HDL regulates TGFß-receptor lipid raft partitioning, restoring contractile features of cholesterol-loaded vascular smooth muscle cells

Abstract

Background: Cholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulates miR-143/145, a master regulator of the contractile state downstream of TGFβ signaling. In vitro, this results in transitioning from a contractile mVSMC to a macrophage-like state. This process likely occurs in vivo based on studies in mouse and human atherosclerotic plaques.

Objectives: To test whether cholesterol-loading reduces VSMC TGFβ signaling and if cholesterol efflux will restore signaling and the contractile state in vitro and in vivo.

Methods: Human coronary artery (h)VSMCs were cholesterol-loaded, then treated with HDL (to promote cholesterol efflux). For in vivo studies, partial conditional deletion of Tgfβr2 in lineage-traced VSMC mice was induced. Mice wild-type for VSMC Tgfβr2 or partially deficient (Tgfβr2+/-) were made hypercholesterolemic to establish atherosclerosis. Mice were then treated with apoA1 (which forms HDL).

Results: Cholesterol-loading of hVSMCs downregulated TGFβ signaling and contractile gene expression; macrophage markers were induced. TGFβ signaling positively regulated miR-143/145 expression, increasing Acta2 expression and suppressing KLF4. Cholesterol-loading localized TGFβ receptors into lipid rafts, with consequent TGFβ signaling downregulation. Notably, in cholesterol-loaded hVSMCs HDL particles displaced receptors from lipid rafts and increased TGFβ signaling, resulting in enhanced miR-145 expression and decreased KLF4-dependent macrophage features. ApoA1 infusion into Tgfβr2+/- mice restored Acta2 expression and decreased macrophage-marker expression in plaque VSMCs, with evidence of increased TGFβ signaling.

Conclusions: Cholesterol suppresses TGFβ signaling and the contractile state in hVSMC through partitioning of TGFβ receptors into lipid rafts. These changes can be reversed by promotion of cholesterol efflux, consistent with evidence in vivo.

Figure 1.. Contractile gene expression is downregulated in cholesterol-loaded hVSMC.

(A-B) hVSMCs were treated with cholesterol (Chol) (5μg/ml) or 0.2% BSA (CT) for 24h and 48h and gene expression of Acta2, Tagln, Cnn1, Myocd, and Srf were determined by qPCR. (C) hVSMC were treated with cholesterol (Chol) (5μg/ml) or 0.2% BSA (CT) for 24h and protein expression of 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. Data are presented as the mean ± S.E. of three independent experiments and p values are as indicated.

Figure 2.. Cholesterol-loading downregulates TGFβ signaling in hVSMC.

hVSMC were treated with cholesterol (Chol) (5μg/ml) or 0.2% BSA (CT; i.e., 0μg/ml cholesterol) for 24h in the presence or absence of TGFβ1 ligand (10pg/ml). Total RNA was isolated and qPCR was performed to determine the pri-miR143/145 transcripts (A&B) or smooth muscle cell markers, Acta2 and Tagln (C&D). hVSMCs were treated as in A&B, but either in the presence or absence of TGFβ1 10pg/ml) and/or non-scrambled (NS) or miR145 mimic (60nM). qPCR was performed to determine expression of Acta2 (E) and (F) Srf mRNA. (G) hVSMCs were treated as in A&B, but either in the presence or in absence of TGFβ1 (10pg/ml) and/or miR145 inhibitor (60nM). qPCR was performed to determine expression of Acta2. (H) Immunofluorescence images of total SMAD2/3 (Green) in hVSMC after 24 h of the indicated treatments. Cytoplasm was stained with phalloidin (Red). Nuclei were determined as phalloidin negative area (scale bar=50μm). (I) hVSMCs were treated as in A&B, but with varying amounts of cholesterol and in the presence or absence of recombinant TGFβ1 (10pg/ml) for 24h. Proteins were extracted for western blotting to detect phosphorylated SMAD2/3 (p-SMAD2/3), and α-SMA. Total SMAD2/3 or GAPDH was used as loading controls. Blots are representative of at least three independent experiments, and the replicates were quantified by densitometry. Data are presented as the mean ± S.E. of three independent experiments and p values are as indicated.

Figure 3.. Cholesterol-loading partitions TGFβ receptors into membrane lipid rafts.

hVSMC were treated with cholesterol (Chol) (5μg/ml) or 0.2% BSA for 24h. (A) Membrane lipid rafts (LR) and non-raft (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 caveolin-1 (CAV1) 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 cholesterol-treated or untreated cells, and the bands of the TGFβ receptors visualized. (D-E) Densitometry was performed to quantify the levels of TGFβR1 and TGFβR2. Blots are representative of three independent experiments. Data are presented as the mean ± S.E. of at least three independent experiments and p values are as indicated.

Figure 4.. HDL treatment in vitro restores TGFβ signaling in cholesterol-loaded hVSMCs.

(A) hVSMC were treated with cholesterol (Chol) (5μg/ml) or 0.2% BSA for 24h, followed by HDL (50μg/ml) treatment for 48h. Then, treatment groups were stimulated with recombinant TGFβ1 (10pg/ml). Western blotting was performed to detect pSMAD2 and total SMAD2, with densitometry used for quantification. (B-E) qPCR was performed to detect expression of miR143/145, Myocd, Acta2, Cnn1 and Hmgcr at the conclusion of the experiment in A. (F) Cholesterol-loaded cells were either treated with HDL alone, HDL + TGFβR1 antagonist (TGFβR1i; 50ng/ml), or left untreated. Western blotting was performed to detect α-SMA. GAPDH was used as loading control. Blots are representative of at three independent experiments. p values are as indicated.

Figure 5.. HDL treatment displaces TGFβ receptor from membrane lipid rafts in cholesterol-loaded hVSMCs and restores its signaling.

hVSMC were treated with cholesterol (5μg/ml) or 0.2% BSA (CT) for 24h, after which they were either left untreated or treated with HDL (50μg/ml) for 24h. (A) At the end of the 48h protocol, lipid rafts (LR) and non-raft (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 caveolin-1 (CAV1). Densitometry was performed to quantify the level of (B) TGFβR1 and (C) TGFβR2. (D) hVSMCs were loaded with cholesterol (48h, 5μg/ml), and were then either treated with HDL (50μg/ml) for 24h, or left untreated. Western blotting was performed to determine pSMAD2, SMAD2, and GAPDH levels. Data are presented as the mean ± S.E. of at least three independent experiments and the p values are as indicated.

Figure 6.. Macrophage markers upregulated in cholesterol-loaded hVSMC are suppressed by HDL through restoration of TGFβ signaling.

(A) hVSMC were treated with cholesterol (5μg/ml) or 0.2% BSA (CT) for 48h. qPCR was performed to determine the expression of macrophage marker (Cd68) and smooth muscle cell marker (Acta2). (B) hVSMCs were with treated as in A for 48h, then qPCR was performed to determine the expression of macrophage differentiation factor Klf4. (C) hVSMCs were treated with cholesterol (5μg/ml) for the indicated times, then KFL4 expression was determined by Western blotting. (D) Klf4 (60nM) or negative control (CT) siRNA were transfected into hVSMCs for 48h. Then, transfected cells were treated as in B, followed by western blotting for CD68 and KLF4. GAPDH was used as loading control (E) Cholesterol-loaded cells (48h, 5μg/ml) were incubated with miR-145 mimic (60nM) or control mimic (60nM; CT) for 24h and the expressions of CD68, KLF4, and α-SMA determined with GAPDH as a loading control. (F-I) hVSMCs were loaded with cholesterol (48h, 5μg/ml), and were then either treated with HDL (50μg/ml) for 24h, or left untreated. Western blotting was performed to determine the expression of (F) KLF4, and (G) CD68. (H) hVSMCs were treated as in F&G, but in the presence or absence of TGFβR1 inhibitor (50ng/ml). Western blotting was performed to determine KLF4 expression. Data are presented as the mean ± S.E. of at least three independent experiments. p values are as indicated.

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 progression (P) and regression mice (R), that were sufficient (Tgfβr2+/+) or haplosufficient (Tgfβr2+/−) for TGFR2, showing the lineage-positive VSMCs (GFP+) expressing macrophage marker or CD68 (red). Yellow color represents GFP-expressing CD68+ cells. (C) Quantification of GFP/CD68 double +. (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 gene expression. Data are presented as the mean ± S.E. (n=5–6 mice per group). p values are as indicated.

Figure 8.. Schematic representation of proposed role of cholesterol mediated regulation of TGFβ signaling.