Luteolin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting TGFBR1 Signaling

Abstract

Vascular smooth muscle cell (VSMC) proliferation and migration play a critical role in the development of arterial remodeling during various vascular diseases including atherosclerosis, hypertension, and related diseases. Luteolin is a food-derived flavonoid that exerts protective effects on cardiovascular diseases. Here, we investigated whether transforming growth factor-β receptor 1 (TGFBR1) signaling underlies the inhibitory effects of luteolin on VSMC proliferation and migration. We found that luteolin reduced the proliferation and migration of VSMCs, specifically A7r5 and HASMC cells, in a dose-dependent manner, based on MTS and EdU, and Transwell and wound healing assays, respectively. We also demonstrated that it inhibited the expression of proliferation-related proteins including PCNA and Cyclin D1, as well as the migration-related proteins MMP2 and MMP9, in a dose-dependent manner by western blotting. In addition, luteolin dose-dependently inhibited the phosphorylation of TGFBR1, Smad2, and Smad3. Notably, adenovirus-mediated overexpression of TGFBR1 enhanced TGFBR1, Smad2, and Smad3 activation in VSMCs and partially blocked the inhibitory effect of luteolin on TGFBR1, Smad2, and Smad3. Moreover, overexpression of TGFBR1 rescued the inhibitory effects of luteolin on the proliferation and migration of VSMCs. Additionally, molecular docking showed that this compound could dock onto an agonist binding site of TGFBR1, and that the binding energy between luteolin and TGFBR1 was -10.194 kcal/mol. Simulations of molecular dynamics showed that TGFBR1-luteolin binding was stable. Collectively, these data demonstrated that luteolin might inhibit VSMC proliferation and migration by suppressing TGFBR1 signaling.

Keywords: Smad2/3; TGFBR1; luteolin; migration; proliferation; vascular smooth muscle cell.

FIGURE 1

Luteolin suppresses vascular smooth muscle cell (VSMC) proliferation without inducing apoptosis. (A,B) A7r5 and HASMC cells were incubated with different concentrations of luteolin (10, 20, and 40 μM) for 48 h, and then VSMC proliferation was tested by MTS assays (n = 6). (C) EdU proliferation assay. The red regions (EdU) in the images represent proliferating cells, and the blue regions (Hoechst 33324) represent the nuclei of all cells. (D,E) Percentage of EdU-positive VSMCs (the proliferation ratio was tested by assessing Hoechst 33324/EdU staining; n = 3). (F) A7r5 and HASMC cells were incubated with luteolin (10, 20, and 40 μM) for 24 h, and the expression of Cyclin D1 and PCNA was tested by western blotting. (G–H) Relative expression levels of Cyclin D1 and PCNA (n = 3). (I) Expression levels of BAX and BCL-2 were tested by western blotting. (J) Relative expression levels of BAX (n = 3). (K) Relative expression levels of BCL-2 (n = 3). Data are presented as the mean ± SD. ^∗P < 0.05, ^∗P < 0.01, compared to the control group.

FIGURE 2

Luteolin suppresses the migration of vascular smooth muscle cells (VSMCs). (A) A7r5 and HASMC cells were incubated with different concentrations of luteolin (10, 20, and 40 μM) for 24 h, and the migration of VSMCs was tested by wound healing assays; the migration distance was estimated based on the width of the scrape at 0 and 48 h. (B,C) Percentage of relative VSMC migration (n = 3). (D) A7r5 and HASMC cells were harvested in the logarithmic growth phase, treated with different concentrations of luteolin (10, 20, and 40 μM), and tested by performing Transwell assays for 12 h. (E,F) The number of cells in each field of view (n = 5). (G) A7r5 and HASMC cells, respectively, were incubated with luteolin (10, 20, and 40 μM) for 24 h, and the expression levels of MMP2 and MMP9 were tested by western blotting. (H–K) Relative expression levels of MMP2 and MMP9 (n = 3). Data are presented as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, compared to the control group.

FIGURE 3

Luteolin suppresses activation of the TGFBR1 signaling pathway. (A) A7r5 and HASMC cells, respectively, were incubated with luteolin (10, 20, and 40 μM) for 1 h, and the expression levels of p-TGFBR1, TGFBR1, p-Smad2, Smad2, p-Smad3, and Smad3 were tested by western blotting. (B–G) Relative phosphorylation levels of TGFBR1, Smad2, and Smad3 (n = 3). Data are presented as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, compared to the control group.

FIGURE 4

Overexpression of TGFBR1 partially rescues TGFBR1 activation after luteolin treatment. (A) A7r5 and HASMC cells, respectively, were incubated with a TGFBR1 overexpression adenovirus vector and GFP control adenovirus for 48 h. Cells were then treated with luteolin (40 μM) for 1 h. The expression levels of p-TGFBR1, TGFBR1, p-Smad2, Smad2, p-Smad3, and Smad3 were tested by western blotting. (B–G) Relative phosphorylation levels of TGFBR1, Smad2, and Smad3 (n = 3). Data are presented as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, compared to the control group.

FIGURE 5

Overexpression of TGFBR1 partially blocks the inhibitory effect of luteolin on vascular smooth muscle cell (VSMC) proliferation. (A,B) A7r5 and HASMC cells, respectively, were incubated with TGFBR1 overexpression adenovirus vector and GFP control adenovirus for 48 h and then treated with luteolin (40 μM) for 48 h. Then, the proliferation of VSMCs was tested by MTS assays (n = 6). (C) EdU proliferation assay. The red regions (EdU) in the images represent proliferating cells, and the blue regions (Hoechst 33324) represent the nuclei of all cells. (D,E) Percentage of EdU-positive VSMCs (n = 3). (F) A7r5 and HASMC cells, respectively, were incubated with TGFBR1 overexpression adenovirus vector and GFP control adenovirus for 48 h and then treated with luteolin (40 μM) for 24 h. the expression levels of Cyclin D1 and PCNA were tested by western blotting. (G–J) Relative expression levels of Cyclin D1 and PCNA (n = 3). Data are shown as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 6

Overexpression of TGFBR1 partially blocks the inhibitory effect of luteolin on vascular smooth muscle cell (VSMC) migration. (A) A7r5 and HASMC cells were incubated with TGFBR1 overexpression adenovirus vector and GFP control adenovirus for 48 h and then treated with luteolin (40 μM) for 24 h. Then, the migration of VSMCs was tested by wound healing assays. (B,C) Percentage of relative VSMC migration (n = 3). (D) A7r5 and HASMC cells were incubated with TGFBR1 overexpression adenovirus vector and GFP control adenovirus for 48 h. Then, Transwell assays were performed after treatment with luteolin (40 μM) for 12 h. (E,F) The number of cells in each field of view (n = 5). Data are shown as the mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 7

Molecular docking and simulation of the molecular dynamics. (A) Three-dimensional crystal structure of luteolin (ZINC18185774) in complex with TGFBR1 (PDB ID: 1PY5). Luteolin is shown in green, and the hydrogen bonds are indicated by sky-blue lines. (B) Surface presentation of the TGFBR1-luteolin complex crystal structure at 0 and 100 ns. (C) Plots of root mean square deviation (RMSD) of heavy atoms of TGFBR1 unbound (blue) and TGFBR1-luteolin complex (red). (D) Potential energy profiles of TGFBR1-free (blue) and TGFBR1-luteolin complex (red) during the 100-ns molecular dynamics simulation.