YAP accelerates vascular senescence via blocking autophagic flux and activating mTOR

Abstract Yes‐associated protein (YAP), a major effector of the Hippo signalling pathway, is widely implicated in vascular pathophysiology processes. Here, we identify a new role of YAP in the regulation of vascular senescence. The inhibition or deficiency and overexpression of YAP were performed in human umbilical vein endothelial cells (HUVECs) and isolated vascular tissues. Cellular and vascular senescence was assessed by analysis of the senescence‐associated β‐galactosidase (SA‐β‐gal) and expression of senescence markers P16, P21, P53, TERT and TRF1. We found that YAP was highly expressed in old vascular tissues, inhibition and knockdown of YAP decreased senescence, while overexpression of YAP increased the senescence in both HUVECs and vascular tissues. In addition, autophagic flux blockage and mTOR pathway activation were observed during YAP‐induced HUVECs and vascular senescence, which could be relieved by the inhibition and knockdown of YAP. Moreover, YAP‐promoted cellular and vascular senescence could be relieved by mTOR inhibition. Collectively, our findings indicate that YAP may serve as a potential therapeutic target for ageing‐associated cardiovascular disease.

the expression of target genes when the Hippo pathway is inactive.
Whereas when the Hippo pathway is active,YAP is phosphorylated, resulting in cytoplasmic retention and proteolytic degradation. [6][7][8] Previous studies have discovered that YAP exerts important effects in the regulation of cellular senescence, in addition to cell proliferation and apoptosis, but the effects vary with different cells. [9][10][11][12] Additionally, YAP is found widely implicated in vascular physiopathology processes, 13,23 such as modulating the activation of endothelial cellular and vascular inflammation, the hallmark of senescence. 14,15 Recently, it has been found that YAP could activate the mammalian target of rapamycin (mTOR), a critical regulator of regulating life span and ageing. 3,16 In addition, many studies indicated that YAP modulates autophagy by interacting with TANK-binding kinase F I G U R E 1 Expression of YAP in young and old vascular tissues. A, SA-β-gal staining was performed to analyse vascular senescence degree from ageing (13 mo) and young (1 mo) rats. B, Western blot was used to analyse the expression of PYAP and YAP and senescence markers P16 and P53 in vascular tissues from ageing and young rats. C, The semi-quantification of YAP. D, The semi-quantification of PYAP. E, The semi-quantification of P16. F, The semiquantification of P53. All experiments were repeated three times, and data are expressed as mean ± SEM. *P < 0.05 vs young group F I G U R E 2 Inhibition of YAP pharmacologically attenuates cellular senescence. A, To build a cellular senescence, HUVECs were cultured for 6 days in 0.5 μg/mL LPS after incubating with DMEM for 12 h. Cellular senescence was assessed by analysis of SA-β-gal in HUVECs treated with 0.1 mg/mL verteporfin for 6 d. B, Western blot was preformed to analyse PYAP, YAP, P16, P21 and P53 in HUVECs treated with or without 0.1 mg/mL verteporfin in the presence of 0.5 μg/mL LPS for 6 d. C, SA-β-gal-positive cell percentage. D-H, The semiquantification of the proteins in panel C, respectively. I, Immunofluorescence attaining was used to ascertain the localization of YAP. The nuclei were labelled with DAPI (blue staining); YAP is red (IgG(H + L)Cy3). J, Expression of indicated proteins was analysed by immunoblot in nuclear and cytoplasmic protein extractions from HUVECs treated with or without 0.1 mg/mL verteporfin in the presence of 0.5 μg/mL LPS for 6 d. All experiments were repeated at least three times, and data are expressed as mean ± SEM, *P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group 1(TBK1) in the cytoplasm or regulating Armus and other members of this family, which plays a major role in senescence process. [17][18][19][20][21][22][23][24] However, whether autophagy and mTOR signalling pathway are involved in the senescence promoting effect of YAP remains unclear.
Here, we identify a novel function of YAP in regulation of vascular senescence and investigate the potential mechanism underlying the senescence promoting effect.
The above 2 solutions were mixed well for 20 minutes at room temperature. Then, cells were incubated with the transfection complex solution for 6 hours at 37°C, and re-incubated in complete medium for an additional 18 hours at 37°C. The synthetic sequence of siYAP is as follows: siYAP#1:GCGTAGCCAGTTACCAACA; siYAP#2:CAGTGGCACCTATCACTCT; siYAP#3:GGTGATACTATCAA CCAAA. Control siRNA is a non-targeting 20-25 nt siRNA (Scramble; Obio technology; China) designed as a negative control. Animas treated with Ad-YAP: 4-week-old SD rats first were randomly assigned to the following four groups (n = 4 for each group): 0 PFU group, 6.32 × 10 4 PFU group, 6.32 × 10 5 PFU group and 12.64 × 10 5 PFU group. Next, the rats in control group were treated with phosphate-buffered saline (PBS), while the rats in the Ad-YAP group were treated with PBS and Ad-YAP in the tail vein. Finally, rats were killed under anaesthesia by pentobarbital sodium and the section of aorta from the ascending aorta to the common iliac arteries was dissected after 4 weeks.

F I G U R E 3
Knockdown of YAP inhibits cellular and vascular senescence. A, SA-β-gal staining was used to analyse senescence degree in HUVECs transfected with YAP siRNA (siYAP#1, siYAP#2, siYAP#3) or negative control siRNA (non-targeting 20-25 nt siRNA) for 6 h. B, SA-β-gal-positive cell percentage. C, Western blot was preformed to analyse PYAP, YAP, P16, P21 and P53 in cells transfected with YAP siRNA (siYAP#1, siYAP#2, siYAP#3) or negative control siRNA for 6 h. D-G, The semi-quantification of the proteins in panel C respectively. H, Western blot was used to analyse YAP and senescence markers P16, P21 and P53 in vascular tissues treated with YAP siRNA (siYAP#1, siYAP#2, siYAP#3) or negative control siRNA for 6 h. I-J, The semi-quantification of the proteins in panel H, respectively. K, Expression of indicated proteins was analysed by immunoblot in nuclear and cytoplasmic protein extractions from in HUVECs treated with or without YAP siRNA (siYAP#1, siYAP#2, siYAP#3) or negative control siRNA for 6 h. L-M, The semi-quantification of the proteins in panel H, respectively. All experiments were repeated at least three times, and data are expressed as mean ± SEM, *P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group

| Western Blot analysis
Total protein was extracted from HUVECs or isolated vascular tissue using RIPA buffer with protease and phosphatase inhibitors. Nuclear and cytoplasmic protein were extracted from HUVECs using Nuclear

| SA-β-gal staining
The senescence Cells Histochemical Staining Kit (Beyotime C0602) was used to evaluate senescence following manufacturer's instructions. Firstly, cells or tissues were incubated with fixation buffer for 10 minutes at room temperature. And then, samples were washed and incubated with staining mixture (containing X-gal) at 37°C for 24-30 hours. Senescence degree was next detected by a fluorescence microscope (Leica Dmi8, Germany). Finally, the numbers of SA-β-galpositive cells were counted by ImageJ software in a blinded manner. fixed with 4% paraformaldehyde, mounted with a reagent containing DAPI as the method in previous study 25 and detected using confocal laser scanning microscope (Leica sp8, Germany). Lastly, the numbers of APs (yellow dots) and ALs (red dot) were counted manually.

| Statistical analysis
All data were analysed by SPSS 21.0 software and expressed as mean ± standard error of the mean (SEM). Statistical differences were evaluated using Student's test or one-way analysis of variance (ANOVA), followed by the Tukey-Kramer HSD post hoc test for multiple comparisons. P < 0.05 was considered as significant statistically differences.

| YAP is highly expressed in old vascular tissues
To determine the difference in YAP expression, the vascular tissues from young and ageing rats were attained. SA-β-gal staining was firstly used to examine the senescence in vascular tissues. As shown in Figure 1A, vascular tissues obtained from ageing rats showed significantly higher β-gal staining than those from young rats. Then, senescence markers P16 and P53 were used to determined vascular senescence by Western blot and the results showed that P16 and P53 were elevated significantly in old vascular tissue ( Figure 1B,E-F). Interestingly, we found YAP was highly expressed in old vascular tissues ( Figure 1B-D). Taken together, our results suggest that YAP expression has positive correlation with vascular senescence.

| Inhibition of YAP pharmacologically or genetically attenuates cellular and vascular senescence
To evaluate the potential roles of YAP in vascular senescence, we first treated HUVECs with YAP inhibitor verteporfin, a compound reported to interfere with YAP binding to the TEAD transcription F I G U R E 4 YAP accelerates cellular and vascular senescence. A, SA-β-gal staining was used to analyse senescence degree in HUVECs treated with 0.5 μg/mL LPS for 6 d or Ad-YAP for 12 h or co-treated with both. B, Western blot was used to analyse PYAP, YAP, P16, P21, P53, TERT and TRF1 in HUVECs treated 0.5 μg/mL LPS for 6 d or Ad-YAP for 12 h or co-treated with both. C, SA-β-gal-positive cell percentage in plane A. D-J, The semi-quantifications of the proteins of plane B, respectively. K, Immunofluorescence attaining was used to ascertain the localization of YAP and P53 in HUVECs treated with Ad-GFP or Ad-YAP for 12 h. The nuclei were labelled with DAPI (blue staining; P53 are red (IgG(H + L)Cy3); YAP is white (Alexa Fluor 647). Scale bar = 25 μm. L, Expression of indicated proteins was analysed by immunoblot in nuclear and cytoplasmic protein extractions from HUVECs treated with 0.5 μg/mL LPS for 6 d or Ad-YAP for 12 h or cotreated with both. M, Rats were treated with YAP adenovirus in different concentration gradients for 4 wk. SA-β-gal staining was used to analyse vascular senescence degree. All experiments were repeated at least three times, and data are mean ± SEM,*P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group factors which interact with coactivator YAP and mediate downstream gene expression. 11,26 To build a model of cellular and organic senescence, the stressor used in this study was LPS, an experimental stressor has been found to induce oxidative stress senescence and accelerate the expression of YAP in human and mouse endothelial cells. 27 Our results showed that HUVECs treated with LPS exhibited significantly increased β-gal-positive cells than the control group, and cells treated with LPS and verteporfin showed significantly decreased β-gal-positive cells than LPS group (Figure 2A Figure 2I-J). Collectively, these results illustrated that inhibiting YAP pharmacological activity decreases cellular senescence.
We next delivered siRNA specifically targeting YAP in HUVECs to further assess the effects of YAP on senescence. We found HUVECs treated with YAP siRNA showed decreased β-gal staining ( Figure 3A

| Overexpression of YAP accelerates cellular and vascular senescence
To ascertain the effect of YAP overexpression on senescence, Ad-YAP was delivered into HUVECs. We found HUVECs co-treated with Ad-YAP showed more β-gal staining ( Figure 4A,C) and displayed significantly higher expression of YAP and senescence markers P53, P21 and P16 ( Figure 4B,D-H,K-L). Additionally, Western blot experiment was performed to determine the telomere activity, a well-accepted marker of cellular senescence, which was affected by YAP. The telomere structure and length stabilization were associated with telomerase reverse transcriptase (TERT) and telomeric-associated protein TRF1. 28 Our results showed HUVECs treated with Ad-YAP displayed significantly lower TERT and TRF1 than LPS group and control group ( Figure 4B,I-J). Furthermore, we added different concentrations of Ad-YAP into rats to determine the effects of YAP overexpression on vascular senescence.
As shown in Figure 4E, β-gal staining fold change was related to Ad-YAP concentrations and the vascular tissue size changed in a concentration-dependent manner ( Figure 4M), which was consistent with prior studies that YAP plays an important role in cell proliferation and organism size. 11 Collectively, those in vitro and in vivo results supported that overexpression of YAP accelerates cellular and vascular senescence.

| Autophagy and mTOR are involved in YAP promoting cellular and vascular senescence
Previous studies have discovered that YAP regulates autophagy by actin depolymerization to control proliferation. 22 Next, we examined the autophagic flux by detecting the numbers of autophagosomes (APs) and autolysosomes (ALs) in the presence and absence of chloroquine, which inhibits lysosomal acidification and prevents autophagosome-lysosome fusion. 30 We found HUVECs treated with Ad-YAP displayed similar effects to LPS, including accumulated APs and few ALs accompanying with higher expression of YAP, P53, P21 and P16. However, cells co-treated with chloroquine showed no alterations with significance in the numbers of APs and ALs and the expression of YAP, P53, P21 and P16 ( Figure 6A-F), suggesting that autophagic flux blockage occurs during YAP promoting senescence. In addition, we found that YAP knockdown attenuated autophagic flux blockage in HUVECs, with more ALs, less APs and decreased expression of YAP, P53, P21 and P16 of ( Figure 6A-B,E). Collectively, these findings showed that autophagic flux blockage occurred during YAP promoting senescence, which can be relieved by YAP inhibition.
Noticeably, prior works had proved that mTOR pathway plays an important role in regulating life span and ageing. 3 In addition, YAP could regulate mTOR pathway by PTEN, an upstream negative regulator of mTOR. 16,[31][32][33] Herein, we investigated mTOR signal activity in YAP promoting senescence and found that HUVECs or F I G U R E 5 Impaired autophagy occurred during YAP promoting cellular and vascular senescence. A, Western blot was preformed to analyse the expression of Beclin1, LC3Ⅱ, LC3Ⅰ and P62 in HUVECs treated with or without 0.1 mg/mL verteporfin in the presence of 0.5 μg/ mL LPS for 6 d. B, The semi-quantifications of the proteins of plane A, respectively. C, Western blot was used to analyse YAP, p-mTOR (Ser2448), mTOR, Beclin1, LC3Ⅱ, LC3Ⅰ and P62 expression in HUVECs with or without YAP siRNA (siYAP#1, siYAP#2, siYAP#3 for 6 h) in the presence of LPS (0.5 μg/mL LPS for 6 d). D, The semi-quantifications of the proteins of plane C, respectively. E, Western blot was preformed to analyse p-mTOR (Ser2448), mTOR, LC3Ⅱ, LC3Ⅰ and P62 in isolated vascular tissue with or without YAP siRNA (siYAP#1, siYAP#2, siYAP#3 for 6 h) in the presence of LPS (0.5 μg/mL for 6 d). F, The semi-quantifications of the proteins of plane E, respectively. All experiments were repeated at least three times, and data are expressed as mean ± SEM, *P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group vascular tissue treated with siRNA-YAP showed down-regulated ratio of p-mTOR to mTOR ( Figure 5C-F Taken together, these findings illustrated that mTOR activity is essential for YAP promoting senescence. Autophagy is a complex intracellular dynamic process that delivers cytoplasmic constituents for degradation into lysosomes, 34 which is essential in mediating proper vascular function, as its role in cell survival and smooth muscle cell phenotype and proliferation. 35 In addition, it was reported that autophagic flux is impaired during senescence-associated disease such as atherosclerosis. 30,36 Moreover, it has been found that YAP regulates autophagy by actin depolymerization to control proliferation. 17 Therefore, the level of autophagy was examined in our study, and we found that autophagic flux blockage occurred during YAP promoting senescence, which could be relieved by YAP inhibition in HUVECs and vascular tissues, suggesting that autophagic flux blockage is involved in the promoting effect of YAP on vascular senescence.

| D ISCUSS I ON
Studies over the past decade have uncovered a critical role for mTOR pathways and Hippo pathways as key players regulating organ size through their respective roles in the modulation of cell growth (size) and cell number (proliferation). In addition, the Hippo-YAP pathway is an upstream regulator of mTOR. 31,[37][38][39] More importantly, increasing mTOR activity perturbs the ability of the whole organism to cope with stress, causing premature senescence and ageing. 37 Here, we investigated the mTOR signalling alteration in YAP-promoted senescence, and we found that YAP deficiency decreased mTOR activity while YAP overexpression increased mTOR activity. We next inhibited mTOR activity with rapamycin to ascertain the effects of mTOR on YAP promoting senescence. We found that, by inhibiting mTOR, YAP-promoted cellular and vascular senescence could be rescued, and the autophagic flux blockage during YAP promoting senescence was also relieved. Collectively, these results illustrated that mTOR signalling pathway plays a vital role in YAP promoting vascular senescence. In addition, YAP-mTOR may mediate vascular senescence by inhibiting autophagic flux. Furthermore, prior studies have found that mutually regulating effects exist between mTOR and YAP, which could be found in YAP-promoted senescence here. 16,31,39,40 Considering the complicated modulation among YAP, mTOR and autophagy, it needs further experiments to prove the clear YAP-mTORautophagic flux signalling axis.
As far as the existing results are concerned, our study indicated that up-regulation of YAP could accelerate vascular senescence. In addition, autophagic flux blockage and mTOR activation were observed during YAP-induced vascular senescence, which could be relieved by YAP inhibition and knockdown. Moreover, YAP-promoted vascular senescence could be rescued by mTOR inhibition. These findings here provide a novel mechanism of cellular and vascular senescence, and suggest YAP may serve as a potential therapeutic target in ageing-associated cardiovascular disease treatment. F I G U R E 6 Autophagic flux was impaired during YAP promoting cellular senescence. A, Western blot was preformed to analyse the expression of LC3Ⅱ, LC3Ⅰ, P62, YAP, P53, P21 and P16 in HUVECs co-treated with or without Chloroquine (CQ, 10 mol/L for 24 h) in the presence of YAP siRNA (siYAP#1, siYAP#2, siYAP#3 for 6 h). Experiments were repeated three times. B, The semi-quantifications of the proteins of plane A, respectively. Data are expressed as mean ± SEM, *P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group. C, Western blot was preformed to analyse the expression of LC3Ⅱ, LC3Ⅰ, P62, YAP, P53 and P16 in HUVECs co-treated with or without CQ (10 mol/L for 24 h) in the presence of Ad-YAP. All experiments were repeated at least three times. D, The semi-quantifications of the proteins of plane C, respectively. Data are expressed as mean ± SEM, *P < 0.05 vs Ctrl group, # P < 0.05 vs Ad-YAP group. E, Tandem fluorescent mRFP-GFP-LC3 adenovirus was subjected to HUVECs to detect the numbers of APs in the presence and absence of CQ (10 mol/L) for 24 h. The nuclei were labelled with DAPI (blue staining); GFP dots are green; mRFP dots are red; YAP is white (Alexa Fluor 647). Scale bar = 10 μm. Experiments were repeated three times. F, Quantitative analysis of APs (yellow dots) and ALs (red dot) in plan E by counting manually; N = 30-50 nuclei per group. Data are expressed as mean ± SEM,*P < 0.05 vs Ctrl group, # P < 0.05 vs LPS group