Smooth muscle FGF/TGFβ cross talk regulates atherosclerosis progression

Abstract The conversion of vascular smooth muscle cells (SMCs) from contractile to proliferative phenotype is thought to play an important role in atherosclerosis. However, the contribution of this process to plaque growth has never been fully defined. In this study, we show that activation of SMC TGFβ signaling, achieved by suppression of SMC fibroblast growth factor (FGF) signaling input, induces their conversion to a contractile phenotype and dramatically reduces atherosclerotic plaque size. The FGF/TGFβ signaling cross talk was observed in vitro and in vivo. In vitro, inhibition of FGF signaling increased TGFβ activity, thereby promoting smooth muscle differentiation and decreasing proliferation. In vivo, smooth muscle‐specific knockout of an FGF receptor adaptor Frs2α led to a profound inhibition of atherosclerotic plaque growth when these animals were crossed on Apoe −/− background and subjected to a high‐fat diet. In particular, there was a significant reduction in plaque cellularity, increase in fibrous cap area, and decrease in necrotic core size. In agreement with these findings, examination of human coronary arteries with various degrees of atherosclerosis revealed a strong correlation between the activation of FGF signaling, loss of TGFβ activity, and increased disease severity. These results identify SMC FGF/TGFβ signaling cross talk as an important regulator of SMC phenotype switch and document a major contribution of medial SMC proliferation to atherosclerotic plaque growth.

A D E B C Figure EV2. FGFR1 knockdown increases smooth muscle marker gene expression via the let-7-TGFb pathway in primary human aortic smooth muscle cells (HASMCs).
A-C Upper panels: Immunoblots of SM-calponin, phosphorylated Smad2 (p-Smad2), and TGFbR1 expression in control and FGFR1-knockdown HASMCs treated with SB431542 (10 lM), TGFbR2, or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SMcalponin and p-Smad2 were normalized to GAPDH, b-tubulin, HSP90, or Smad2/3 and expressed as a fraction of a control value. D Images of phase-contrast and GFP fluorescence signals showed the expression of let-7b at day 0. Images are representative of three independent experiments. E Control and FGFR1-knockdown HASMCs were cultured in the growth medium (M231+ SMGS) at day 0 and then switched from growth conditions to differentiation medium (M231+ SMDS) for 6 days with or without let-7b lentiviruses. Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFbR1 expression in control and FGFR1-knockdown HASMCs with or without let-7b lentiviruses. Blots are representative of three independent experiments.
Source data are available online for this figure. A qRT-PCR analysis of Frs2a expression in mouse aorta. b-actin was used for sample loading normalization. n = 3 control and 3 Frs2a SMCKO mice were analyzed. B Immunoblot analysis of FRS2a expression in mouse aorta. In each group, aorta was pooled from 4 mice/group. C Representative images of FRS2a immunofluorescence staining of control and Frs2a SMCKO aorta. Endothelial cells are visualized by CD31 (green). Black arrows indicate endothelial cells. L: lumen. Nuclei were stained with DAPI (blue). Images are representative of 3 mice/group. Scale bar: 10 lm. D Gross appearance of aorta in 8-week-old control and Frs2a SMCKO mice. Asc: Ascending; Desc: Descending. Images are representative of 3 mice/group. Scale bar: 5 mm. E 5-lm cross sections of control and Frs2a SMCKO mouse brachiocephalic artery were stained with EVG (elastic Van Gieson), anti-SM a-actin, anti-SM22a, and anti-Notch3 antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 10 lm. Images are representative of 3 mice/group. F Left: Histological analysis of control and Frs2a SMCKO mouse brachiocephalic artery with anti-CD31 (green) and anti-p-Smad2 (red) antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 10 lm. Right: Percentage of p-Smad2 + cells in the media. Images are representative of 6 mice/group. G, H Left: Representative images of vascular structure in heart and skeletal muscle in control and Frs2a SMCKO mice. Scale bar: 62 lm for 100× and 16 lm for 400×.
Right: Vascular density was quantified. Images are representative of 5 mice/group.
Data information: All data represent the mean AE SD (NS: not significant compared to control; ***P < 0.001 compared to control; unpaired two-tailed Student's t-test).
Source data are available online for this figure.
EMBO Molecular Medicine ª 2016 The Authors A B C D Figure EV4. Frs2a SMCKO /Apoe À/À mice have normal body weight, lipid profiles, and heart function.
Data information: All data represent the mean AE SD (NS: not significant compared to Apoe À/À ; unpaired two-tailed Student's t-test). A full table of P-values for this figure is shown in Appendix Table S1. A Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch of Apoe À/À or Frs2 SMCKO /Apoe À/À mice after 2 months of high-fat diet or normal diet. Images are representative of 3 mice/group. Scale bar: 5 mm. B (Left) Microphotographs of aortas (en face) from Apoe À/À and Frs2 SMCKO /Apoe À/À mice after 2 months of high-fat diet after staining with Oil Red O. (Right) Lesion area quantification. n = 6 mice per group. C Quantification of SM a-actin area in the plaque from Apoe À/À and Frs2 SMCKO /Apoe À/À mice after 4 months of high-fat diet. Apoe À/À mice N = 9, Frs2 SMCKO /Apoe À/À mice N = 12. Nuclei were counterstained with DAPI (blue). Scale bar: 62 lm. D Measurement of collagen 1 area from Apoe À/À and Frs2 SMCKO /Apoe À/À mice after 4 months of high-fat diet (**P < 0.01 compared to Apoe À/À ; unpaired two-tailed Student's t-test). Data expressed as the ratio of collagen 1 signal to the total vessel area. Apoe À/À mice N = 9, Frs2 SMCKO /Apoe À/À mice N = 12. Nuclei were counterstained with DAPI (blue). Scale bar: 62 lm.
Data information: All data shown as mean AE SD (*P < 0.05, **P < 0.01 compared to Apoe À/À ; unpaired two-tailed Student's t-test). A full table of P-values for this figure is shown in Appendix Table S1.
EMBO Molecular Medicine ª 2016 The Authors