Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Contractile Gene Expression Is Downregulated in Chol-Loaded hVSMCs (A, B) Human vascular smooth muscle cells (hVSMCs) were treated with cholesterol (Chol) (5 μg/mL) or 0.2% bovine serum albumin (control [CT]) for 24 hours and 48 hours and gene expression of Acta2 , Tagln , Cnn1, Myocd, and Srf were determined by quantitative polymerase chain reaction. (C) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 24 hours and protein expression of α–smooth muscle actin (α-SMA) and CNN1 were determined by Western blotting (representative blots shown). Densitometry showing the (D) α-SMA and (E) CNN1 band intensities normalized to GAPDH. For data analysis, unpaired Student’s t -testing was performed for comparing the means of 2 groups. For 2 or more independent groups, 1-way analysis of variance followed by Dunnett post hoc test was performed. A P value of ≤0.05 was considered significant. Data are presented as the mean ± SEM of 3 independent experiments, and P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001).

Article Snippet: The primary antibodies used were as follows: ACTA2 (#A2547, Sigma); CNN1 (#M3556, DAKO); SRF (#5147, Cell Signaling); p38MAPK (#sc-535, Santa Cruz Biotechnology); SMAD2/3 (#8685, Cell Signaling); TUBA (#T-5168, Sigma); and SMAD2 (#3103, Cell Signaling), phospho-SMAD2 (#3101S, Cell Signaling), phospho-p38MAPK (#9211S, Cell Signaling), SMAD4 (#9515, Cell Signaling); CD68 (#MCA1815, AbD Serotec, Bio-Rad); KLF4 (#12173, Cell Signaling); PU.1 (#sc-352, Santa Cruz Biotechnology); TGFβR1 (#3712, Cell Signaling); TGFβR2 (#sc-400, Santa Cruz Biotechnology); Caveolin (#610059, BD Transduction Laboratories); CD71 (#13113, Cell Signaling); GAPDH (#AM4300, Ambion).

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Chol-Loading Downregulates TGFβ Signaling in hVSMC hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT; ie, 0 μg/mL cholesterol) for 24 hours in the presence or absence of TGFβ1 ligand (10 pg/mL). Total RNA was isolated and quantitative polymerase chain reaction (qPCR) was performed to determine the pri-Mir143/145 precursor transcripts (A,B) or SMC markers, Acta2 and Tagln (C,D). hVSMCs were treated as in A and B, but either in the presence or absence of TGFβ1 10 pg/mL) and/or nonscrambled (NS) or Mir145 mimic (60 nmol/L). qPCR was performed to determine expression of Acta2 (E) and (F) Srf mRNA. (G) hVSMCs were treated as in A and B, but either in the presence or in absence of TGFβ1 (10 pg/mL) and/or Mir145 inhibitor (60 nmol/L). qPCR was performed to determine expression of Acta2. (H) Immunofluorescence images of total SMAD2/3 (green) in hVSMCs after 24 hours of the indicated treatments. Cytoplasm was stained with phalloidin (red). Nuclei were determined as phalloidin negative area (bar = 50 μm). (I) hVSMCs were treated as in A and B, but with varying amounts of Chol and in the presence or absence of recombinant TGFβ1 (10 pg/mL) for 24 hours. Proteins were extracted for Western blotting to detect phosphorylated (p) SMAD2/3, and α-SMA. Total SMAD2/3 or GAPDH was used as loading CT proteins. Blots are representative of at least 3 independent experiments, and the replicates were quantified by densitometry. For data comparisons of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of 3 independent experiments, and P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). ns = not significant; other abbreviations as in .

Article Snippet: The primary antibodies used were as follows: ACTA2 (#A2547, Sigma); CNN1 (#M3556, DAKO); SRF (#5147, Cell Signaling); p38MAPK (#sc-535, Santa Cruz Biotechnology); SMAD2/3 (#8685, Cell Signaling); TUBA (#T-5168, Sigma); and SMAD2 (#3103, Cell Signaling), phospho-SMAD2 (#3101S, Cell Signaling), phospho-p38MAPK (#9211S, Cell Signaling), SMAD4 (#9515, Cell Signaling); CD68 (#MCA1815, AbD Serotec, Bio-Rad); KLF4 (#12173, Cell Signaling); PU.1 (#sc-352, Santa Cruz Biotechnology); TGFβR1 (#3712, Cell Signaling); TGFβR2 (#sc-400, Santa Cruz Biotechnology); Caveolin (#610059, BD Transduction Laboratories); CD71 (#13113, Cell Signaling); GAPDH (#AM4300, Ambion).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Recombinant, Western Blot