Journal: PLoS ONE

Article Title: Instruction of Circulating Endothelial Progenitors In Vitro towards Specialized Blood-Brain Barrier and Arterial Phenotypes

doi: 10.1371/journal.pone.0084179

Figure Lengend Snippet: Accession numbers of TaqMan® (Applied Biosystems) assays used for the quantitative-PCR.

Article Snippet: LMOD1 , Hs00201704_m1.

Techniques:

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Role of LMOD1 actin nucleation domain on CAD phenotype in Lmod1 SMKO mice. ( A ) LRR domain of mouse LMOD1 bound to an actin subunit at the pointed end of the actin filament, showing the mutated amino acids in LMOD1 ND (K444D, H448D, R455D, R470D, and R473D). Model produced based on superimposition of PDB codes 4Z79, 5WFN, and 8F8S. , Also shown at right is an SDS-PAGE gel of E. coli -expressed human LMOD1 WT and LMOD1 ND (K449D, H453D, R460D, R475D, R478D). ( B ) Time course of polymerization of 2 μM actin (6% pyrene-labelled), measured as the fluorescence increase upon incorporation of pyrene-actin into filaments. Curves represent the average of three independent experiments, color-coded by protein construct and concentration, with standard deviations (SD) shown. ( C ) Lmod1 SMKO MASMC incubated with oxLDL in the presence of Lentivirus carrying each indicated construct with quantitation of oxLDL area/nuclei. ( D ) Western blot of indicated proteins from similar experiment as in panel C . ( E ) Strategy for making the inducible Lmod1 iND mouse model. The wildtype mouse ( top ) expresses normal levels of LMOD1 from the minigene placed in first intron which is recombined out upon Cre exposure ( bottom ) enabling expression of the mutated second exon (E2-iND) which contains the five amino acid substitutions. Validation studies demonstrate the loss of Lmod1 WT and induction of Lmod1 iND mRNA ( F ) and protein ( G ) with Tagln-Cre . ( H ) CIFM of LMOD1 ( Hi, Hiii ) and ORO staining of coronary artery ( Hii, Hiv ) in Lmod1 WT and Lmod1 iND (induced with Tagln-Cre ) mice treated for 10 weeks with PCSK9/HFD. n ≥ 5 mice per genotype. Scale bars, 100 μm for CIFM and 20 μm for ORO staining.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Produced, SDS Page, Fluorescence, Construct, Concentration Assay, Incubation, Quantitation Assay, Western Blot, Expressing, Biomarker Discovery, Staining

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: LMOD1 omics analyses in human coronary artery atherosclerosis. UMAP (A) and attending feature plot of LMOD1 (B) derived from indicated scRNA-seq study of human coronary atherosclerosis. UMAP (C) and attending feature plot of LMOD1 (D) derived from indicated scATAC-seq study of human coronary atherosclerosis. (E) Open chromatin occupancy map across the LMOD1 locus in indicated cell types.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Derivative Assay

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Baseline LMOD1 knockout phenotyping. ( A ) Nucleotide substitutions (red) in exon 2 of Lmod1 generate the orthologous R365* premature stop codon previously reported in a human patient. ( B ) Gastroparesis (yellow arrow) and megabladder (below) in Lmod1 R365* mouse phenocopies the same visceral myopathy described in a human. ( C ) Design of floxed Lmod1 mouse. Quantitative Lmod1 mRNA ( D ) and LMOD1 protein ( E ) expression in mice with indicated genotypes (n=3 mice). ( F ) Percent recombination of floxed Lmod1 locus with Itga8-CreER T2 driver ( Lmod1 SMKO ) using conventional PCR ( top ) and quantitative PCR ( bottom ) of genomic DNA derived from mouse aorta. ( G ) Quantitative RT-PCR of Lmod1 mRNA in aorta with primers depicted in panel C (n=3 mice). The reduced signal with primers F2+R2 signify the absence of an internal promoter yielding a mature Lmod1 mRNA, thus validating this conditional knockout as a true null allele. 4 ( H ) Intestinal myopathy (arrow) in conditional knockout of Lmod1 using Myh11-CreER T2 (labeled as Lmod1 KO-Myh11 ), but not in mice where the Itga8-CreER T2 driver was used (labeled Lmod1 KO-Itga8 ). ( I ) Body weights of homozygous floxed Lmod1 / Itga8Cre-ER T2 mice treated with oil (black) or tamoxifen (Tmx; red) beginning at eight weeks of age. The number of mice in each arm of the experiment is indicated in parentheses. ( J ) Systolic blood pressure, assessed by tail cuff method in oil (black) versus Tmx (red) homozygous floxed Lmod1 / Itga8Cre-ER T2 mice (n=6 mice). ( K ) Serum lipopolysaccharide (LPS) levels in conditional Lmod1 knockout mice using the indicated Cre drivers.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Knock-Out, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Labeling

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) Confocal immunofluorescence microscopy (CIFM) of LMOD1 and ACTA2 in aorta and intestine of the indicated mice. Scale bars are 20 μm. Western blotting for LMOD1 in aorta ( B ) and intestine ( C ) of indicated mouse genotype. ( D ) Survival curves for Oil versus Tamoxifen (Tmx) treated floxed Lmod1 / Myh11-CreER T2 (left) and floxed Lmod1 / Itga8-CreER T2 (right) mice. The black arrow indicates the time of Oil or Tmx administration. Western blotting ( E ) and quantitation ( F ) of indicated proteins after six months of Oil or Tmx administration; n=5 independent mice per genotype ( Lmod1 WT is Lmod1 fl/fl ).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Immunofluorescence, Microscopy, Western Blot, Quantitation Assay

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Baseline vascular phenotype in Lmod1 SMKO mice. (A) Gross images of wildtype and Lmod1 SMKO heart/aorta showing obvious thickened aortic wall in KO and areas (white dotted lines) sampled for thoracic and abdominal aortic sections. (B) H&E cross-sections of ascending aorta (AsAo), thoracic aorta (ThAo), and abdominal aorta (AbdAo) from Lmod1 WT and Lmod1 SMKO mice (n=3 mice per region). (C) Quantitation of aortic diameter from gross images of Lmod1 WT and Lmod1 SMKO aortae (n=6 mice per region). (D) H&E images of carotid artery and (E) quantitative measures of circumference and medial thickness from Lmod1 WT and Lmod1 SMKO carotids (n ≥ 6 mice). (F) Schematic of sampling (in 1mm levels) from base to apex of heart. (G) H&E images of coronary artery from Lmod1 WT and Lmod1 SMKO mice and (H) quantitative measures of circumference at Levels 1 and 2 (n=6 mice). (I) Baseline blood pressure before and 10 days after Tamoxifen (Tmx) administration in male and female WT and Lmod1 SMKO mice (n=8 mice). (J) Western blots of indicated proteins of each genotype and (J) quantitative data (n=3 mice per genotype).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Quantitation Assay, Sampling, Western Blot

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Cardiovascular changes in Lmod1 SMKO mice subjected to an athero-aneurysm regimen. ( A ) Experimental design of study. Note, Lmod1 WT mice here were of genotype Lmod1 fl/fl and received the same schedule of PCSK9/HFD and angiotensin II as Lmod1 SMKO mice. ( B ) Total cholesterol levels two weeks after the start of PCSK9/HFD in Lmod1 WT (black dots, n=18) and Lmod1 SMKO (red dots, n=28) mice. ( C ) Body weights in male ( top ) and female ( bottom ) mice of same genotype as above. ( D ) AAA in Lmod1 WT but not Lmod1 SMKO mice. ( E ) Survival curve of Lmod1 WT versus Lmod1 SMKO mice. ( F ) Heart weight (HW) to tibial length (TL) in Lmod1 WT (n=16) and Lmod1 SMKO (n=15) mice at the termination of study. ( G ) Gross heart images showing tofu-like coronary arteries (yellow arrows) in Lmod1 SMKO mice. Hematoxylin ( H ) and Masson trichrome ( I ) stained coronary arteries; note small lumen (Lu) in Lmod1 SMKO mice. ( J ) Masson trichrome stained heart tissue shows interstitial fibrosis in Lmod1 WT but not Lmod1 SMKO mice (n=3 mice). ( K ) Transmission electron microscopy of coronary arteries. Both Lmod1 WT and lipid droplet-containing Lmod1 SMKO coronary arteries show characteristic peripheral dense bodies (arrows) and basement membrane (arrowheads). Note the nearly 100% occluded Lmod1 SMKO coronary artery with lipid-laden foam cells containing lipid droplets of variable color within residential SMC of the medial wall (magnified red boxed region at bottom).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining, Transmission Assay, Electron Microscopy, Membrane

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) Experimental study starting at seven weeks of age. Beginning here, all Lmod1 WT mice carried the Itga8-CreER T2 allele and were treated with the same atherogenic regimen as Lmod1 SMKO mice. ( B ) Total serum cholesterol in Lmod1 WT (black dots) and Lmod1 SMKO (red dots) mice at indicated ages. ( C ) Total serum triglycerides and HDL and LDL. ( D ) Cumulative survival curves for Lmod1 WT and Lmod1 SMKO mice across multiple cohorts. Oil-Red-O (ORO) staining ( E ) and quantitation of percent ORO staining ( F ) across the aorta of Lmod1 WT (n=5) and Lmod1 SMKO (n=9) mice. ( G ) Gross cardiac images showing tofu-like appearance of coronary arteries (yellow arrows) in Lmod1 SMKO mice. Masson trichrome staining ( H ) and CIFM imaging of LMOD1 and ACTA2 protein ( I ) in sections of coronary artery from Lmod1 WT (top panels) and Lmod1 SMKO mice (bottom) under the PCSK9/HFD regimen for 52 days. Note the prominent ACTA2 positive fibrous cap in Lmod1 SMKO section. Scales bars, 50 μm.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining, Quantitation Assay, Imaging

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Representative staining of coronary vessels from Lmod1 WT (n=5 females and 10 males) and Lmod1 SMKO (n=6 females and 11 males) mice after 8 ( A-C ), 16 ( D-F ), and 63 ( G-I ) days of PCSK9/HFD. Quantitative measures of ORO staining ( J ), plaque area ( K ), and circumference ( L ) of coronary arteries from Lmod1 WT and Lmod1 SMKO mice after 63 days of PCSK9/HFD. Data in panel J represents 10 and 12 coronary arteries from 5 Lmod1 WT and 5 Lmod1 SMKO mice, respectively. Data in panels K and L represent 26 and 18 coronary arteries from 5 Lmod1 WT and 5 Lmod1 SMKO mice, respectively. Scales bars (located in upper right corner of each panel) are 20 μm save lower panel H (50 μm). Transmission electron micrographs (TEM) showing a presumptive medial SMC migrating through a fenestra (yellow arrows) of the IEL (marked in red asterisks) at 8 days ( M ); two intimal foam cells (black arrows), two recently migrated intimal SMC (yellow arrows, one of which has a lipid droplet), and a medial SMC foam cell (white arrow) at 16 days ( N ); and a more complex plaque with lipid rich foam cell core and foam cell-containing fibrous cap at 63 days ( O ). See Figures S15-S17 for additional electron micrographs of Lmod1 WT and Lmod1 SMKO coronary arteries. Lu, lumen.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining, Transmission Assay

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Picrosirius red and Alizarin red staining of coronary arteries. Picrosirius red stained coronaries from Lmod1 WT ( A, B ) and Lmod1 SMKO ( C, D ) mice. Note lumen (Lu) and necrotic core of atheroma (arrows) in Lmod1 SMKO coronary arteries. ( E ) Percent plaque area stained for picrosirius red (PR) in Lmod1 WT and Lmod1 SMKO coronary arteries. Data represent measures of coronary arteries from seven independent mice per genotype. Alizarin red staining was not observed in plaques of Lmod1 SMKO mice ( F ), but was readily observed in a calcified human coronary artery ( G ). All scale bars are 50 μm.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Coronary artery SMC lineage tracing in Lmod1 SMKO mice. Lmod1 WT ( A-C ) and Lmod1 SMKO ( D-J ) coronary arteries from mice carrying the membrane tomato/membrane GFP (mTmG) reporter. Red fluorescence ( A,B,D,E,G,H ) indicates surrounding cardiomyocytes and endothelial and immune cells as well as fibroblasts whose mTmG reporter did not undergo recombination (i.e., Itga8-CreER T2 was inactive in these cells). Conversely, green fluorescence represents medial and plaque cells of SMC origin (i.e., Itga8-CreER T2 was active in these cells and recombined out the tomato reporter allowing for membrane GFP expression). Note GFP+ cells beginning to populate early atheroma ( D,E ; see also, 20F ) and comprising a large portion of advanced plaques ( G,H ). White asterisks indicate necrotic core. Immunogold electron microscopy lineage tracing (IEMLT) of a Lmod1 WT control coronary artery ( C ); an Lmod1 SMKO coronary from 16-day lesion with two intimal core-centric GFP+ cells (labeled 1 and 2) ( F ); and an advanced, 63-day lesion showing several GFP+ cells at the fibrous cap (I). ( J ) Higher magnification of red boxed region in panel I showing three fibrous cap cells (labeled 1-3), two of which (1 and 2) are GFP+. ( K ) Percentage of GFP+ cells in plaques of seven independent Lmod1 SMKO coronaries (red bar) and the percentage of Core (green bar) and Cap (blue bar) derived foam cells that were GFP+. Scale bars are 50 μm. Adv, adventitia; CM, cardiomyocyte; EC, endothelial cell; Int, intima; Med, media; RBC, red blood cell. See Supplemental Figures 19 and 20 for additional IEMLT images.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Membrane, Fluorescence, Expressing, Electron Microscopy, Control, Labeling, Derivative Assay

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Additional IEMLT in Lmod1 SMKO mice. Lmod1 SMKO coronary arteries from four independent female mice subjected to PCSK9/HFD for six weeks showing (A) three GFP+ SMC-derived intimal cells (yellow arrows pointing to membrane-bound gold particles here and below) with one located just below a GFP-EC (black arrow) in an evolving cap and two containing lipid droplets in the core; (B) two intimal cells near the cap with one GFP+ (left yellow arrow) and the other GFP-(right yellow arrow), both located subjacent to two negative EC (black arrows); (C) three SMC-derived intimal cells (yellow arrows), with one (upper) containing lipid droplets; and (D) a non-lipid containing SMC-derived cap cell (yellow arrow) subjacent to a GFP-EC (black arrow). In a separate cohort of mice treated for only six days with PCSK9/HFD (E, F), GFP+ SMC could be seen moving through the fenestra (yellow arrows) of the internal elastic lamina (red asterisks here and elsewhere). The more intense gold particle staining in panels E and F relates to the timing for enhancement of gold particles in this cohort (see Methods). EC, endothelial cell; Le, leukocyte.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Derivative Assay, Membrane, Staining

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) UCSC genome browser screenshot of human LMOD1 gene structure and position of annotated SNV. The approximate position of sgRNAs used to generate an 11.35 kb intronic deletion in mice is shown. Note that this deletion removes the rs34091558 SNV and an SRF-binding consensus CArG box (indicated by ChIP-seq peak in human coronary artery SMCs and an arrow pointing to a red [conserved CArG] line on the “CArGs track”). This region also coincides with active chromatin marks. Western blot ( B ) and CIFM ( C ) of aortic LMOD1 in mice homozygous for the intronic deletion. Western blot ( D ) and quantitation ( E ) of aortic LMOD1 and ITGA8 in the indicated genotypes. Oil-Red-O staining ( F,H ) and CIFM of LMOD1 ( G,I ) in coronary arteries from Lmod1 WT ( F,G ) and Lmod1 SMKO ( H,I ) mice. Scale bars, 50 μm.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Quantitation Assay, Staining

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Baseline and PCSK9/HFD bulk RNA-seq of aorta. A, PCA plot displaying variance of wild type animals compared with Lmod1 SMKO on chow diet and, B, high fat diet (HFD). Volcano plot showing differentially expressed genes (DEGs) under C, baseline and, D, HFD. Overrepresentation pathway analysis with upregulated pathways in Lmod1 SMKO over Lmod1 WT mice shown under E, chow diet and, F, HFD.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: RNA Sequencing

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) Schematic for scRNA-seq (10x-FLEX, blue) and spatial (10x-Visium, red). Libraries interrogate midline cross-sections of right and left ventricles of mouse hearts. ( B ) scRNA-seq modality: ( i ) UMAP with accompanying stacked bar chart for cell type distribution across Lmod1 WT and Lmod1 SMKO ; and ( ii ) dot plot of top 5 gene markers per cell type. ( C ) Spatial modality: ( i ) spatial feature plot of Myh11 (used as proxy for coronary artery-enriched spatial libraries); ( ii ) volcano plot of differentially expressed genes (DEG) for Lmod1 WT and Lmod1 SMKO Myh11 + libraries (cutoffs: average log2 fold change ≥ 1, p ≤ 0.05);, ( iii ) spatial feature plot of up-regulated Thbs1 showing coronary-artery enriched expression. ( D ) Spatial spot deconvolution (purple) reflects the integration of scRNA-seq (red) and spatial (blue) modalities. Coronary artery-specific spatial feature plot of Myh11 expression with corresponding spatial spot deconvolution with cell type composition (pie-chart) using genotype-matched scRNA-seq reference.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Expressing

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Multi-modal transcriptomic workflow for ambient RNA correction, dataset integration, and spatial deconvolution. (A) Pre-CellBender ( top ) dot plot of annotated cell-type markers with representative UMAPs (cardiomyocytes: Mb , Cryab ; endothelial cells: Egfl7 , Shank3 ) demonstrates ambient RNA artifacts in scRNA-seq data, indicated by red dotted boxes and diffuse low-level expression across clusters. Post-CellBender ( bottom ) ambient RNA correction restricts marker expression to appropriate cell types in both dot plots and UMAPs. (B) UMAP visualization of integrated scRNA-seq data with samples overlaid and (C) split by genotype, demonstrating correction for batch effects. (D, E) Genotype-matched spatial voxel deconvolution for Lmod1 WT (three coronary arterial regions, CA) and Lmod1 SMKO (seven CA regions) shown with corresponding H&E-stained cross sections, with CA regions highlighted in yellow. Each CA region has Myh11 spatial feature plots paired with deconvolution voxels, done by genotype-matched scRNA-seq and spatial modalities.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Expressing, Marker, Staining

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: THBS1 in mouse and human atherosclerosis. (A) Smooth muscle cell SCPA activity shown by volcano plots with log₂ fold change (x-axis) over q-value (y-axis). First plot displays all pathways, and second plot highlights lipid-associated pathways (red) relative to non-lipid pathways (grey). (B) Scatterplot of genes within GOBP Regulation of Lipid Transport comparing Lmod1 WT over Lmod1 SMKO with Thbs1 highlighted in red. (C) Dot plot of up-regulated genes in vascular for scRNA-seq modality. (D) Venn diagram illustrating shared up-regulated genes in Lmod1 SMKO across two bulk RNA-seq datasets and spatial modality. ( E ) THBS1 protein colocalization with GFP+ cells in the media and plaque of Lmod1 SMKO coronary artery. The tdTomato reporter was pseudo-colored white (marking mainly cardiomyocytes and endothelial cells). Scale bars, 20 μm. ( F ) Two sections of human coronary artery stained with H&E ( top ) or for the indicated proteins ( below ) magnified from H&E boxed regions. Scale bars, 50 μm. ( G ) Western blots of indicated proteins in cultured Lmod1 WT or Lmod1 SMKO and quantitation. ( H ) Representative histological analysis of the human aortic arch. Hematoxylin and eosin (H&E) staining shows overall tissue morphology. Oil-Red-O staining highlights lipid accumulation, particularly in the inner curvature (IC). Scale bars are 1 cm for the aorta and 400 mm for the ORO stained IC/IC. ( I ) Western blot analysis of THBS1 in segments of the descending aorta (DA), IC, and OC of the human atherosclerotic aortic arch from two patients. Quantification of THBS1 expression normalized to ACTB. Data are presented as mean ± SEM (n = 3). p <0.01. ( J ) OxLDL staining of primary mouse aortic SMC (MASMC) from each genotype with quantitation below. ( K ) Lmod1 SMKO MASMC transduced with shRNA-control or shRNA- Thbs1 and treated with oxLDL. Similar findings were observed in an independent experiment. ( L ) Western blot validation of the Lentiviral-mediated knockdown of THBS1 protein with shRNA- Thbs1 in MASMC.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Activity Assay, RNA Sequencing, Staining, Western Blot, Cell Culture, Quantitation Assay, Expressing, Transduction, shRNA, Control, Biomarker Discovery, Knockdown

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Effect of Lmod1 iND on CAD phenotype in Lmod1 SMKO mice. (A) Western blot of LMOD1 protein t1/2 in Lmod1 WT (top) and Lmod1 iND (bottom) MASMCs following various times of Cycloheximide (CHX) exposure. (B) Quantitation of LMOD1 bands in panel A (n=3 independent experiments). (C) Electropherogram demonstrating sequence fidelity of the five DNA sequence-encoding amino acid substitutions (indicated in red at bottom with arrows). (D) IFM of LMOD1 and ORO staining of coronary arteries from each mouse model after 10 weeks of PCSK9/HFD. (E) Western blotting and quantitation of aortic LMOD1 from each genotype. (F) Total cholesterol measures in Lmod1 iND mice crossed with Tagln-Cre (black dots) or Itga8-CreER T2 (red dots) to induce the Lmod1 iND mutant.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Western Blot, Quantitation Assay, Sequencing, Staining, Mutagenesis

Journal: bioRxiv

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

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ORO-staining of aortic root and large versus small coronary arteries. Lmod1 WT (A-C) and Lmod1 SMKO (D-F) microvascular coronaries (A, D), branches of large coronary arteries near base of heart (B, E), and aortic root (C, F) stained for ORO. Each panel is representative of similarly-stained vessels (>50) in at least 10 mice of each genotype. Scale bars are 20 μm for panels A and D and 100 μm for panels B, C, E, and F.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining