Six-week-old male C57BL/6 mice were purchased from the Beijing Charles River Animal Laboratory (Beijing, China). All animal experiments complied with the international standards stated in the Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee of Shenyang Medical College (approved animal protocol #: 17-25). The mice were randomly divided into the following three groups and treated for 3 weeks: (1) control (Ctr) group: the mice underwent sham procedures with the implantation of an empty osmotic mini-pump (n = 6); (2) AngII hypertensive group (AngII): the mice were implanted with an osmotic mini-pump (Alzet model 1002D, DURECT Inco., Cupertino, CA, United States) with the infusion of AngII (1.1 mg/kg/day, Sigma Aldrich, St. Louis, MO, United States, n = 6); (3) AngII with verteporfin treatment (AngII/Ve): the mice were implanted with an osmotic mini-pump with AngII plus verteporfin (Selleck, Houston, TX, United States) treatment (n = 6). We have previously shown that the mice that received the pressor dose of AngII for 5 days can develop hypertension and cardiovascular injury (Zhang et al., 2021). Verteporfin was dissolved in 10% of dimethylsulfoxide (DMSO) saline solution and administrated at a dosage of 60 mg/kg into mice by intraperitoneal injection every other day. The mice in Ctr and AngII groups were administrated with an intraperitoneal injection of the same amount (0.1 ml) of 10% of DMSO solvent every other day to eliminate the possible effects of this solvent in the mice. Verteporfin is an inhibitor of the YAP-TEAD complex and has been shown to inhibit the translocation of YAP/TAZ into the nuclei and decrease the protein expression of YAP/TAZ (Szeto et al., 2016; Wei et al., 2017). The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by using the tail-cuff method (Softron Biotechnology Co., Ltd., Beijing, China) in the conscious mice as previously described (Huang et al., 2018). The mice were trained daily for 5 consecutive days before the beginning of the experiment. Blood pressure was measured at baseline (before mini-pump implantation) and thereafter once a week. At least five successive readings were recorded and averaged for each mouse. The mice were euthanized by an overdose of anesthesia (5% chloral hydrate, administrated at a dosage of 0.4 ml/100 g body weight, I.P.). The aorta was harvested and weighted.

The thoracic aorta (1 cm below the highest point of the aortic arch) was isolated and fixed in 4% of paraformaldehyde in phosphate-buffered saline. The tissue samples were cut into 4 μm-thick sections and stained with H&E (Sigma Aldrich, St. Louis, MO, United States). The slides were photographed using a Leica DM4B fluorescence microscope. Four images in four non-consecutive slides per animal were acquired and analyzed using the Image J 1.48V software system, and the radial thickness of the media was measured. Trichrome staining (Masson, Sigma Aldrich, St. Louis, MO, United States) was used to assess aortic fibrosis. The semiquantitative analysis was performed, using Image J 1.48V software system, and aortic fibrosis was expressed as a percentage of positive staining area with the total selected area. The image quantitation and representative photomicrographs were taken in a blinded fashion that the reviewers were without the knowledge of the experimental groups.

The aorta was cleaned out of the adherent connective tissues and cut into 3-mm rings. Acetylcholine-induced endothelium-dependent vasorelaxation in aortic rings was determined by using an organ bath chamber (4-channel Tissue Bath System, DMT Inc., Denmark), as previously described (Zhou et al., 2010). Aortic rings were precontracted with 70% of norepinephrine-induced (about 30 nmol/L, Sigma Aldrich, St. Louis, MO, United States) maximal constriction, and then an accumulative concentration of acetylcholine (10^–9 to 10^–5 mol/L, Sigma Aldrich, St. Louis, MO, United States) was added into an organ chamber.

Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, United States), supplemented with 10% of fetal bovine serum (FBS), 100 U/ml of streptomycin, and 100 U/ml of penicillin at 37°C with 5% of CO₂. The cells were incubated with AngII (100 nmol/L) for 24 h. In some experiments, the cells were preincubated with YAP-TAZ inhibitor verteporfin (0.5 μM) or protein phosphatase 2A C (PP2Ac) inhibitor LB-100 (0.1 μM) 1 h before AngII was applied. Veterporfin was dissolved in DMSO solution and further diluted by using the DMEM culture medium. The final concentration of DMSO is <0.01% in the cultured medium. LB100 was dissolved with the culture medium.

The aortic sections (4 μm) were cut from paraffin-embedded tissues and microwaved for 30 min at 60°C for antigen retrieval with citrate buffer (pH 6.0, ZSGB-BIO, Beijing, China). The sections were incubated with primary mouse anti-YAP [1:200 dilution with Tris-buffered saline + Tween-20 (TBST) buffer, sc-101199, Santa Cruz Biotech., Santa Cruz, CA, United States] or mouse anti-F4/80 (1:300 dilution with TBST buffer, 123101, BioLegend) overnight at 4°C, followed by the incubation with fluorescein (FITC)-conjugated goat anti-mouse secondary antibody for YAP or F4/80 (1:500 dilution with TBST buffer, Beyotime Biotechnology, Shanghai, China) at 37°C for 1 h. The nuclei were stained by counterstaining by using 4’,6-diamidino-2-phenylindole (DAPI). YAP fluorescence intensity was visualized and photographed using a Leica DM4B fluorescence microscope (Leica Microsystems Inc., Mannheim, Germany). The relative fluorescence intensity (RFI) of nuclear YAP was determined using the Image J 1.48V software system and normalized by the Ctr group. Monocytes/macrophages (F4/80 positive cells) were viewed, using a fluorescence microscope, and the number of F4/80 positive cells was counted by experienced reviewers who did not know the experimental group of the mice. The images that were acquired from the incubation with lgG without primary antibodies were the negative control. In addition, monocyte and macrophage marker 2 (MOMA2), another marker for monocyte and macrophage, was determined by immunohistochemistry. The slides were incubated with primary antibody against MOMA2 (1:100 dilution with TBST buffer, MOMA2, ab33451, Abcam, Cambridge, United Kingdom) at 4°C overnight, followed by the incubation with the horseradish peroxidase-conjugated goat anti-rat lgG secondary antibody (1:200 dilution with TBST buffer, SA00001-15, Proteintech Group, Danvers, MA, United States). A Vectastain Elite ABC Kit (Vector Laboratories, Burlingham, CA, United States) was used. At least three images per sample for immunofluorescence and immunohistochemistry were acquired, and a semiquantitative analysis was performed, using the Image J 1.48V software system. The data were expressed as a percentage of MOMA2 positive areas with total selected areas.

The aorta or HUVECs were lysed in RIPA lysis buffer supplemented with 1 mmol/L of phenylmethylsulfonyl fluoride, 10 μg/ml of aprotinin, and 10 μg/ml of leupeptin at 4°C for 60 min. In some experiments, the nuclear and cytoplasmic protein fractions were separated and extracted using the nucleoprotein extraction kit (Beyotime Biotechnology, Shanghai, China) according to the protocol of the manufacture. After the homogenization, protein concentrations were measured using a BCA protein assay kit (Beyotime Biotech., Shanghai, China). Of note, 30 μg of total proteins were separated by 8 or 12% of polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membrane. The membranes were incubated with blocking solution (5% of milk in TBST buffer) at room temperature for 2 h and then incubated with primary antibodies against YAP (Sc-101199, Santa Cruz Biotech., Santa Cruz, CA, United States), p-YAP (13008T, Cell Signaling, Chicago, IL, United States), TAZ (4883S, Cell Signaling, Chicago, IL, United States), p-TAZ (59971S, Cell Signaling, Chicago, IL, United States), PP2Ac (2038S, Cell Signaling, Chicago, IL, United States), p-PP2Ac (Sc-271903, Santa Cruz Biotech., Santa Cruz, CA, United States), monocyte chemoattractant protein (MCP)-1 (Sc-52701, Santa Cruz Biotech., Santa Cruz, CA, United States), tumor necrosis factor (TNF)α (Sc-52746, Santa Cruz Biotech., Santa Cruz, CA, United States), HDAC1 (Sc-81598, Santa Cruz Biotech., Santa Cruz, CA, United States), phosphor-endothelial nitric oxide synthase (p-eNOS, AF3247, Affinity, Jiangsu, China), and GAPDH (60004-1-Ig, Proteintech Group, Danvers, MA, United States) at 4°C overnight (1:500 dilution with blocking solution). The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution using blocking solution) for 2 h at room temperature. The signals of luminal chemiluminescence were detected by using an Aplegen Omega Lum G Gel Documentation System (Aplegen Inc., Pleasanton, United States) and quantified using Image J 1.48V software system. HDAC1 is used as a loading control for nuclear lysates and GAPDH as a loading control for cytosolic lysate or whole lysates. Data were normalized by the Ctr group.

The PP2Ac activity was measured using the V2460 kit from Promega (Madison, WI, United States) according to the instruction of the manufacturer. The cells were homogenized in 300 μl of phosphatase storage buffer at 4°C for 30 s and centrifuged at 28,000g at 4°C for 1 h, and 250 μl of supernatant was added to the provided spin columns to remove endogenous free phosphate. The filtrate was collected, 35 μl of the filtrate was added to the wells containing the appropriate reaction solution, and incubated for 10 min at 30°C. The reaction was stopped by adding 50 μl of molybdate dye/additive mixture. The optical density was read at 630 nm using a plate reader. The enzyme activity of PP2Ac was calculated based on a standard curve.

The results were expressed as mean ± SE of the mean. Statistical analyses were performed using GraphPad statistical software package, and the statistical significance of difference was determined by one-way or two-way ANOVA with Bonferroni correction for multiple comparisons. Maximal response to an agonist (Emax) and the concentration of the agonist required for a half-maximal response curve (EC50) were determined and calculated from the concentration-response curve, using the best fit to a logistic sigmoid function. The Kruskal-Wallis non-parameter analysis was used for EC50. Values were considered significant when p < 0.05.

Yes-associated protein and TAZ are major downstream effectors of the Hippo pathway, phosphorylated YAP (p-YAP) and phosphorylated TAZ (p-TAZ) are an inactive form and retained in the cytosolic apartment, and YAP/TAZ activation is initiated by dephosphorylation and promoted the relocation into the nucleus from the cytosolic apartment (Totaro et al., 2018). To determine YAP/TAZ activation, the nuclear and cytoplasmic fractions in the aortic tissues were separated. AngII increased the expressions of nuclear YAP and TAZ and decreased cytosolic p-YAP and p-TAZ expressions, and treatment with verteporfin reversed AngII-induced changes in nuclear YAP/TAZ and cytosolic p-YAP/p-TAZ expressions (Figures 1A–D). Immunofluorescence staining showed that the total immunofluorescence intensity of YAP and its colocalization with the nucleus were significantly increased in the aorta of AngII mice, which was reduced in verteporfin-treated hypertensive mice (Figures 1E,F). The results suggest that YAP/TAZ can be activated in the vasculature of AngII hypertensive mice.

The infusion of AngII for 3 weeks significantly increased SBP (169 ± 7 vs. 112 ± 5 mm Hg in Ctr group) and diastolic blood pressure (DBP) in the mice, and treatment with verteporfin significantly attenuated the elevation of SBP (148 ± 8 vs. 169 ± 7 mmHg in AngII group, p < 0.05, Figure 2A) and DBP (Table 1) in AngII hypertensive mice. There was no significant difference in heart rate among Ctr, AngII, and AngII/Ve groups (Table 1). H&E staining showed that AngII increased the aortic thickness and hypertrophy (49.5 ± 4.9 vs. 31.7 ± 2.7 μm in Ctr group, p < 0.05), which was reduced in verteporfin-treated hypertensive mice (41.7 ± 1.7 vs. 49.5 ± 4.9 μm in AngII group, p < 0.05, Figures 2B,C). Masson-Trichrome staining revealed that AngII significantly increased positive staining areas of collagen, which were partially reduced in AngII/Ve mice (Figures 2D,E). Endothelium-dependent relaxation (EDR) to acetylcholine was significantly impaired in the aorta of AngII mice, compared with the Ctr group. The Emax was 67.5 ± 6.7% in AngII mice vs. 91.9 ± 1.2% in control mice (p < 0.05); EC50 was 252 ± 87 nmol/L in AngII mice vs. 39 ± 3 nmol/L in control mice (p < 0.05). Treatment with verteporfin significantly improved EDR to acetylcholine (Emax: 84.1 ± 4.2%, p < 0.05) in hypertensive mice. EC50 was 252 ± 87 nmol/L in AngII mice vs. 81 ± 14 nmol/L in control mice (p > 0.05). There was no significant difference in EC50 between AngII group and AngII/Ve group (Figure 3A and Table 1). Furthermore, we determined aortic p-eNOS expression, as shown in Figure 3B, and p-eNOS (ser 1177) was significantly decreased in AngII mice. Treatment with verteporfin prevented AngII-induced decrease in p-eNOS.

AngiotensinII increases immune cell infiltration in the vascular wall, which may release proinflammatory cytokines and causes vascular inflammation (Piqueras and Sanz, 2020). We determined aortic MOMA2 expression by immunohistochemistry and F4/80 expression by immunofluorescence. As shown in Figures 4A–D, AngII increased aortic MOMA2 expression and the number of F4/80 positive cells. Treatment with verteporfin significantly reduced the MOMA2 expression and the number of F4/80 positive cells in AngII hypertensive mice. Furthermore, AngII significantly increased the expressions of proinflammatory cytokines MCP-1 and TNFα in the aorta, which were reduced in verteporfin-treated hypertensive mice (Figures 4E,F).

Consistent with the findings in vivo, AngII significantly increased the protein expressions of YAP and TAZ in the nucleus fraction (Figures 5A,C) associated with reduced expressions of p-YAP and p-TAZ in the cytosolic fraction of HUVECs in vitro (Figures 5B,D), and immunofluorescence staining showed that AngII increased YAP immunofluorescence intensity in the nucleus of HUEVCs. Treatment with verteporfin reduced AngII-induced nucleus relocation of YAP (Figures 5E,F). Consistent with the findings in vivo, the inhibition of YAP/TAZ with verteporfin in HUVECs in vitro prevented a decrease in AngII-induced p-eNOS expression (Figure 6A) and an increase in the expressions of proinflammatory cytokines MCP-1 and TNFα (Figures 6B,C). These results suggest that AngII can activate YAP or TAZ in the endothelium, which may contribute to AngII-induced endothelial activation. PP2A is a multifunctional serine/threonine phosphatase. Phosphorylated PP2A is an inactive form, and after dephosphorylation, PP2A is activated. PP2Ac is a major catalytic subunit, and it has been shown that MST1/2 kinases are dephosphorylated by PP2Ac, thus inhibiting the Hippo pathway and activating YAP (Hata et al., 2013). We determined the PP2Ac activity in HUVECs, using a V2460 kit from Promega (Madison, WI, United States) (Ding et al., 2020). As shown in Figure 7A, AngII significantly increased PP2Ac activity, and treatment with PP2Ac inhibitor LB-100 reduced PP2Ac activity in AngII-treated cells. Phosphor-PP2Ac (an inactive form of PP2Ac) but no PP2Ac in AngII-treated cells was significantly decreased, which was significantly reversed in the LB-100-treated group (Figures 7B,C). To investigate whether PP2Ac is involved in AngII-induced YAP activation in the endothelium, HUEVCs were incubated with AngII with or without LB-100. As shown in Figures 7D–G, the inhibition of PP2Ac with LB-100 prevented a decrease in AngII-induced cytoplasmic YAP/TAZ phosphorylation at ser127 and an increase in nucleus YAP/TAZ expression, suggesting that AngII activates YAP/TAZ signaling via the stimulation of PP2Ac. To further investigate whether PP2Ac activation of YAP/TAZ participates in the regulation of AngII-induced endothelial activation, we determined the protein expressions of proinflammatory cytokines TNFα and MCP-1 in AngII-treated HUVECs. AngII significantly increased the expressions of TNFα and MCP-1, and treatment with LB-100 reversed the expressions of AngII-induced proinflammatory cytokines (Figures 7H,I).

This study has several limitations. First, we may miss a group in normal mice with verteporfin treatment, and although the results of this group may not affect our conclusion, it may help us understand the vascular effects of verteporfin on normal mice. Then, hypertension and AngII can affect different vascular beds, resistance artery plays an important role in the regulation of vascular tone and blood pressure, and this study only analyzes verteporfin effects on aorta but not resistance artery. Finally, it has been shown that T lymphocytes and different macrophage subsets play the role in hypertensive vascular injury (Guzik et al., 2007; Carnevale et al., 2020), we did not detect the markers of T cells and macrophage subsets in the vascular system, and it is unclear whether the activation of the YAP/TAZ pathway involves these immune cells in AngII hypertensive mice, which is worthy to be investigated in future.

This study demonstrates that the infusion of AngII activates the endothelial YAP/TAZ pathway, which importantly contributes to vascular inflammation and endothelial dysfunction in hypertensive mice. AngII may activate YAP/TAZ by PP2Ac-dependent dephosphorylation of YAP/TAZ. Our results suggest that YAP/TAZ play a critical role in hypertensive vascular injury and may be a new target molecule for hypertension.