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. 2014 Jul 1;103(1):111-20.
doi: 10.1093/cvr/cvu105. Epub 2014 Apr 17.

Muscle-derived Follistatin-Like 1 Functions to Reduce Neointimal Formation After Vascular Injury

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Free PMC article

Muscle-derived Follistatin-Like 1 Functions to Reduce Neointimal Formation After Vascular Injury

Megumi Miyabe et al. Cardiovasc Res. .
Free PMC article

Abstract

Aims: It is well-established that exercise diminishes cardiovascular risk, but whether humoral factors secreted by muscle confer these benefits has not been conclusively shown. We have shown that the secreted protein follistatin-like 1 (Fstl1) has beneficial actions on cardiac and endothelial function. However, the role of muscle-derived Fstl1 in proliferative vascular disease remains largely unknown. Here, we investigated whether muscle-derived Fstl1 modulates vascular remodelling in response to injury.

Methods and results: The targeted ablation of Fstl1 in muscle led to an increase in neointimal formation following wire-induced arterial injury compared with control mice. Conversely, muscle-specific Fstl1 transgenic (TG) mice displayed a decrease in the neointimal thickening following arterial injury. Muscle-specific Fstl1 ablation and overexpression increased and decreased, respectively, the frequency of BrdU-positive proliferating cells in injured vessels. In cultured human aortic smooth muscle cells (HASMCs), treatment with human FSTL1 protein decreased proliferation and migration induced by stimulation with PDGF-BB. Treatment with FSTL1 enhanced AMPK phosphorylation, and inhibition of AMPK abrogated the inhibitory actions of FSTL1 on HASMC responses to PDGF-BB. The injured arteries of Fstl1-TG mice exhibited an increase in AMPK phosphorylation, and administration of AMPK inhibitor reversed the anti-proliferative actions of Fstl1 on the vessel wall.

Conclusion: Our findings indicate that muscle-derived Fstl1 attenuates neointimal formation in response to arterial injury by suppressing SMC proliferation through an AMPK-dependent mechanism. Thus, the release of protein factors from muscle, such as Fstl1, may partly explain why the maintenance of muscle function can have a therapeutic effect on the cardiovascular system.

Keywords: AMPK; Fstl1; Myokine; Smooth muscle cell; Vascular remodelling.

Figures

Figure 1
Figure 1
Fstl1-KO mice show enhanced neointimal formation following vascular injury. (A) Fstl1 protein levels in plasma and skeletal muscle tissue in muscle-specific Fstl1-KO and control littermate mice. Fstl1 levels in plasma (1.0 μl) and skeletal muscle were determined by western blot analysis. (B) Assessment of neointimal thickening in injured arteries in control and Fstl1-KO mice. Upper panels show the representative haematoxylin–eosin sections of femoral arteries from control and Fstl1-KO mice at 21 days after wire injury. Scale bar shows 50 μm. Lower panels show the quantitative analyses of I/M ratio (the ratio of intimal thickness/medial thickness) and luminal narrowing ratio (the ratio of intimal area/medial area). n = 8 in each group. (C) Assessment of vascular proliferation in the neointima after vascular injury. Upper panels show the representative BrdU staining of femoral arteries from control and Fstl1-KO mice at 7 days after surgery. Scale bar shows 10 μm. Quantitative data of BrdU labelling index are shown in the lower panel. The BrdU labelling index was calculated by dividing the number of BrdU-labelled cells by the number of total cells. n = 7 in each group. Data are presented as mean ± SE.
Figure 2
Figure 2
Fstl1-TG mice exhibit attenuated neointimal hyperplasia after arterial injury. (A) Plasma Fstl1 level in muscle-specific Fstl1-TG and littermate wild-type (WT) mice. Fstl1 level in plasma (1.0 μl) was determined by western blot analysis. n = 4 in each group. (B) Evaluation of neointimal thickening in wire-injured vessels in WT and Fstl1-TG mice. Upper panels show the representative haematoxylin–eosin sections of femoral arteries from WT and Fstl1-TG mice at 21 days after surgery. Scale bar shows 50 μm. Lower panels show the quantitative analyses of I/M ratio (the ratio of intimal thickness/medial thickness) and luminal narrowing ratio (the ratio of intimal area/medial area). n = 8 in each group. (C) Measurement of neointimal vascular proliferation in response to injury. Upper panels show the representative BrdU staining of femoral arteries from WT and Fstl1-TG mice at 7 days after injury. Scale bar shows 10 μm. The lower panel shows quantitative data of BrdU labelling index as calculated by dividing the number of BrdU-labelled cells by the number of total cells. n = 7 in each group. Data are presented as mean ± SE.
Figure 3
Figure 3
FSTL1 suppresses PDGF-BB-induced proliferation and migration of HASMCs. (A) Effect of FSTL1 on DNA synthesis of HASMCs. HASMCs were treated with or without recombinant human FSTL1 protein (100 or 250 ng/ml) for 10 h followed by stimulation with PDGF-BB (10 ng/ml), HB-EGF (10 ng/ml), or vehicle for 24 h. Cells were labelled with BrdU during the last 22 h of the culture period. (B) Effect of FSTL1 on the number of HASMCs. HASMCs were treated with or without recombinant FSTL1 protein (100 or 250 ng/ml) for 10 h followed by stimulation with PDGF-BB (10 ng/ml) or vehicle for 20 h. The numbers of HASMCs were assessed by MTS-based assay. (C) Effect of FSTL1 on the migratory activity of HASMCs. HASMCs were cultured on fibronectin-coated glass dishes, pre-treated with FSTL1 protein (250 ng/ml) or vehicle for 10 h, and scratched with a pipette tip. Wounded cells were incubated with PDGF-BB (10 ng/ml) or vehicle for 10 h. n = 3 in each group. (D) Effect of FSTL1 on the chemotactic activity of HASMCs. HASMCs were pre-incubated in the transwell in the absence or presence of FSTL1 protein (250 ng/ml) for 10 h followed by treatment with PDGF-BB (10 ng/ml) or vehicle for 4 h. n = 6 in each group. Data are presented as mean ± SE.
Figure 4
Figure 4
FSTL1 inhibits PDGF-BB-induced ERK activation HASMCs were pre-incubated in the presence or absence of FSTL1 (250 ng/ml) for 12 h and treated with PDGF-BB (10 ng/ml) or vehicle for 15 min. Phosphorylation levels of ERK (P-ERK) were determined by western blot analysis. Representative blots are shown in upper panels. ERK phosphorylation levels were quantified by using Image J, and quantitative data are shown in the lower panel. n = 3 in each group. Data are presented as mean ± SE.
Figure 5
Figure 5
AMPK is essential for the suppressive action of FSTL1 on HASMC growth. (A) Time-dependent changes in the phosphorylation of AMPK and ACC in HASMCs following stimulation with FSTL1 (250 ng/ml). The phosphorylation levels of AMPK (P-AMPK) and ACC (P-ACC) were determined by western blot analysis. (B) Quantitative analysis of phosphorylation levels of ACC and AMPK. Phosphorylation levels were quantified by using Image J, and quantitative data are shown. n = 3 in each group. *P < 0.05 vs. basal. **P < 0.01 vs. basal. (C) Inhibition of AMPK activation reverses FSTL1-stimulated ACC phosphorylation. HASMCs were transduced with c-Myc tagged-Ad-dn-AMPK or Ad-β-gal at an MOI of 10 for 24 h and treated with FSTL1 (250 ng/ml) or vehicle for 5 min. The phosphorylation levels of ACC (P-ACC) were assessed by western blot analysis. (D and E) Effect of AMPK inactivation on FSTL1-induced inhibition of HASMC growth. HASMCs were transduced with Ad-dn-AMPK or Ad-β-gal for 24 h, and treated with or without FSTL1 (250 ng/ml) for 10 h followed by stimulation with PDGF-BB (10 ng/ml) or vehicle for 20 h. DNA synthesis and number of HASMCs were quantified by using BrdU cell proliferation (D) and MTS-based (E) assays, respectively. n = 8 in each group. (F) Involvement of AMPK in the inhibitory effect of FSTL1 on PDGF-BB-induced ERK phosphorylation. HASMCs were transduced with Ad-dn-AMPK or Ad-β-gal for 24 h and treated with or without FSTL1 (250 ng/ml) for 10 h followed by stimulation with PDGF-BB (10 ng/ml) or vehicle for 15 min. Data are presented as mean ± SE.
Figure 6
Figure 6
Fstl1 ameliorates neointimal thickenings via AMPK-dependent manner. (A and B) Phosphorylation of AMPK and ACC in injured arteries. Phosphorylation levels of ACC (P-ACC) and AMPK (P-AMPK) in injured arteries (pooled samples, n = 3) from control and Fstl1-KO mice (A), and from WT and Fstl1-TG mice (B), at 3 days after surgery were evaluated by western blot analysis. Representative blots are shown. Right panels show quantitative analyses of phosphorylation levels of ACC (P-ACC/ACC) and AMPK (P-AMPK/AMPK). (C) Role of AMPK signalling in Fstl1-mediated attenuation of neointimal thickenings. AMPK inhibitor compound C or vehicle was intraperitoneally injected into WT or Fstl1-TG mice. Quantitative analysis of I/M ratio (the ratio of intimal area/medial area) is shown. n = 6 in each group. N.S., not significant. Data are presented as mean ± SE.

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