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: Macrophage Markers Upregulated in Chol-Loaded hVSMCs Are Suppressed by HDL Through Restoration of TGFβ Signaling (A) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 48 hours. qPCR was performed to determine the expression of macrophage marker ( Cd68 ) and SMC marker ( Acta2 ). (B) hVSMCs were with treated as in A for 48 hours, then qPCR was performed to determine the expression of macrophage differentiation factor Klf4 . (C) hVSMCs were treated with Chol (5 μg/mL) for the indicated times, then KFL4 expression was determined by Western blotting. (D) Klf4 (60 nmol/L) or negative CT small, interfering RNA (siRNA) were transfected into hVSMCs for 48 hours. Then, transfected cells were treated as in B, followed by Western blotting for CD68 and KLF4. GAPDH was used as loading CT. (E) Chol-loaded cells (48 hours, 5 μg/mL) were incubated with Mir145 mimic (60 nmol/L) or CT mimic (60 nmol/LM) for 24 hours and the expressions of CD68, KLF4, and α-SMA determined with GAPDH as a loading CT. The P values for the comparisons between CT and Mir145 mimics are CD68 (0.025), KLF4 (0.018), and α-SMA (0.01). (F-I) hVSMCs were loaded with Chol (48 hours, 5 μg/mL) and were then either treated with HDL (50 μg/mL) for 24 hours or left untreated. Western blotting was performed to determine the expression of (F) KLF4 and (G) CD68. (H) hVSMCs were treated as in F and G, but in the presence or absence of TGFβR1i (50 ng/mL). Western blotting was performed to determine KLF4 expression. For data analysis, unpaired Student’s t -test was performed for comparing the means of 2 groups. For 2 or more independent groups, 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments. P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). Abbreviations as in , , and .

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: Expressing, Marker, Western Blot, Small Interfering RNA, Transfection, Incubation