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. 2020 Feb 21;11(2):141.
doi: 10.1038/s41419-020-2343-1.

Laminar flow inhibits the Hippo/YAP pathway via autophagy and SIRT1-mediated deacetylation against atherosclerosis

Ping Yuan  1 , Qiongying Hu  2 , Xuemei He  3 , Yang Long  4 , Xueqin Song  4 , Fei Wu  5 , Yanzheng He  5 , Xiangyu Zhou  6
Affiliations

Laminar flow inhibits the Hippo/YAP pathway via autophagy and SIRT1-mediated deacetylation against atherosclerosis

Ping Yuan et al. Cell Death Dis. .

Abstract

Atherosclerosis is a multifactorial disease of the vasculature, and shear stress is a crucial regulator of its process. Disturbed flow promotes atherosclerotic effects, while laminar flow has a protective action on the endothelium. Hippo/YAP is a major cascade that senses various mechanical cues and mediates the expression of pro-inflammatory genes. However, the mechanism modulating the transcription factor YAP in response to different patterns of blood flow remains unclear. In this study, we provide evidence that shear stress modulates YAP activity via autophagy in endothelial cells. Laminar flow promoted the expression of the autophagic markers BECLIN 1 and LC3II/LC3I. Autophagy blockade using a chemical inhibitor repressed YAP degradation under laminar flow. Conversely, the induction of autophagy under disturbed flow partially antagonized the nuclear import and transcriptional activation of YAP. In parallel, laminar flow led to the increased expression of SIRT1 protein, a NAD+-dependent deacetylase. Further investigation showed that SIRT1-mediated YAP deacetylation. The forced expression of SIRT1 under disturbed flow effectively attenuated YAP activation and nuclear accumulation, thereby downregulating the expression of pro-inflammatory genes. In atheroprone vessels of mice receiving rapamycin to induce autophagy, the enhanced expression of SIRT1 was observed together with YAP repression. Altogether, these results show that endothelial autophagy and SIRT1 expression induced by laminar flow contribute to the inhibition of Hippo/YAP signaling and interrupt atherosclerotic plaque formation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Shear flow regulates YAP phosphorylation, subcellular localization, and downstream gene expression.
a HUVECs were exposed to UF (12 dyn/cm2) or DF (0.5 ± 6 dyn/cm2, 1 Hz) for the indicated times. Cells with static treatment (ST) were a control. After treatment, cells were underwent immunofluorescence staining with YAP (red) and DAPI (blue). b Western blot analysis showed YAP expression and YAP subcellular distribution (NP, nuclear protein; CP, cytoplasmic protein) under UF and DF flow condition in HUVEC cells. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane (0 h) was served as relative control. c Analyzed the relative total YAP protein level in nuclear and cytoplasm using gray density analysis for five independent experiments. GAPDH and Lamin B were used as a loading control for cytoplasmic and nuclear fractions. d HUVECs were transfected with 8 × GTIIC reporter and renilla-luc plasmids and were subjected to shear flow 48 h post transfection. Then the relative firefly/renilla luciferase activity was determined. e The expression of YAP target genes CTGF and CYR61 was determined by real-time PCR. Data represent mean ± SD from three independent experiments (**P < 0.01, *P < 0.05, vs. the control).
Fig. 2
Fig. 2. Different degradation levels of YAP protein in response to shear flow.
a HUVECs were treated with UF or DF for the indicated times in the presence of 20 μM CHX. The half-life of endogenous YAP protein was determined by western blot. b Calculation of half-life (T1/2) of the YAP protein in laminar flow and disturbed flow. c Cytoplasmic and nuclear YAP proteins from HUVECs treated with DF were extracted sequentially for western blot assay to detect the protein degradation. NP and CP represent nuclear and cytosolic proteins, respectively. d HUVECs were treated by UF in the presence of the proteasome inhibitor MG132 (10 μM) or autophagy inhibitors bafilomycin A1 (Baf, 1 μM) and chloroquine (CQ, 10 μM) for the indicated times, and then western blot analysis was employed to assess YAP abundance. e After UF treatment combined with 10 μM of chloroquine, cytoplasmic and nuclear fractions from HUVECs were extracted for western blot analysis to assess YAP amount. Representative images from at least three independent experiments are shown. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane was served as relative control.
Fig. 3
Fig. 3. Laminar flow induces autophagy.
a HUVECs were exposed to shear stress for 12 h and then subjected to immunofluorescence staining. Endothelial cells were labeled with anti-CD31 antibody. LC3 puncta indicated that UF significantly increased the number of autophagosomes in comparison with DF. Representative images from at least three independent experiments are shown. b, c Western blot analysis was performed to show the expression of BECLIN, LC3II/ LC3I, and p62 under UF or DF. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane (ST) was served as relative control. HUVEC cells were treated with UF (d) or (e) with autophagy inhibitors, real-time PCR was performed to determine the expression of ICAM1, MCP-1, and KLF2. Data represent mean ± SD of three independent experiments (**P < 0.01, *P < 0.05, vs. the control).
Fig. 4
Fig. 4. Autophagy modulates YAP.
a After DF treatment together with 100 nM rapamycin administration for 12 h, western blot analysis was performed to show the expression of BECLIN 1, LC3II/ LC3I, and p62. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane (Mock) was served as relative control. b Immunofluorescence staining showed the total abundance and nuclear enrichment of YAP protein in the presence of 100 nM rapamycin for 12 h. c Luciferase assay showed the activity of 8 × GTIIC reporter in HUVECs exposed to 100 nM of rapamycin treatment under DF for 12 h, and d real-time PCR showed the expression levels of YAP downstream genes CTGF and CYR61. e Immunofluorescence assay showed that chloroquine treatment enhanced YAP staining in the cytoplasm under UF. f Luciferase assay showed the activity of 8 × GTIIC reporter in HUVECs exposed to chloroquine under UF. Data represent mean ± SD of three independent experiments (**P < 0.01, *P < 0.05, vs. the control).
Fig. 5
Fig. 5. UF upregulated SIRT1 to deacetylate YAP.
a Immunoblotting showed YAP phosphorylation at S127 after HUVECs were exposed to UF or DF. b Cell lysates from HUVECs treated by DF and UF shear flow were subjected to immunoprecipitation with anti-YAP antibody, and bound proteins were analyzed using specific antibody against acetylated-lysine (AcK), the YAP-flag was used as endogenous control. c Western blot showed the expression of SIRT1 under UF and DF. d Immunoprecipitation assay showed the acetylation level of YAP protein from HUVECs treated by UF together with 10 μM sirtinol. e 293T cells were transfected with FLAG-SIRT1 and pcDNA4/HisMaxB-YAP1 plasmids. Forty-eight hours later, immunoprecipitation assay was performed to show the acetylation status of YAP. Representative images from at least three independent experiments are shown. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane (ST) was served as relative control.
Fig. 6
Fig. 6. SIRT1 promotes the nuclear export and degradation of YAP under UF.
a HUVECs were infected with lentivirus overexpressing SIRT1 for 72 h and the total abundance and nuclear enrichment of YAP protein in the presence of SIRT1 overexpression was detected using western blot. N-YAP represented nuclear YAP. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane was served as relative control. b Luciferase assay showed the activity of 8 × GTIIC reporter in SIRT1-overexpressing HUVECs exposed to DF. Upper panel, western blot to confirm the SIRT1 level after overexpression. c Real-time PCR showed the expression levels of YAP downstream genes CTGF and CYR61 in SIRT1-overexpressing HUVECs. d HUVECs were treated with 10 μM sirtinol and exposed to UF. Then, western blot showed the total abundance of YAP protein under UF. e Real-time PCR showed the expression levels of YAP downstream genes CTGF and CYR61 under the treatment of 10 μM sirtinol together with UF.
Fig. 7
Fig. 7. Rapamycin attenuates atherosclerotic progression in mice.
a Oil Red O staining of mice aortas in the ApoE−/− mice fed a diet supplemented with cholesterol (Vehicle group) or with cholesterol plus Rapamycin (Rapamycin) for 8 weeks, and yellow dashed line indicates the size of plaque area as a percentage of total area. b Quantification of plaque size, Oil Red O-positive area in Vehicle group (n = 10) and rapamycin group (n = 10). Data are mean ± SEM. c Immunofluorescence staining for YAP and SIRT1 proteins in the atherosclerotic vessels from the vehicle group and rapamycin group. Nuclei are counterstained with DAPI (blue). White frame indicates endothelia membrane. Representative images are shown, n = 10. d Western blotting showed the total YAP protein level and SIRT1 level in tissues from vehicle group and rapamycin group. Three samples of each group were shown. Numbers under the blots were mean ± SD of three biologically independent experiments, and the first lane was served as relative control.
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