Ministry of Health and Care through the research project DigiMed Bayern. Author Contributions Y.X., K.A.M., C.F.-H., and L.M. designed and analyzed the experiments. Y.X. and K.A.M. wrote the manuscript. Y.X., H.W. and N.R. performed the experiments. All co-authors contributed to analyzing and interpreting data and editing the manuscript. Competing Interests statement The authors declare no competing interests. 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Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"","lastName":"Winter","suffix":""},{"id":84016361,"identity":"82fb1ad7-06bf-47ba-8cec-354fa268cf8e","order_by":2,"name":"Noemi Rotlan","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Noemi","middleName":"","lastName":"Rotlan","suffix":""},{"id":84016362,"identity":"3b6ce1c1-fb22-46ce-9984-ed287e54fbee","order_by":3,"name":"Allison Ostriker","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Allison","middleName":"","lastName":"Ostriker","suffix":""},{"id":84016363,"identity":"1b65d35d-5212-4328-bb58-4c4d1cbd2595","order_by":4,"name":"Raja Chakraborty","email":"","orcid":"","institution":"Yale 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Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Fernández-Hernando","suffix":""},{"id":84016367,"identity":"0fcf1ef0-788e-4e9b-82a2-d894dafde33d","order_by":8,"name":"Lars Maegdefessel","email":"","orcid":"","institution":"Technical University Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Maegdefessel","suffix":""},{"id":84016368,"identity":"54215d28-861f-4b7a-bf9c-318e808d058f","order_by":9,"name":"Kathleen Martin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIie3QsUoDMQDG8e84OBfRTVKCrY8QOXCp+CwJB83SitDlhg6RwnV0rW9x4AtcCdTl4NYODlcKNx90cZDWnNCCQzzcBPMfjiTcLyEBXK4/GAEClMcpQxfwlRn5PxA/AD/MMoYQ8H5FAKHaSGdWLEse397jQlfb+uFNpq+LxxJxXygLoafRCeP5YAw6COcZq0ZpLqYMubSSLqKAiEQLRXlo7qJHaSYS4jUrNnK+MWS3N0RuGyJZsZ69ezs7oaQ5RWWGDL9O4WwlEvMCdtKZb24IX0YiocMxcqavn1frqVmRoY2QQlSkntyJJypfEH/o3lkRLep60r+0kWPBYXCVmQ9v+/1bvdbdXS6X67/1CagdX0j7O2qEAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1748-0034","institution":"Yale University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kathleen","middleName":"","lastName":"Martin","suffix":""}],"badges":[],"createdAt":"2022-02-14 20:15:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1359588/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1359588/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18256081,"identity":"c3ec2a49-5fc0-413d-a9f2-93441a00a1df","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2401104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of LMO7 in SMC induces feature of stable plaque in atherosclerosis.\u003c/strong\u003e \u003cem\u003eGt(ROSA26)-ZsGreen, Lmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Myh11_creER\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Apoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e (ZsGreen\u003c/em\u003e\u003csup\u003e\u003cem\u003eGt\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Lmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Apoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e­\u003c/em\u003e) and \u003cem\u003eGt(ROSA26)-ZsGreen, Lmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Myh11_creER\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Apoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (control) mice were injected with 1mg/kg tamoxifen at 6-week old for 5 days, followed by washout for 5 days. The mice were then fed with HFD for 12 weeks. (a) The plasma was harvested, and cholesterol and triglyceride levels were measured as shown in the Methods. (b) The aorta and the three branches of aortic arch were harvested. The BCA was dissected for histological analysis and the rest was subjected to \u003cem\u003een face\u003c/em\u003e Oil Red O (ORO) staining. The positive area was quantified using ImageJ in the whole aorta or in the aortic arch, thoracic aorta and abdominal aorta region, separately, and normalized to the whole area of the corresponding region. (c) The cross-sections of BCA were subjected to ORO staining and the positive area was quantified against the whole plaque area. (d) The cross-sections of BCA were subjected to Haematoxylin \u0026amp; Eosin (H\u0026amp;E) staining and the area of plaque and necrotic core and thickness of fibrous cap were quantified using ImageJ. The necrotic core was defined as the area without intact cells and normalized to the whole plaque area. The thickness of fibrous cap was defined as the shortest distance from lumen to the largest necrotic core in the plaque. (e) The cross-sections of BCA were subjected to immunohistochemistry for Ter-119, and counterstained with Methyl Green. The percentage of mice with a positive staining of Ter-119 in the BCA was shown on the right. (f) The cross-sections of BCA were subjected to Picro-Sirius Red (PSR) staining. The positive area was quantified using ImageJ and normalized to the whole plaque area. (n=11 for control, n=10 for \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e) (g) The cross-sections of BCA were immunostained for ACTA2 (red) and CD68 (Cy5). ACTA2\u003csup\u003e+\u003c/sup\u003e and CD68\u003csup\u003e+\u003c/sup\u003e area was quantified using ImageJ and normalized to the whole plaque area. In the fibrous cap area, ACTA2\u003csup\u003e+\u003c/sup\u003e and CD68\u003csup\u003e+ \u003c/sup\u003ecells were counted and normalized all fibrous cap cells (DAPI\u003csup\u003e+\u003c/sup\u003e). (n=10 for control, n=7 for \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e) Scale bar, 50 μm. Data are expressed as mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page01.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/f4644f1472c4b707cb928a72.png"},{"id":18256083,"identity":"2118954a-c105-4914-b062-bfc76dcdda9d","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2990132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of LMO7 in SMC suppresses phenotypic modulation and promotes cell viability \u003cem\u003ein vivo\u003c/em\u003e.\u003c/strong\u003e (a) The cross-sections of BCA were immunostained for ACTA2 (red) and LGALS3 (Cy5). The ZsGreen\u003csup\u003e+\u003c/sup\u003e, ZsGreen\u003csup\u003e+\u003c/sup\u003e ACTA2\u003csup\u003e+\u003c/sup\u003e, ZsGreen\u003csup\u003e+\u003c/sup\u003e LGALS3\u003csup\u003e+\u003c/sup\u003e, ZsGreen\u003csup\u003e-\u003c/sup\u003e LGALS3\u003csup\u003e+\u003c/sup\u003e cell populations were counted and normalized to all the cells (DAPI\u003csup\u003e+\u003c/sup\u003e) or ZsGreen\u003csup\u003e+\u003c/sup\u003e cells in the fibrous cap area as indicated. (b) The cross-sections of BCA were immunostained for cleaved Caps3 (cCasp3) (Cy5). The ZsGreen\u003csup\u003e+\u003c/sup\u003e cCasp3\u003csup\u003e+\u003c/sup\u003e and LGALS3\u003csup\u003e+\u003c/sup\u003e cCasp3\u003csup\u003e+\u003c/sup\u003e cell populations were counted and normalized to all the cells (DAPI\u003csup\u003e+\u003c/sup\u003e) or ZsGreen\u003csup\u003e+\u003c/sup\u003e cells in the fibrous cap area. (n=10 for control, n=8 for \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e) (c, d) Mouse aortic SMCs were starved as in the Methods and treated with 40 µg/ml methyl-β-cyclodextrin cholesterol for 72 hrs. The cell lysates were harvested and mRNA was purified for real time qPCR analysis of (c) \u003cem\u003eMyh11\u003c/em\u003e, \u003cem\u003eLgals3\u003c/em\u003e and (d) \u003cem\u003eKlf4 \u003c/em\u003e(n=4). (e) The serial sections as shown in Fig. 1g were immunostained for ACTA2 (red) and KLF4 (Cy5). (f) Mouse ASMCs were starved and treated with cholesterol as in (c, d) and the cell lysates were harvested for western analysis of MYH11, KLF4 and LGALS3, using β-Tubulin as loading control. (n=5) Scale bar, 50 μm. Data are expressed as mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page02.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/587973493641a155ea7780c1.png"},{"id":18256082,"identity":"cf1b49e7-57cb-460a-a9b0-a81fee321e3f","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2188025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLMO7 regulates ER stress via cholesterol metabolism. \u003c/strong\u003e(a) Mouse aortic SMCs were starved as in the Methods and treated with 40 µg/ml methyl-β-cyclodextrin cholesterol for 72 hrs, and the cell lysates were harvested for western analysis of ATF4 and CHOP, using β-Tubulin as loading control (n=4). (b) The cross-sections of BCA were immunostained for ATF4 (red) and LGALS3 (Cy5). (c, d) Mouse aortic SMCs were starved as in the Methods and treated with 40 µg/ml methyl-β-cyclodextrin cholesterol for 72 hrs, and cells were fixed for (c) filipin staining or (d) ORO staining (n=4). (e) Mouse aortic SMCs were starved as in the Methods and treated with 10 µg/ml methyl-β-cyclodextrin cholesterol for 72 hrs. The cells were scrapped in PBS and cholesterol was extracted as shown in the Methods and subjected to analysis using Amplex® Red Cholesterol Kit (n=4). Scale bar, 50 μm. Data are expressed as mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page03.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/eb4077973820abbde1b4d1f9.png"},{"id":18256137,"identity":"998f7b32-531c-4b3b-8159-1a00d071a259","added_by":"auto","created_at":"2022-02-15 20:53:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1662256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLMO7 regulates SOAT1 expression by mediating its ubiquitination and proteasomal degradation.\u003c/strong\u003e (a) Mouse aortic SMCs were starved as in the Methods and treated with 40 µg/ml methyl-β-cyclodextrin cholesterol for 72 hrs, and the cell lysates were harvested for western analysis of SOAT1, using β-actin as loading control (n=4). (b) The cross-sections of BCA were immunostained for SOAT1 (red) and LGALS3 (Cy5). (c) Endogenous SOAT1 protein was pulled down by specific antibody and the immunoprecipitates were subjected to western analysis of LMO7 (Representative of 3 biological repeats). (d) mASMCs were starved in 0.5% FBS and treated with Cycloheximide (CHX). Cell lysates were harvested at various time points as indicated and subjected to western analysis for SOAT1, using β-actin as loading control (n=4). (e) Human aortic SMCs were transfected with control siRNA or siRNA for LMO7 for two days, followed by vehicle or MG-132 treatment for another 24 hrs. The cell lysates were harvested for SOAT1 immunoprecipitation and blotted for ubiquitin and LMO7 (n=2). (f) Mouse aortic SMCs were starved for 24 hrs as in the Methods and were treated with 40 µg/ml methyl-β-cyclodextrin cholesterol with or without 10 μM K-604 for 72 hrs, and the cell lysates were harvested for western analysis of various markers as shown, using β-actin as loading control (n=6). Scale bar, 50 μm. Data are expressed as mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page04.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/237ab43dedf41a89882b0ff0.png"},{"id":18256087,"identity":"7a7da8d3-248e-4d07-8282-cf3041b3c574","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":826682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle cell RNA sequencing analysis revealed distinct cell populations of SMC-derived cells in \u003cem\u003eLmo7\u003c/em\u003e\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003e(a) t-SNE visualization of cell clusters present in the atherosclerotic aortic root, aortic arch and its three main branches from control and \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e mice combined (2 mice per genotype). (b) Heatmap visualization of marker gene expression in each cell cluster. (c) t-SNE visualization of control and \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e group separately. Pie charts show the proportion of each cluster within SMC-derived population or all the harvested cells.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page05.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/d64a7d0905fb5e1befb00b26.png"},{"id":18256085,"identity":"41c730e6-83c0-44f1-b157-5081af2a30c3","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":285489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLMO7 RNA is induced in human atherosclerosis. \u003c/strong\u003e(a) The diseased and non-diseased portion of human atherosclerotic carotid arteries were separated and subjected to qPCR analysis for \u003cem\u003eLMO7\u003c/em\u003e (n=10). (b) The human stable atherosclerotic carotid arteries and ruptured ones were analyzed for \u003cem\u003eLMO7\u003c/em\u003e RNA (n=15 for each group). (c) Schematic illustrating how cholesterol-induced LMO7 modulates SMC phenotypic modulation via regulating SOAT1 protein stability, cholesterol esterification, ER stress and KLF4/LGALS3 expression.\u003c/p\u003e","description":"","filename":"Lmo7AtherosclerosisFigures21322Page06.png","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/d6586a1f46af9b9777628919.png"},{"id":19278732,"identity":"a3701119-402b-4386-b5af-c81d683d4065","added_by":"auto","created_at":"2022-03-16 09:26:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3757768,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/c5486ee8-2fbc-452c-959d-9481f0a07a23.pdf"},{"id":18256086,"identity":"b76ad103-9a4f-4faf-94c9-670cae394666","added_by":"auto","created_at":"2022-02-15 20:50:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15973,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u003c/p\u003e","description":"","filename":"SupplementaryInformatoin.docx","url":"https://assets-eu.researchsquare.com/files/rs-1359588/v1/286d303c28d918e56c7bb735.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"LMO7 regulates smooth muscle cell cholesterol esterification to influence plaque stability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerosis is the main cause of morbidity and mortality in the US and Europe. \u0026nbsp;Despite widespread use of lipid lowering agents that help slow the progression, complications of atherosclerotic plaque rupture, including myocardial infarction and stroke, remain major challenges\u003csup\u003e1\u003c/sup\u003e. Accordingly, plaque stability is a key factor in prognosis\u003csup\u003e2, 3\u003c/sup\u003e. New approaches to reduce the incidence of thrombotic events would significantly improve morbidity and mortality consequent to atherosclerosis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCholesterol accumulation in the vessel wall leads to plaque formation. Macrophages that engulf the excess cholesterol become foam cells and contribute to a pro-inflammatory milleu\u003csup\u003e4\u003c/sup\u003e. Excessive intracellular cholesterol promotes apoptosis and formation of a necrotic core in the lesions. Smooth muscle cells (SMC) form a protective fibrous cap, providing an essential stabilizing function to separate the pro-thrombotic necrotic core from the lumen\u003csup\u003e5\u003c/sup\u003e. Medial SMC migrate from the plaque shoulder to cover the plaque core at early stages\u003csup\u003e6\u003c/sup\u003e. These SMC secrete copious extracellular matrix (ECM) to form the fibrous cap. Because the thickness and stability of the fibrous cap predicts the vulnerability to rupture\u003csup\u003e7\u003c/sup\u003e, SMC are thought to play a protective role in plaques. Recent fate mapping studies have expanded our understanding of the heterogeneity and roles of SMC-derived populations in atherosclerosis. Notably, SMC transdifferentiation to a macrophage-like state is now appreciated to be a major source of foam cells in mouse and human lesions, with 50% of foam cells derived from SMC in human coronary lesions in one analysis\u003csup\u003e8-10\u003c/sup\u003e. The finding that SMC undergo phenotypic modulation to macrophage-like cells in hyperlipidemic conditions\u003csup\u003e11\u003c/sup\u003e suggests a new paradigm where SMC-derived cells can play distinct roles within lesions: the macrophage-like SMC can contribute to the inflammatory lesion core, whereas matrix synthesizing SMC-derived cells act as a protective component in the fibrous cap. Single cell RNA sequencing (scRNAseq) analyses have demonstrated that SMC can give rise to distinct populations\u003csup\u003e12\u003c/sup\u003e including inflammatory, matrix-producing and osteogenic subtypes\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral studies have recently shown that Kruppel Like Factor 4\u0026nbsp;(KLF4), a transcription factor implicated in pluripotency, is a key factor promoting SMC dedifferentiation\u003csup\u003e9, 13, 14\u003c/sup\u003e. KLF4 has been shown to play a fundamental role in the SMC phenotypic switching, including in the transition to the pro-inflammatory macrophage-like state\u003csup\u003e9\u003c/sup\u003e. Although expressed in many hematopoietic cells including macrophages, LGALS3 has been identified as a KLF4-regulated marker of phenotypically modulated SMC\u003csup\u003e13\u003c/sup\u003e. LGALS3 expression may represent an early-stage transition that subsequently gives rise to multiple distinct phenotypes during atherosclerosis progression\u003csup\u003e13\u003c/sup\u003e. Early signals through which hypercholesterolemia influences SMC transcription factors and phenotypic switching in atherosclerosis are still poorly understood.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe have recently identified LIM domain only protein 7 (LMO7) as a potent modulator of SMC phenotype\u003csup\u003e15\u003c/sup\u003e. LMO7 is a scaffolding protein containing multiple protein-protein interaction domains. LMO7 loss of function in SMC enhances cell proliferation and ECM deposition following vascular injury\u003csup\u003e15\u003c/sup\u003e. Given the roles for SMC phenotypic modulation, proliferation, and ECM synthesis in atherosclerosis, we hypothesized that LMO7 loss of function may lead to more stable lesions with a thicker fibrous cap.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eMice.\u003c/b\u003e\u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice on a C57BL/Swiss mixed background were a gift from Dr. Ju Chen (UCSD). \u003cem\u003eMyh11\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were purchased from Jackson Laboratory. Mouse strain with \u003cem\u003eGt(ROSA26)-ZsGreen\u003c/em\u003e reporter was a generous gift of Dr. Daniel Greif (Yale). For lineage tracing studies, the \u003cem\u003eGt(ROSA26)-ZsGreen\u003c/em\u003e reporter strain was crossed with \u003cem\u003eMyh11-CreER\u003c/em\u003e\u003csup\u003e\u003cem\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e mice on the \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e background. Recombination was induced by injecting 6 week old mice with 1 mg/kg tamoxifen for 5 days, followed by 5 day washout. Inducible, smooth muscle-specific \u003cem\u003eLmo7\u003c/em\u003e knockout mice were generated by crossing the \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e line with the above-mentioned \u003cem\u003eROSA26-ZsGreen\u003c/em\u003e/\u003cem\u003eMyh11-CreER\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2\u003c/em\u003e\u003c/sup\u003e/\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e reporter line. Genotyping of all the mice was performed by PCR. Mice were housed in pathogen-free conditions at Yale University. All experiments were approved by the institutional animal care and use committee of Yale University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman tissue studies.\u003c/b\u003e Human carotid arteries of the Munich Vascular Biobank\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e were taken during carotid endarterectomy (CEA) followed by fixation for 48 hours in 2% zinc-formalin and paraffin embedding. Per specimen, four 5\u0026micro;m thick sections were stained with hematoxylin and eosin (HE) and Elastin van Gieson\u0026rsquo;s staining. Based on the histochemical stains, plaques were characterized according to their features as stable or unstable/ruptured following the American Heart Association (AHA) classification after Stary \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and Redgrave \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e to determine critical fibrous thickness. Plaque and the non-diseased control portion of the carotid biopsy (as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) were separated and lysed for RNA analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal experiments.\u003c/b\u003e After tamoxifen injection and washout, mice were fed with high fat diet (HFD, 40% kcal%, 1.25% cholesterol, Research Diets D12108C) starting at 8 weeks of age for 0\u0026ndash;12 weeks as indicated. For blood lipid assays, mice were starved overnight and anesthetized with 100mg/kg ketamine and 10mg/kg xylazine to collect blood by retro-orbital bleeding before and after HFD feeding. The blood samples were centrifuged at 10,000 rpm for 10 min at R.T. and the plasma (upper phase) was collected for total cholesterol and triglyceride measurement using Cholesterol Assay Kit (Abcam, ab65390) and L-Type Triglyceride M Enzyme Kit (Fujifilm), respectively. Mice were sacrificed at indicated time points post HFD feeding and were immediately perfused first with sodium nitroprusside (SNP) in PBS then 4% paraformaldehyde (PFA). The aortic root (sinus) and the whole aorta as well as brachiocephalic artery (BCA), left common carotid artery, and left subclavian artery branches were dissected and immersed in cold 4% PFA for overnight followed by dehydration in 30% sucrose for another 24 hours. Then the vessels were embedded in OCT compound (Tissue Tek, Elkhart, IN) for cryosectioning.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMouse single cell RNA sequencing sample preparation and sequencing.\u003c/b\u003e After 12 weeks HFD, mice were sacrificed and perfused with PBS. The aortic root, aortic arch and the three major branches, including brachiocephalic artery, left common carotid artery and left subclavian artery were dissected. Tissues were briefly digested in adventitia stripping mix (175U/ml Collagenase II and 1.25U/ml elastase in HBSS) at 37\u0026deg;C for 10 min, and adventitia was peeled off. Then the vessels were cut into 1-2mm long rings and digested in digestion mix (600U/ml collagenase II and 2.5U/ml elastase in HBSS) for 1 h at 37\u0026deg;C with shaking at 700 rpm. Cell suspensions were filtered through 70 \u0026micro;m cell strainer and pelleted at 500 x g for 5 min at 4\u0026deg;C. Cells were then stained in viability staining buffer (0.01 \u0026micro;g/ml DAPI in PBS\u0026thinsp;+\u0026thinsp;2% FBS) for 5 min at 4\u0026deg;C and maintained in this buffer for cell sorting. DAPI negative Cells were sorted using FACSAria II (BD Biosciences) into DMEM\u0026thinsp;+\u0026thinsp;10% FBS. Cell numbers were counted and ~\u0026thinsp;10,000 cells per sample were used immediately for single-cell library preparation at Yale Keck Biotechnology Resource Laboratory. Cells were loaded into a 10x Genomics microfluidics chip and encapsulated with barcoded oligo-dT-containing gel beads using the 10x Genomics Chromium controller according to the manufacturer\u0026rsquo;s instructions. Single-cell 3\u0026rsquo; V3.1 libraries were then constructed according to the manufacturer\u0026rsquo;s instructions. Libraries from control and \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e samples were multiplexed into one lane and sequenced with setting of paired-end 150bp on an Illumina HiSeq 4000 instrument.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of scRNASeq data.\u003c/b\u003e Fastq files from each mouse genotype were aligned to the reference genome containing \u003cem\u003eZsGreen1\u003c/em\u003e gene individually using CellRanger Software (10x Genomics). The dataset was then analyzed using the Partek Flow platform. The dataset was trimmed of cells with read count fewer than 600 or more than 15,000, cells expressing fewer than 500 genes or more than 4,000 genes, and cells with more than 10% mitochondrial counts. The gene expression values then underwent library-size normalization, where raw gene counts from each cell were normalized relative to the total number of read counts present in that cell. Principal component analysis (PCA) was used for dimensionality reduction, followed by cell clustering using a graph-based clustering approach. t-distributed stochastic neighbor embedding (t-SNE) was then used for two-dimensional visualization of the resulting clusters. Contractile SMC, modulated SMC, fibromyocyte, macrophage, endothelial cell, T cell, neutrophil and fibroblast populations were classified based on the gene expression profile. Cells within the specific classification were combined for further analysis for differential gene expression analysis between control and \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological staining.\u003c/b\u003e Haemotoxylin \u0026amp; Eosin (H\u0026amp;E) staining was performed at Yale Pathology Tissue Service using standard protocols. For Picro-Sirius Red (PSR) staining, tissue sections were washed with water to remove OCT and immersed in staining solution (0.1% Direct Red 80 (Sigma, #365548) in a saturated aqueous solution of picric acid) for 1h. The sections were then washed with two changes of 0.5% acetic acid solution, dehydrated with three changes of 100% ethanol, cleared with Histoclear (National Diagnostics, HS-200) and mounted with DPX mounting media (Electron Microscopy Sciences). For \u003cem\u003een face\u003c/em\u003e Oil Red O (ORO) staining of aortas, the dissected aorta was cut longitudinally and pinned on a silicon plate. The aorta was fixed with 4% PFA for 1h at RT, briefly rinsed with 78% methanol and immersed in the ORO staining solution (0.2% w/v ORO solution in methanol mixed with 1M NaOH at 7:2 ratio) for 1h. Then the aorta was washed with 78% methanol twice and mounted with OCT medium between two microscope slides. Tissue sections and cell ORO staining follows the same protocol as above. Micrographs of ORO staining was taken with a Nikon Eclipse 80i microscope using NIS Elements software. For filipin staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at home temperature, followed by incubation with 1.5 mg/ml glycine in PBS for 10 min at room temperature (R.T) to quench the paraformaldehyde. Cells were then stained with filipin working solution (0.05 mg/ml filipin (Sigma, F-9765) in PBS\u0026thinsp;+\u0026thinsp;10% FBS) for 2 hrs at R.T. Cells were rinsed with PBS twice and were imaged on a Nikon Eclipse Ti Confocal Microscope.\u003c/p\u003e \u003cp\u003e\u003cb\u003eImmunohistochemistry and immunofluorescence.\u003c/b\u003e For Immunohistochemistry, cryosections were air-dried completely and fixed/permeabilized in cold acetone for 10 min. OCT was removed by distilled H\u003csub\u003e2\u003c/sub\u003eO washing. For TGF-β (MAB1835, clone #1D11, R\u0026amp;D Systems, 1: 25), staining was performed using Mouse-on-Mouse (M.O.M.\u0026trade;, BMK-2202, Vector Laboratories) kit and developed using DAB kit (SK-4100, Vector Laboratories) per manufacturer\u0026rsquo;s protocol. For Ter-119 (14-5921-81, Invitrogen, 1: 100), staining was performed using goat anti-rat HRP (31470, Invitrogen, 1: 500) and developed using DAB kit, followed by Methyl Green counterstain. The sections were mounted in DPX mounting media (Electron Microscopy Sciences) and imaged on a Nikon Eclipse 80i microscope using NIS Elements software. For Immunofluorescence, cryosections were air-dried completely and washed with distilled H\u003csub\u003e2\u003c/sub\u003eO to remove OCT. The sections were then permeabilized/blocked in blocking buffer (4% goat normal serum, 1% BSA and 0.5% Triton X-100 in PBS) at room temperature (R.T.) for 1 hr, incubated with primary antibodies at 4\u0026deg;C for 20 hrs, and secondary antibodies at R.T. for 1 hr in blocking buffer. Finally, the sections were stained with DAPI for nuclei at R.T. for 5 min and mounted with Vectashield Mounting Media (Vector Laboratories). The following primary antibodies were used: LMO7 (sc-98422x, Santa Cruz, 1: 100), LGALS3 (CL8942AP, Cedarlane, 1: 100), ACTA2 (sc-32251 AF546, Alexa 546-conjucated, Santa Cruz, 1: 300), CD68 (MCA1957, Bio-Rad, 1: 100), Ki67 (MA5-14520, Thermo, 1: 200), KLF4 (#4038, Cell Signaling, 1: 100), p-SMAD3 (S423/425) (ab52903, Abcam, 1: 100), ATF4 (#11815, Cell Signaling, 1: 100), SOAT1 (ab39327, Abcam, 1: 100). Appropriate fluorescent secondary antibodies were purchased from Molecular Probes and diluted 1: 300 in blocking buffer. For cleaved Caspase 3 (cCasp3) staining (#9664, Cell Signaling, 1: 150), VECTASTAIN\u0026reg; Elite ABC kit (PK-6101, Vector Laboratories) was used and developed by Tyramide signal amplification (NEL705A001KT, Akoya Biosciences) steps. Micrographs were captured on a Nikon Eclipse Ti Confocal Microscope using Volocity software.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrimary tissue or cell isolation and cell culture.\u003c/b\u003e To isolate primary SMC for cell culture mice were sacrificed, perfused with PBS, and the descending aortas were isolated by microdissection. The vessels were briefly digested in adventitia stripping mix (175U/ml Collagenase II and 1.25U/ml elastase in HBSS) for 10 min, and the adventitia was peeled off. Then the vessels were cut into 1-2mm long rings and digested in digestion mix (400U/ml collagenase II, 2.5U/ml elastase and 0.2mg/ml soybean trypsin inhibitor in HBSS) for 1hr at 37\u0026deg;C with shaking at 700rpm. The cell suspension was centrifuged at 20 X g for 5 min, washed once with DMEM\u0026thinsp;+\u0026thinsp;20% FBS\u0026thinsp;+\u0026thinsp;Penicillin/Streptomycin (Pen/Strep) and plated in culture dish in the same media. Cells were subcultured 1: 3\u0026thinsp;~\u0026thinsp;4 upon becoming 90% confluent. Primary human aortic SMCs were purchased from Lonza at passage 3 and propagated in M199 media with 10% FBS, 2.7 ng/ml EGF, 2 ng/ml FGF and Pen/Strep (complete media). Cells were used for experiments between passages 4\u0026ndash;7. Drugs used in the experiments: Cycloheximide (Sigma, C7698-1G), MG132 (Selleckchem, S2619), methyl-β-cyclodextrin cholesterol (Sigma, C4951), K-604 (Biovision, B-2926).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient transfection of siRNA.\u003c/b\u003e Transient transfection of small interfering RNA (siRNA) in hCASMC was performed using Lipofectamine\u0026reg; RNAiMAX (Life Technologies). Cells were plated the day before transfection to reach a confluency of 70%. Transfection reagent and siRNA were mixed in OPTI-MEM (Gibco) and added to the cells as per the Lipofectamine instructions. M199 (human ASMC) or DMEM (mouse ASMC) media with 20% FBS was added to the cells after 6 hours. After another 18 hours, the media was changed to M199 complete media (human ASMC) or DMEM\u0026thinsp;+\u0026thinsp;20% FBS (mouse ASMC). siRNA for LMO7 or SOAT1 and nonsilencing siRNAs (siControl) were purchased from Invitrogen.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCholesterol biochemical assay.\u003c/b\u003e Mouse ASMCs were starved for 16 hrs and treated with 10 ug/ml cholesterol for 72 hrs. Cells were washed with PBS and scraped in 1ml PBS. Cells were spun down at 200 X g for 5 min. 1/10 of the cells were lysed in RIPA buffer and used for protein quantification and the remainder were used for cholesterol assay. Cholesterol was extracted from these cells with 200 ul of chloroform: isopropanol: NP40 (7:11:0.1). The pellet was spun at 15,000xg for 10 min and the supernatant containing lipid was transferred to a clean glass bottle and dried under nitrogen gas. Dried lipid was dissolved with 200 ul 1X Reaction Buffer provided in the Amplex\u0026reg; Red Cholesterol Assay Kit (Invitrogen, A12216). Total cholesterol (TC) was measured using the kit with esterase in the reaction, while free cholesterol (FC) was measured without esterase. Cholesterol ester (CE) level was calculated by subtracting FC from TC. The mass of each cholesterol fraction was normalized to the mass of protein of the corresponding sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell lysis and western blotting.\u003c/b\u003e Cells were briefly washed with PBS and scraped in cold 1X RIPA buffer supplemented with Protease inhibitor cocktail and PhosSTOP (Roche) on ice. Cell lysates were centrifuged at 20,000 x g for 3 min at 4\u0026deg;C and the supernatant was mixed with loading buffer and heated at 95\u0026deg;C for 5 min. Equal amounts of protein from different samples were separated on SDS-PAGE gel, transferred onto PVDF membrane and immunoblotted with primary antibodies at 4\u0026deg;C for 16 hrs and secondary antibodies (Pierce) at R.T. for 1 h. Blots were developed using SuperSignal\u0026reg; Chemiluminescence Substrate (Pierce). Digital images were taken with Gel Doc\u0026trade; XR\u0026thinsp;+\u0026thinsp;System using ImageLab software. Primary antibodies used: LMO7 for human samples (ab224113, Abcam, 1: 1000), LMO7 for mouse samples (sc-98422x, Santa Cruz, 1: 2000), LGALS3 (CL8942AP, Cedarlane, 1: 1000), MYH11 (M7786, Sigma, 1:1000), KLF4 (ab129473, Abcam, 1: 1000), ATF4 (#11815, Cell Signaling, 1: 1000), CHOP (#2895, Cell Signaling, 1: 500), SOAT1 (ab39327, Abcam, 1: 1000), Ubiquitin (13724, Cayman, 1: 1000), β-Tubulin (sc-9104, Santa Cruz, 1: 500), GAPDH (#2118, Cell Signaling, 1: 2000), β-actin (#3700, Cell Signaling, 1: 1000). Secondary antibodies conjugated with HRP were purchased from Thermo Fisher and used at 1: 3000 dilution. Densitometry of bands was quantitated using ImageJ.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprecipitation.\u003c/b\u003e Cells were washed and scraped as above except using 1X Cell Lysis Buffer (Cell Signaling). After centrifugation, equal amount of cell lysate from each sample was mixed with primary antibodies with gentle rotation at 4\u0026deg;C for 16 hrs. Then magnetic beads (Bio-Rad) were added and rotated for another 2 hrs to pull down antibody-protein complexes. Beads were washed with Cell Lysis Buffer twice and heated in the presence of loading buffer at 95\u0026deg;C for 5 min. Primary antibodies used for IP: SOAT1 (ab39327, Abcam, 1: 100), normal Rabbit IgG (#2729, Cell Signaling). Western analysis was the same as above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR.\u003c/b\u003e Cells were briefly washed with PBS and scraped in TRIzol\u0026trade; (Invitrogen) and RNA was extracted and purified using Direct-zol\u0026trade; RNA Miniprep Plus kit (ZYMO Research). cDNA was reverse transcribed using 5X All-In-One RT MasterMix (G486, ABM) with equal amount of RNA from each sample. cDNA was then diluted 1: 10 and used for quantitative real-time PCR analysis (Bio-Rad CFX96 Real-Time System) using BrightGreen qPCR MasterMix (MasterMix-S, ABM). For miRNA analysis, miRNAs were purified using miRNeasy Kit (217004, Qiagen) and reverse transcribed using miRNA cDNA Synthesis Kit (G902, ABM), followed by real-time qPCR analysis using BrightGreen miRNA qPCR MasterMix (MasterMix-mS, ABM). qPCR was analyzed using the delta delta CT method. All samples were run in duplicate, and signals were normalized to the indicated housekeeping genes/microRNAs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analysis was performed using Graph-Pad (Prism7). Comparisons between two samples were performed using the nonparametric Mann-Whitney U test. Data sets with three or more groups were analyzed by 2-way ANOVA followed by Holm-Sidak multiple comparisons testing. Comparisons between two curves were performed by Nonlinear Regression with Extra sum-of-squares F test. P-value smaller than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLMO7 is induced in mouse atherosclerosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined LMO7 expression in SMC and other cells during murine atherosclerosis development. We generated a smooth muscle-specific, tamoxifen-inducible, ZsGreen reporter mouse in the \u003cem\u003eApoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e background (\u003cem\u003eMyh11_CreER\u003csup\u003eT2\u003c/sup\u003e\u003c/em\u003e; \u003cem\u003eGt(ROSA26)-ZsGreen;\u0026nbsp;\u003c/em\u003e \u003cem\u003eApoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e). Tamoxifen treatment results in fluorescent labeling of SMCs and their daughter cells (Suppl Fig. 1a). We subjected these mice to high fat diet (HFD) feeding, harvested the brachiocephalic artery (BCA) at multiple time points, and immunostained for LMO7 and LGALS3\u003csup\u003e13\u003c/sup\u003e. LMO7 expression was relatively low prior to HFD feeding but was greatly induced in the plaque by HFD, although not in medial layer (Suppl Fig. 1a). We found that cholesterol loading of cultured SMC was sufficient to induce robust expression of LMO7 \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(Suppl Fig. 1b). LMO7 staining was found in ZsGreen\u003csup\u003e+\u003c/sup\u003e and ZsGreen\u003csup\u003e-\u003c/sup\u003e cells and was most prominent after 4-weeks HFD feeding in ZsGreen\u003csup\u003e-\u003c/sup\u003e/LGALS3\u003csup\u003e+\u003c/sup\u003e cells, which are likely macrophages. However, from 8 weeks onward, there was extensive LMO7 staining in ZsGreen\u003csup\u003e+\u003c/sup\u003e cells, suggesting potential roles in SMC as well as in macrophages. Intriguingly, LGALS3 staining directly correlated with the LMO7 staining pattern in ZsGreen\u003csup\u003e+\u003c/sup\u003e cells, suggesting a potential role for LMO7 in transdifferentiating SMC (Suppl Fig. 1a, arrows).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlobal Knockout of LMO7 induces larger plaque but with a stabilized structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the function of LMO7 during atherosclerosis progression, we compared \u003cem\u003eLmo7\u003c/em\u003e global knockout mice (\u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e)\u003c/em\u003e crossed with\u003cem\u003e\u0026nbsp;Apoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice to \u003cem\u003eApoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e control after 12 weeks HFD. There was no significant difference in total plasma cholesterol or triglycerides before and after HFD feeding between the two groups (Suppl Fig. 2a). We observed a significantly larger Oil Red-O (ORO) positive area in the whole aorta \u003cem\u003een face\u003c/em\u003e, in segments of the aortic arch, and in the brachiocephalic artery (BCA) in the \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice compared to the controls (Suppl Fig. 2b-c).\u003c/p\u003e\n\u003cp\u003eWe further examined the histological structure of BCA plaques. Lesion size was larger in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, but the necrotic core was smaller, and fibrous cap was thicker (Suppl Fig. 2d), suggesting that LMO7 deletion enhanced plaque stability. We also note a reduced fraction of \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice with erythrocyte infiltration in the plaque (Ter-119 staining), a surrogate for spontaneous plaque rupture (Suppl Fig. 2e). Consistent with this, we observed a larger collagen\u003csup\u003e+\u003c/sup\u003e area in the \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eBCA plaques using polarized microscopy of Picro-Sirius Red (PSR) stained sections (Suppl Fig. 2f). These data reveal that global LMO7 knockout yielded a larger but more stabilized plaque.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockout of LMO7 in SMC alters SMC phenotype and promotes features of plaque stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the function of LMO7 specifically in SMC during atherosclerosis progression, we generated \u003cem\u003eGt(ROSA26)-ZsGreen\u003c/em\u003e\u003cem\u003e, Lmo7\u003csup\u003efl/fl\u003c/sup\u003e, Myh11_creER\u003csup\u003eT2\u003c/sup\u003e, Apoe\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e(Lmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e) mice. These mice, along with \u003cem\u003eGt(ROSA26)-ZsGreen, Lmo7\u003csup\u003e+/+\u003c/sup\u003e, Myh11_creER\u003csup\u003eT2\u003c/sup\u003e, Apoe\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e (control) mice, were injected with tamoxifen and fed with HFD for 12 weeks. There was no significant difference in total cholesterol or triglycerides between the two groups (Fig. 1a). There was no difference in ORO staining in the whole aorta \u003cem\u003een face\u0026nbsp;\u003c/em\u003ebetween the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e and control mice, but we observed a significantly larger ORO positive area in the aortic arch and BCA plaques in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 1b-c). H\u0026amp;E staining in BCA plaques showed no difference in lesion size between the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e and control mice; however, we noted multiple features of enhanced stability in the\u003cem\u003e\u0026nbsp;Lmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice, including a smaller necrotic core and thicker fibrous cap (Fig. 1d), lower fraction of mice with Ter-119\u003csup\u003e+\u003c/sup\u003e staining (Fig. 1e), and a larger collagen\u003csup\u003e+\u003c/sup\u003e area (Fig. 1f).\u003c/p\u003e\n\u003cp\u003eCell composition is an important determinant of plaque stability\u003csup\u003e19, 20\u003c/sup\u003e, with a greater SMC to macrophage ratio in the lesion, and especially in the fibrous cap area, correlating with plaque stability\u003csup\u003e21\u003c/sup\u003e. We observed a larger ACTA2\u003csup\u003e+\u003c/sup\u003e (SMC) area and smaller CD68\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e(macrophage) area in the plaques of \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice compared to controls, and a higher percentage of ACTA2\u003csup\u003e+\u003c/sup\u003e cells and a lower percentage of CD68\u003csup\u003e+\u003c/sup\u003e cells in the fibrous cap area of \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 1g), indicating a more favorable cellular composition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe noticed a marked increase in SMC-derived (ZsGreen\u003csup\u003e+\u003c/sup\u003e) cells in the BCA lesions of \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice compared to controls (Fig. 1g, 2a). To quantitatively assess the phenotypes of these SMC-derived cells, we immunostained for ACTA2 and LGALS3. Similar to CD68 (Fig. 1g), there was a reduction in LGALS3\u003csup\u003e+\u003c/sup\u003e cells in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e compared to controls (Fig. 2a). In the fibrous cap, we noted more ZsGreen\u003csup\u003e+\u003c/sup\u003e cells in \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e versus control lesions, and a higher percentage of these cells were ACTA2\u003csup\u003e+\u003c/sup\u003e (Fig. 2a). There was also a trend toward a lower percentage of ZsGreen\u003csup\u003e+\u003c/sup\u003e LGALS3\u003csup\u003e+\u003c/sup\u003e cells relative to total DAPI\u003csup\u003e+\u003c/sup\u003e nuclei in the cap (Fig. 2a). In the ZsGreen\u003csup\u003e+\u003c/sup\u003e population, there were more ACTA2\u003csup\u003e+\u003c/sup\u003e and fewer LGALS3\u003csup\u003e+\u003c/sup\u003e cells, indicating that the phenotypic modulation of SMC was skewed toward more protective and less inflammatory states in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice. Moreover, the percentage of ZsGreen\u003csup\u003e-\u003c/sup\u003e LGALS3\u003csup\u003e+\u003c/sup\u003e cells relative to DAPI\u003csup\u003e+\u003c/sup\u003e nuclei was also reduced (Fig. 2a), indicating that the recruitment, or retention of macrophages in the lesion was also suppressed in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether the increased number of SMC-derived (ZsGreen\u003csup\u003e+\u003c/sup\u003e) cells in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e lesions was due to altered SMC proliferation and/or apoptosis, we immunostained for Ki67, a proliferation marker, and cleaved caspase 3 (cCasp3), an apoptosis marker, in the BCAs. There were more ZsGreen\u003csup\u003e+\u003c/sup\u003e Ki67\u003csup\u003e+\u003c/sup\u003e cells in the fibrous cap of the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Suppl Fig. 3), indicating enhanced SMC proliferation. This is consistent with our prior report of enhanced SMC proliferation in\u003cem\u003e\u0026nbsp;Lmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice following vascular injury\u003csup\u003e15\u003c/sup\u003e. We found that there were fewer ZsGreen\u003csup\u003e+\u003c/sup\u003e cCasp3\u003csup\u003e+\u003c/sup\u003e cells within fibrous cap cells (DAPI\u003csup\u003e+\u003c/sup\u003e) and in the ZsGreen\u003csup\u003e+\u003c/sup\u003e SMC (Fig. 2b), revealing that LMO7 limits proliferation and promotes apoptosis in plaque SMCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic modulation by cholesterol treatment is suppressed in LMO7-deficient SMC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next determined whether LMO7 could regulate cholesterol-induced SMC transdifferentiation \u003cem\u003ein vitro\u003c/em\u003e by treating primary mouse aortic SMC isolated from WT or\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice with cyclodextrin-balanced cholesterol (as in\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e). As expected, qPCR analysis indicated that cholesterol treatment inhibited \u003cem\u003eMyh11\u003c/em\u003e and induced \u003cem\u003eLgals3\u003c/em\u003e RNA in WT SMC (Fig. 2c). Cholesterol-induced changes in these lineage markers were significantly reduced in \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e SMC (Fig. 2c), consistent with an inhibited transdifferentiation in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 2a). Cholesterol treatment reduces SMC collagen expression \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. We noted higher RNA levels of several collagen genes in \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003ecells compared to WT both at baseline and after cholesterol treatment (Suppl Fig. 4), consistent with the higher ECM content in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 1f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further evaluated KLF4, a master regulator of SMC phenotypic modulation\u003csup\u003e9\u003c/sup\u003e. Similar to \u003cem\u003eLgals3\u003c/em\u003e, cholesterol treatment induced \u003cem\u003eKlf4\u003c/em\u003e mRNA in both WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC, but to a lesser extent in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003ecells (Fig. 2d). These differences were also observed in the protein levels of KLF4, LGALS3 and MYH11 in WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e SMC lysates (Fig. 2f). \u0026nbsp;Immunostaining for KLF4 was also reduced in BCA lesions in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 2e). Collectively, these data indicate that LMO7 is required for optimal KLF4 expression and SMC phenotypic modulation in response to cholesterol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMO7 regulates lineage markers and KLF4 expression via TGF-\u0026beta;-miR-145 axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous findings demonstrated that loss of LMO7 in SMC induces stronger TGF-\u0026beta; signaling in vascular injury models\u003csup\u003e15\u003c/sup\u003e. Here, in atherosclerosis, we also observed stronger immunostaining for TGF-\u0026beta; as well as p-SMAD3, the major signal-transducer of this pathway, in BCAs of \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice compared to WT lesions (Suppl Fig. 5a, b). Considering the protective role of the TGF-\u0026beta; pathway in SMC in atherosclerosis\u003csup\u003e23\u003c/sup\u003e, we tested whether TGF-\u0026beta; contributes to the anti-transdifferentiation effect of LMO7 depletion. Co-treatment of cultured SMC with cholesterol and SB431542, an inhibitor of TGF\u0026beta;RI, largely eliminated the difference between WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e in expression of \u003cem\u003eMyh11,\u003c/em\u003e \u003cem\u003eLgals3,\u003c/em\u003e and \u003cem\u003eKlf4\u003c/em\u003e (Suppl Fig. 5c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that TGF-\u0026beta; suppresses KLF4 expression by inducing the miR-143/145 microRNA cluster\u003csup\u003e24\u003c/sup\u003e. Moreover, cholesterol treatment induces KLF4 by inhibiting miR-145 in SMC\u003csup\u003e25\u003c/sup\u003e. Therefore, we assessed miR-145 expression in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC after cholesterol and SB431542 treatment. We noticed higher levels of miR-145 at baseline in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC (Suppl Fig. 5d), consistent with increased TGF-\u0026beta; signaling\u003csup\u003e15\u003c/sup\u003e. Cholesterol treatment inhibited miR-145 expression in both WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e SMC, likely due to the suppression of TGF-\u0026beta; signaling, however, miR-145 expression remained significantly higher in \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC compared to WT SMC (Suppl Fig. 5d). Lastly, co-treatment of SMC with TGF\u0026beta;RI inhibitor and cholesterol further suppressed miR-145 levels in \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC down to the expression observed in WT cells (Suppl Fig. 5d). The regulation of miR-145 is consistent with that of KLF4 by LMO7. These data suggest that the TGF-\u0026beta;-miR-145-KLF4 axis could, at least in part, mediate the LMO7-regulated SMC phenotypic modulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMO7 regulates ER stress via cholesterol metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndoplasmic reticulum (ER) stress is elevated in SMC in hyperlipidemic conditions\u003csup\u003e26\u003c/sup\u003e, and has also been implicated in KLF4 regulation: \u0026nbsp;ER stress activates ATF4, which in turn transactivates the \u003cem\u003eKLF4\u003c/em\u003e gene and inhibits KLF4 protein degradation\u003csup\u003e27\u003c/sup\u003e. We therefore hypothesized that LMO7 may influence KLF4 expression by modifying cholesterol-induced ER stress. Cholesterol treatment induced a significant upregulation of ATF4 and its downstream pro-apoptotic gene CHOP in WT SMC, but not in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC (Fig. 3a). Additionally, immunostaining showed less ATF4 expression in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice compared to control (Fig. 3b), indicating attenuated ER stress.\u003c/p\u003e\n\u003cp\u003eExcess of free cholesterol (FC) is a major inducer of ER stress\u003csup\u003e26, 28\u003c/sup\u003e. We determined that FC levels as measured by filipin staining were lower in the \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e SMCs compared to WT cells following cholesterol treatment (Fig. 3c). Differential cholesterol uptake does not likely explain this difference in FC as our cell-based transdifferentiation assays employ a water-soluble cholesterol formulation. Furthermore, there was no difference in \u003cem\u003eLrp1\u003c/em\u003e mRNA, the major receptor for LDL uptake in SMC\u003csup\u003e29\u003c/sup\u003e between WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e SMC (Suppl Fig. 6a). We also found no differences in mRNA for critical genes in the cholesterol biosynthesis pathway, including \u003cem\u003eSqle, Cyp51\u003c/em\u003e, \u003cem\u003eDhcr24\u003c/em\u003e and \u003cem\u003eHmgcr\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e between WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC either at baseline or with cholesterol (Suppl Fig. 6b). There was also no change in ABCA1, the main membrane transporter which mediates cholesterol efflux\u003csup\u003e31\u003c/sup\u003e (Suppl Fig. 6c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe hypothesized that FC was reduced in \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC through enhanced conversion of FC to cholesterol ester (CE), an important mechanism shown to reduce cholesterol cytotoxicity\u003csup\u003e26, 32\u003c/sup\u003e. This could potentially explain the \u003cem\u003ein vivo\u003c/em\u003e data showing higher ORO positive area in the aortic arch (Fig. 1b) and BCA (Fig. 1c) of \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice, as ORO stains specifically neutral lipids including CE but not FC. We performed ORO staining on cholesterol-treated mouse SMC in culture and observed more ORO\u003csup\u003e+\u003c/sup\u003e lipid droplets in the \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC (Fig. 3d). This observation correlated with increased cholesterol esters found in LMO7-deficient SMC (Fig. 3e). Total cholesterol (TC) was similar in WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC but FC reduced in cells lacking Lmo7 (Fig. 3e). Together, these data suggested that the reduced level of FC in \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003eSMC is likely due to its accelerated conversion to CE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMO7 regulates SOAT1 expression by mediating its ubiquitination and proteasomal degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSterol o-acyltransferase (SOAT; also called ACAT) catalyzes the conversion of FC to CE. Notably, we observed an increase expression of SOAT1, the major SOAT isoform in SMC, in \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC compared to WT SMC (Fig. 4a). Concordantly, \u003cem\u003ein vivo\u003c/em\u003e analysis showed stronger SOAT1 immunofluorescence in ZsGreen\u003csup\u003e+\u003c/sup\u003e cells in the BCA of the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 4b), suggesting that LMO7 may attenuate SOAT1 expression. We did not see a significant difference in \u003cem\u003eSoat1\u003c/em\u003e mRNA between WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC (Suppl Fig. 6d), suggesting that LMO7 might control SOAT1 expression at the post-transcriptional level. Thus, we analyzed whether LMO7, an F-box protein that mediates proteasomal degradation of target proteins\u003csup\u003e15\u003c/sup\u003e, interacts directly with SOAT1. Our results show that SOAT1 and LMO7 co-immunoprecipitate (Fig. 4c) and suppression of protein synthesis using cycloheximide (CHX) revealed a slower rate of SOAT1 protein decay (enhanced protein half-life) in the \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC compared to WT cells (Fig. 4d). These results suggest that LMO7 might regulate SOAT1 protein stability and degradation. Indeed, we observed that the proteasome inhibitor MG-132 enhanced the SOAT1 ubiquitin signal in control but not LMO7 depleted cells (Fig. 4e), indicating that LMO7 can mediate the ubiquitination of SOAT1 protein.\u003c/p\u003e\n\u003cp\u003eTo verify the function of SOAT1 in LMO7-mediated SMC phenotypic modulation, we next assessed cholesterol-induced SMC transdifferentiation in the presence of the SOAT1 inhibitor K-604. In both WT and \u003cem\u003eLmo7\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eSMC, SOAT1 inhibition enhanced the induction of KLF4, LGALS3, and the ER stress markers ATF4 and CHOP (Fig. 4f). Importantly, SOAT1 inhibition abrogated the effect of \u003cem\u003eLmo7\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e, such that levels of KLF4 and LGALS3 were comparable to WT controls (Fig. 4f). These data suggest that modulation of cholesterol-induced SMC transdifferentiation, by LMO7, requires SOAT1-dependent regulation of cholesterol metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle cell RNA sequencing revealed a distinct cellular composition of SMC-derived cells in the \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively investigate the changes in plaque composition and SMC phenotype with loss of LMO7, we performed single cell RNA sequencing (scRNAseq) analysis of cells isolated from aortic root, aortic arch and its three major branches which were harvested from control and \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (both on Apoe\u003csup\u003e-/-\u003c/sup\u003e background) fed a HFD for 12 weeks. We combined cell data points from WT and \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e groups for analysis and identified 11 populations by graph-based clustering (Fig. 5a). Based on the gene expression profile, we identified all the major cell types in the atherosclerotic lesion, including SMCs, fibroblasts, macrophages (including classically activated- \u0026ldquo;Macrophage 1\u0026rdquo;, and alternatively activated- \u0026ldquo;Macrophage 2\u0026rdquo; clusters), endothelial cells, neutrophils, and T lymphocytes (Fig. 5b). Five SMC-derived ZsGreen+ clusters were identified (Fig. 5b). Based on the published defining markers for various subpopulations\u003csup\u003e12, 13\u003c/sup\u003e, they were classified as \u0026ldquo;SMC\u0026rdquo; (classical contractile phenotype SMC), cap-stabilizing \u0026ldquo;Phactr1\u003csup\u003e+\u003c/sup\u003e SMC\u0026rdquo;\u003csup\u003e13\u003c/sup\u003e, \u0026ldquo;Transitioning SMC\u0026rdquo; (\u003cem\u003eKlf4\u003csup\u003e+\u003c/sup\u003e Lgals3\u003csup\u003e+\u003c/sup\u003e Myh11\u003csup\u003elow\u003c/sup\u003e\u003c/em\u003e), inflammatory \u0026ldquo;Macrophage-like cell\u0026rdquo; (expressing both SMC and macrophage markers) or rupture-protective \u0026ldquo;Fibromyocyte\u0026rdquo;\u003csup\u003e12\u003c/sup\u003e (Fig. 5a, defining genes shown in Fig. 5b). Interestingly, WT and \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice displayed distinct profiles of SMC-derived populations: WT mice contained 52.2% transitioning SMC, while this population was dramatically reduced (8.0%) in \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 5c). Conversely, there were many more contractile SMC and slightly increased fibromyocytes in \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e mice (Fig. 5c). Interestingly, the macrophage1 cluster was also reduced in \u003cem\u003eLmo7\u003csup\u003ei\u0026Delta;SM\u003c/sup\u003e\u003c/em\u003e plaques, suggesting reduced inflammation (Fig. 5c). These data were consistent with the plaque characterization based on immunostaining and lineage tracing in Fig. 1-2.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMO7 is induced in human atherosclerosis and enriched in ruptured lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether these roles of LMO7 may translate to human atherosclerosis, we evaluated LMO7 mRNA levels in human atherosclerotic arteries from patients undergoing carotid endarterectomy. These samples included normal control regions (Fig. 6a). There was an increase in LMO7 mRNA in all atherosclerotic regions (average of 3-fold induction) compared to a non-diseased control segment of the same artery in each patient (Fig. 6b). Additionally, there was 5.7-fold more LMO7 mRNA in ruptured human plaque samples compared to stable plaques (Fig. 6b). These data reveal a positive correlation between SMC LMO7 expression and atherosclerotic plaque vulnerability in patients.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe most salient finding of our study is the identification of LMO7 as a key molecule that integrates cellular cholesterol metabolism and SMC transdifferentiation affecting atherosclerotic plaque composition and stability. We demonstrate that cholesterol-induced LMO7 promotes SOAT1 ubiquitination and degradation leading to accumulation of FC. LMO7 depletion stabilizes SOAT1, promoting cholesterol esterification in SMC. This alleviates ER stress-induced induction of KLF4 and inflammatory SMC fate transitions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Finally, our study also reveals that enhancing cholesterol esterification dramatically alters SMC phenotypic modulation and promotes features of plaque stability.\u003c/p\u003e \u003cp\u003eThe pathogenesis of atherosclerosis involves the actions of multiple cell types. The complex role of SMCs in this disease was underestimated until recently. Previously, SMCs were thought to de-differentiate to a synthetic phenotype and participate in fibrous cap formation. While SMC transitions are indeed necessary for fibrous cap formation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, recent studies showed that SMC can also transform into a range of diverse phenotypes in lesions, including macrophage-like cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, fibromyocytes\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, osteoblasts and chondrocytes\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Since these different phenotypic transitions likely exert distinct or even opposite effects with respect to lesion growth and stability, it is crucial to understand the mechanisms controlling cell fate. In the current study, we demonstrated that loss of LMO7 in SMC led to alteration of intracellular cholesterol processing and phenotypic modulation. A comprehensive scRNAseq analysis showed that the \u0026ldquo;transitioning\u0026rdquo; SMC state (\u003cem\u003eKlf4\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eLgals3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMyh11\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e) was substantially reduced in the \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e mice, consistent with analysis of lesion cell composition by immunostaining. Moreover, the LMO7-deficient SMC-derived cells presented enhanced proliferation and viability and deposited larger amounts of ECM, leading to features of stable plaque.\u003c/p\u003e \u003cp\u003eWe demonstrated that LMO7 modulates cholesterol-induced SMC phenotypic switching \u003cem\u003ein vitro\u003c/em\u003e via regulation of KLF4 and LGALS3 expression, the two key players in this process\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We further showed that suppression of KLF4 and LGALS3 expression in \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e SMC was due to the attenuated ER stress and ATF4 activity. Previous studies showed that unresolved ER stress leads to apoptosis in macrophages and SMCs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. ER stress could also induce dramatic phenotypic modulation in SMCs by activating ATF4, thus inducing KLF4 expression\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The Milewicz group recently published a study demonstrating that inhibiting the PERK-eIF2α-ATF4 pathway suppresses SMC transition to macrophage-like cells by cholesterol treatment\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This study supports our finding that cholesterol-induced ER stress is a critical mediator of adverse SMC phenotypic modulation in atherosclerosis, and approaches to suppress SMC ER stress may have therapeutic utility.\u003c/p\u003e \u003cp\u003eIn addition to cholesterol metabolism and ER stress, our data suggest that LMO7 may also regulate KLF4 and LGALS3 expression via TGF-β1 signaling. Our previous study showed that knockout of LMO7 in SMC induced elevated TGF-β1 expression and signaling, contributing to enhanced collagen deposition in injury-induced vascular remodeling\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Interestingly, cholesterol treatment could suppress TGF-β1 signaling and downstream effectors\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We now report that SMC LMO7 depletion enhances TGF-β1 expression and signaling in atherosclerotic plaques, and that TGF-β1 inhibition blunts the protective effect of LMO7 depletion on cholesterol-induced SMC transdifferentiation \u003cem\u003ein vitro\u003c/em\u003e. These data indicate that LMO7 can influence KLF4 expression through a TGF-β1-dependent miR-145 axis. It is likely that the ability of LMO7 to regulate both TGF-β1 signaling and cholesterol esterification accounts for the potent effects of this protein on SMC fate transitions in atherosclerosis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLmo7\u003c/em\u003e global knockout or only in SMC-derived cells elicited a similar phenotype of smaller necrotic core and thicker fibrous cap, although \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had larger lesions compared to control, while the SMC \u003cem\u003eLmo7\u003c/em\u003e deficient mice did not, indicating that LMO7 may also play roles in other relevant cell types. For example, TGF-β1-induced endothelial-mesenchymal transitions can contribute to atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Macrophages may also play a role in the phenotype of \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Intriguingly, in our scRNAseq analysis with \u003cem\u003eLmo7\u003c/em\u003e\u003csup\u003e\u003cem\u003eiΔSM\u003c/em\u003e\u003c/sup\u003e mice, we observed a dramatic alteration of macrophage subpopulations, indicating that SMC exert a paracrine effect on macrophages, potentially through TGF-β1 signaling, which has been shown to influence macrophage classical versus alternative activation patterns\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our findings suggest that targeting LMO7 specifically in SMC has beneficial effects on lesion composition without enhancing lesion size.\u003c/p\u003e \u003cp\u003eIn atherosclerosis, monocyte-derived macrophages were thought to be the major cell type to process excessive cholesterol, which internalize modified lipoproteins and become foam cells. However, recent studies in human and mouse lesions revealed that majority of the foam cells are derived from SMCs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This finding highlights the role of SMC in cholesterol storage and its contribution to the control of lipid content in the plaque. In our study, loss of LMO7 in SMC enhanced the conversion of FC to CE, a critical step of lipid droplet formation in foam cells, and increase in foam cell number is associated with reduced SMC transition to macrophage-like cells, as well as with a more stable plaque. Interestingly, previous studies showed that foamy cells derived from macrophages are less inflammatory than non-foamy cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, suggesting that foam cell formation for lipid storage is a protective mechanism in hyperlipidemic disease.\u003c/p\u003e \u003cp\u003eCurrent treatments targeting atherosclerotic vascular diseases, including statins and anti-PCSK9 antibodies, aim to lower circulating lipid levels. These therapies have proven to be effective, but there are still an estimated 805,000 myocardial infarctions per year, and stroke accounts for \u0026gt;\u0026thinsp;5% of all deaths in the US\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, indicating that additional approaches are required to prevent the occurrence of atherothrombotic events. Enhancing plaque stability may be a beneficial strategy. In our study, when cholesterol esterification was enhanced via elevated SOAT1 by manipulating LMO7 expression globally, or in a SMC-specific manner, it induced features of stabilized plaque, with smaller necrotic cores and thicker fibrous caps. This change in cholesterol storage did not affect circulating cholesterol and triglyceride or cellular TC levels, indicating that promoting cholesterol storage in the esterified form can by itself enhance lesion stability, or potentially combine with the existing lipid-lowering therapies to further reduce incidence of myocardial infarction and stroke.\u003c/p\u003e \u003cp\u003eCholesterol esterification is catalyzed by SOAT, which may therefore serve as a potential drug target. SOAT inhibition has been evaluated in mouse atherosclerosis with conflicting results\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Studies from the Chang lab showed that myeloid-specific knockout of ACAT1/SOAT1 in \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice reduced lesional macrophage content and suppressed atherosclerosis progression\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Additionally, administration of an ACAT/SOAT inhibitor in \u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice reduced atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. However, Accad et al. reported that global knockout of SOAT1/ACAT1 reduced neutral lipids but resulted in more macrophages in the lesions\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Further, Fazio et al. demonstrated that transplantation of ACTA1/SAOT1-deficient bone marrow to the \u003cem\u003eLDLR\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exacerbated atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, suggesting a beneficial role of macrophage SOAT1. Importantly, in patients receiving standard care for secondary prevention, administration of ACAT/SOAT inhibitors elicited less normalized atheroma volume regression compared to placebo group (ATIVATE trial\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e), and increased the incidence of major cardiovascular events in patients with familial hypercholesterolemia (CAPTIVATE trial\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e), indicating a protective role of SOAT activity in human cardiovascular disease. Our results that loss of LMO7 in SMC promoted plaque stability by induction of SOAT1 expression provide additional insights into the beneficial role of SOAT1. This assertion is strengthened by the finding that inhibition of SOAT activity in mouse ASMC enhanced their transition to macrophage-like cells by cholesterol treatment\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and abolished the protective effect of LMO7 depletion on this process. These findings suggest that SOAT1 in SMCs attenuates the cholesterol-induced SMC phenotypic transitions that destabilize atherosclerotic plaques, such that enhancing SMC SOAT1 activation and cholesterol esterification may be a potential therapeutic strategy.\u003c/p\u003e \u003cp\u003eWe report that LMO7 is induced by cholesterol in SMC in plaques, and regulates cholesterol processing, which, in turn, influences SMC phenotypic transitions, plaque composition, and features of stability. Notably, LMO7 is also induced in human atherosclerosis compared to healthy vessel and is expressed at higher levels in ruptured or unstable compared to stable lesions. Interestingly, an LMO7 SNP (rs2273996) has been associated with human coronary artery disease\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. These studies suggest that LMO7 expression and binding partners and cholesterol esterification specifically in SMC could be of interest for targeting lesion stability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the NIH to K.A.M. (R01HL151222,\u0026nbsp;R01HL142090,\u0026nbsp;R01 HL146101), to J.H. (R01HL115247A, R01HL122815), and to CF-H (R35HL135820).\u0026nbsp;Research in L.M.\u0026rsquo;s laboratories is funded by the Swedish Heart-Lung-Foundation (20210519), the Swedish Research Council (Vetenkapsr\u0026aring;det, 2019-01577), the German Center for Cardiovascular Research (DZHK), the SFB1123 and TRR267 of the German Research Council (DFG), the National Institutes of Health (NIH; 1R011HL150359-01), the Bavarian State Ministry of Health and Care through the research project \u003cem\u003eDigiMed\u003c/em\u003e Bayern.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eY.X., K.A.M., C.F.-H., and L.M. designed and analyzed the experiments. Y.X. and K.A.M. wrote the manuscript. Y.X., H.W. and N.R. performed the experiments. All co-authors contributed to analyzing and interpreting data and editing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR, Delling FN, Djousse L, Elkind MSV, Ferguson JF, Fornage M, Jordan LC, Khan SS, Kissela BM, Knutson KL, Kwan TW, Lackland DT, Lewis TT, Lichtman JH, Longenecker CT, Loop MS, Lutsey PL, Martin SS, Matsushita K, Moran AE, Mussolino ME, O'Flaherty M, Pandey A, Perak AM, Rosamond WD, Roth GA, Sampson UKA, Satou GM, Schroeder EB, Shah SH, Spartano NL, Stokes A, Tirschwell DL, Tsao CW, Turakhia MP, VanWagner LB, Wilkins JT, Wong SS, Virani SS, American Heart Association Council on E, Prevention Statistics C and Stroke Statistics S. 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Circ Res. 2016;118:83\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1359588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1359588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSmooth muscle cells (SMC) can undergo distinct fate transitions that influence atherosclerotic plaque phenotype: SMC make up the protective fibrous cap that prevents plaque rupture, but can also transdifferentiate to a destabilizing pro-inflammatory phenotype. We found that LMO7, a scaffolding protein that modulates SMC phenotype, is induced in atherosclerosis. SMC LMO7 deletion produced a paradoxical phenotype with enhanced indices of plaque stability despite greater lipid content. We determined that LMO7 promotes SOAT1 ubiquitination and degradation. LMO7 depletion thus favored cholesterol esterification and “safe” storage, relieving ER stress and subsequent induction of KLF4 and SMC transdifferentiation. Immunostaining and single cell sequencing confirmed that SMC LMO7 depletion enhanced cap stabilizing fate transitions while substantially reducing inflammatory transdifferentiation. Similarly, LMO7 was induced in human atherosclerosis, but at lower levels in stable versus ruptured plaques. These findings reveal that SMC cholesterol esterification, rather than total cholesterol content, influences plaque composition and stability.\u003c/p\u003e","manuscriptTitle":"LMO7 regulates smooth muscle cell cholesterol esterification to influence plaque stability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-15 20:50:02","doi":"10.21203/rs.3.rs-1359588/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c8988a3-8acc-443c-91e0-fb0737d67dcd","owner":[],"postedDate":"February 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-03-16T09:26:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-15 20:50:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1359588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1359588","identity":"rs-1359588","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}