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

Abstract

Many cells identified as macrophage-like in human and mouse atherosclerotic plaques are thought to be of vascular smooth muscle cell (VSMC) origin. We identified cholesterol-mediated down-regulation of TGFβ signaling in vitro in human (h)VSMCs by localization of TGFβ receptors in membrane lipid rafts, which was reversed by high-density lipoprotein (HDL)-mediated cholesterol efflux. This restored VSMC contractile marker (Acta2) and suppressed macrophage marker (CD68) expression by promoting TGFβ enhancement of Mir145 expression. In vivo, administration of ApoA1 (which forms HDL) to atherosclerotic mice also promoted VSMC Acta2 expression and reduced CD68 expression. Because macrophage-like VSMCs are thought to have adverse properties, our studies not only show mechanistically how cholesterol causes their transition, but also suggest that efflux-competent HDL particles may have a therapeutic role by restoring a more favorable phenotypic state of VSMCs in atherosclerotic plaques.

Keywords: HDL; lipid rafts. TGFβ signaling; vascular smooth muscle cells.

Graphical abstract

Figure 1

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).

Figure 2

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 Figure 1.

Figure 3

Cholesterol-Loading Partitions TGFβ Receptors Into Membrane Lipid Rafts hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin for 24 hours. (A) Membrane lipid raft (LR) and nonraft (NR) fractions were isolated, and Western blotting was performed using each of these fractions to determine the expressions of TGFβR1 and TGFβR2, as well as CAV1, flotillin, and transferrin receptor (CD71). (B) Densitometry was performed to quantify the levels of TGFβR1 and TGFβR2 in the LR and NR fractions. (C) Western blotting was performed from total cell lysates of Chol-treated or untreated cells, and the bands of the TGFβ receptors visualized. (D and E) Densitometry was performed to quantify the levels of TGFβR1 and TGFβR2. Blots are representative of 3 independent experiments. 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, and P values are as indicated (∗P < 0.05, ∗∗P < 0.01). Abbreviations as in Figure 1.

Figure 4

HDL Treatment In Vitro Restores TGFβ Signaling in Cholesterol-Loaded hVSMCs (A) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin for 24 hours, followed by high-density lipoprotein (HDL) (50 μg/mL) treatment for 48 hours. Then, treatment groups were stimulated with recombinant TGFβ1 (10 pg/mL). Western blotting was performed to detect pSMAD2 and total (t) SMAD2, with densitometry used for quantification. (B to E) qPCR was performed to detect expression of Mir143/145, Myocd, Acta2, Cnn1, and Hmgcr at the conclusion of the experiment in A. (F) Chol-loaded cells were either treated with HDL alone, HDL + TGFβR1 antagonist (TGFβR1i; 50 ng/mL), or left untreated. Western blotting was performed to detect α-SMA. GAPDH was used as loading CT protein. For data analysis of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Blots are representative of 3-5 independent experiments (mean ± SEM). P values are as indicated (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). Abbreviations as in Figures 1 and 2.

Figure 5

HDL Treatment Displaces TGFβ Receptor From Membrane Lipid Rafts in Chol-Loaded hVSMCs and Restores its Signaling hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 24 hours, after which they were all treated with HDL (50 μg/mL) for 24 hours. (A) At the end of the 48-hour protocol, LR and NR fractions were isolated, and Western blotting was performed using each of these fractions to determine the expressions of TGFβR1 and TGFβR2, as well as CAV1, and flotillin. Densitometry was performed to quantify the level of (B) TGFβR1 and (C) TGFβR2. (D) 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 pSMAD2, SMAD2, and GAPDH levels. For data analysis of 2 or more independent groups, 2-way analysis of variance followed by Šídák multiple comparisons post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments, and the P values are as indicated (∗P < 0.05, ∗∗P < 0.01). Abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4.

Figure 6

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 Figures 1, 2, and 4.

Figure 7

HDL Increases the Expression of Acta2 Relative to That of CD68 in Atherosclerotic Mice (A) Schematic representation of experimental design. Note that ApoA1, which forms HDL particles in vivo, was injected after atherosclerosis progression (P) to induce regression (R). (B) Representative images from P and R mice that were sufficient (Tgfβr2^+/+) or haplosufficient (Tgfβr2^+/−) for TGFβR2, showing the lineage-positive VSMCs (GFP⁺) expressing macrophage marker or CD68 (red). Yellow color represents GFP-expressing CD68⁺ cells. (C) Quantification of GFP⁺/CD68⁺. (D) Aortic digestion followed by cell sorting of GFP⁺ cells was performed using flow cytometry to capture lineage-positive cells (GFP) expressing macrophage markers (CD11b and F4/80). (E) Total RNA was isolated from sorted cells and qPCR was performed to identify Acta2 expression. For data analysis of 2 or more independent groups, 2-way analysis of variance followed by Šídák multiple comparisons post hoc test was performed. Data are presented as the mean ± SEM (n = 5-6 mice per group). P values are as indicated (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). DAPI = 4ʹ,6-diamidino-2-phenylindole; other abbreviations as in Figures 1, 2, and 4.

Figure 8

HDL Promotes pSMAD2 Levels in TGFβR2^+/− Mice In Vivo (A) Representative image from P and R mice (TGFβR2^+/+ and TGFβR2^+/−) showing the lineage-marked SMCs (GFP⁺ green cells) and pSMAD2 level (white color) (media [M], plaque [P], lumen [L]). pSMAD2⁺ cells were quantified using Image J software in the (B) plaque and (C) media. For data analysis of 2 or more independent groups, 2-way analysis of variance followed by Šídák multiple comparisons post hoc test was performed. Data are presented as the mean ± SEM (n = 3 mice per group). P values are as indicated (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). Abbreviations as in Figures 1, 2, and 7.