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. 2023 Jan 11;80(2):38. doi: 10.1007/s00018-022-04623-5

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice

Zheng-Da Pang 1,2,#, Xia Sun 1,4,#, Ru-Yue Bai 1, Meng-Zhuan Han 1, Yong-Jian Zhang 1,3, Wei Wu 1, Yu Zhang 1, Bao-Chang Lai 1, Yi Zhang 1, Yan Wang 1, Xiao-Jun Du 1,✉, Xiu-Ling Deng 1,2,✉
PMCID: PMC11072047  PMID: 36629913

Abstract

Background

Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium.

Methods

Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3−/−) mice and cultured endothelial cells.

Results

The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3−/− mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II.

Conclusions

Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-022-04623-5.

Keywords: Hypertension, Angiotensin II, Galectin-3, YAP, Endothelial dysfunction, Oxidative stress

Introduction

Vascular endothelial cells play a key role in the maintenance of vascular homeostasis in response to various stimuli. Endothelial dysfunction is characterized by the loss of bioavailability of nitric oxide (NO), overproduction of reactive oxygen species (ROS), and inflammatory responses, forming a fundamental abnormality in settings of hypertension, coronary artery disease or diabetes [1]. However, the underlying mechanisms responsible for endothelial dysfunction remain incompletely defined.

Galectin-3 (Gal-3) is a β-galactoside-specific lectin that binds to glycoproteins thereby regulating their function particularly under pathophysiological conditions [2]. Being a potential biomarker and/or disease mediator, it has been shown that elevated circulating Gal-3 is associated with increased risk for heart failure [3], arrhythmias [4] or cardiovascular mortality [5]. In a mouse model of angiotensin II (Ang II) induced hypertension, Gal-3 gene deletion (Gal-3−/−) significantly reduced the expression of inflammatory cytokines in myocardial tissue. Aldosterone, a downstream hormone of Ang II, mediates vascular fibrosis and inflammatory response in part through upregulated Gal-3 [6, 7]. However, the regulatory mechanism of Gal-3 expression by Ang II and the role of Gal-3 in mediating endothelial dysfunction in hypertension are poorly understood.

Recently, Yes-associated protein (YAP) is implicated as a crucial factor in various biological processes in endothelial cells, including angiogenesis and atherosclerosis [8, 9]. Studies have also shown that the transcriptional activity of YAP is regulated by G protein-coupled receptors (GPCRs) [10]. Whereas being inhibited by Gs-coupled receptors [2, 11], YAP transcriptional activity is enhanced by stimulation of Gq-protein coupled GPCRs [11–13]. In vascular smooth muscle cells (VSMCs), Ang II, through activating angiotensin receptor type 1 (AT1R), mediates dephosphorylation of YAPSer127 with increased nuclear localization thereby facilitating phenotypic changes of VSMCs and hypertensive vascular remodelling [14]. Furthermore, in endothelial cells, tyrosine kinase (Src or c-Ab1) is required for the phosphorylation of YAP tyrosine-357 (YAPY357) with increased nuclear localization and hence transcriptional regulation [15]. Src or c-Abl inhibitor markedly attenuated the fibronectin- or oscillatory shear stress-induced phosphorylation of YAPY357 [15]. However, it remains unknown whether Ang II-Src-YAP signaling pathway regulates the expression of Gal-3 in vascular endothelium.

The aim of the present study was to determine the transcriptional regulation of Gal-3 expression in vascular endothelium by Src-YAP signaling, and the role of Gal-3 in mediating endothelial dysfunction in the early phase of hypertension by Ang II infusion in mice.

Materials and methods

Materials

Please see the Major Resources Supplemental Table 1 in the Supplemental information.

Animals and Ang II administration

Male C57BL/6J mice were obtained from Xi’an Jiaotong University Animal Experiment Center. Gal-3 knockout (Gal-3−/−) mice (C57BL/6J background) were obtained from Jackson Laboratories. Subsequent crossing of both strains generated Gal-3−/− and wild-type littermates (WT). Animals were housed in a temperature and 12/12-h light/dark cycle controlled facility with free access to food and water, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA.

To induce early-phase hypertension, WT and Gal-3−/− male mice (8-week old) were infused with Ang II (0.75 mg/kg/day) or normal saline for 2 weeks through osmotic minipump (ALZET model 2002, Cupertino, CA) subcutaneously implanted under isoflurane anesthesia. The model and the dose of Ang II were chosen according to our previous study [16] and preliminary experiments showing that the treatment increased systolic blood pressure (SBP) without overt vascular structure remodeling. During the study period, the SBP was measured weekly using tail-cuff system (BP-300A; Chengdu Taimeng Company, Sichuan, China). At the end of experiment, blood and aortas were collected for further assays.

Human subjects

Hypertensive patients and age, sex and race-matched healthy controls participated in this study were recruited from the hospital health examination center, the First Affiliated Hospital, Xi’an Jiaotong University and given their informed consents in advance, and all procedures were performed in accordance with the guidelines in the Declaration of Helsinki and were approved by the Ethics Committee of Xi’an Jiaotong University Health Science Center (No. 2020-607).

Enzyme-linked immunosorbent assay (ELISA)

Mouse Gal-3 level in serum was measured by DuoSet ELISA kit following the manufacturer’s protocol. Commercial ELISA kits were used to determine serum levels of Gal-3 (Human), Nitric oxide (NO), interleukin-6 (IL-6) or monocyte chemoattractant protein-1 (MCP-1). Each sample was examined in duplicate.

Cell isolation and culture

Mouse thoracic aorta endothelial cells (MAECs) were isolated as described previously [17]. The thoracic aorta was dissected free of adhesive fat and connective tissues and immersed into collagenase II solution (2 mg/ml). After incubation for 45 min at 37 °C, endothelial cells were collected by centrifugation at 1200 rpm for 5 min. Then the precipitate was gently re-suspended in 2 ml of 20% fetal bovine serum (FBS, Invitrogen, Massachusetts, USA) and 2% endothelial cell growth supplement-Dulbecco’s modified Eagle’s medium (DMEM). After 2-h incubation at 37 °C in a humidified atmosphere with 5% CO2, the medium was changed to remove smooth muscle cells. Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM/F12 (Gibco) containing 10% FBS, 2% endothelial cell growth supplement, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere with 5% CO2. Cells between 5 to 8 passages were used.

Isometric force measurements in wire myograph

Mice were sacrificed and their thoracic aortas and mesenteric arteries were rapidly removed and placed in oxygenated ice-cold Krebs–Henseleit solution (KHS; pH = 7.4), dissected free of adhesive fat and connective tissues. For experiments with Ang II pre-incubation, isolated thoracic aortas were immediately placed in DMEM/F12 without or with Ang II (10−9 M) and incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. The thoracic aorta or mesenteric artery (150–250 μm in diameter) was cut into 2-mm rings and then mounted to a Multi Wire Myograph System (model 610M; Danish Myo Technology, Aarhus, Denmark). The vessels were allowed to equilibrate for at least 60 min with the bath solution changed every 20 min. After the equilibration, the reactivity of the rings was checked thrice by administration of 6 × 10−2 M KCl (achieved by substitution of NaCl in KHS with an equimolar concentration of KCl). To assess the integrity of endothelium, thoracic aortas or mesenteric arteries were pre-contracted with phenylephrine (Phe; 10−6 M), and a high dose of acetylcholine (ACh; 10−5 M) was used to relax the arterial rings. ACh-induced relaxation was >80% of the pre-contracted tone in all cases, indicating that the endothelium was functionally intact. Endothelium-dependent relaxations were determined by cumulative addition of ACh (10−9–10−5 M) in Phe (10−6 M) pre-contracted segments. Some segments were incubated with 10−4 M N-nitro-l-arginine (l-NAME), a nonselective NOS inhibitor for 15 min before measurement of endothelium-dependent relaxation. Sodium nitroprusside (SNP; 10−10–10−6 M), an exogenous NO donor, was used in darkness to test endothelium-independent relaxation.

In mesenteric arteries, NO-mediated endothelium-dependent relaxation was expressed as the differences in the area under the concentration–response curve (AUC): AUCACh+L-NAME–AUCAch.

Determination of NADPH oxidase activity

Mouse thoracic aortas were rapidly removed and put in liquid nitrogen. The frozen tissue was ground into powder in liquid nitrogen and lysis buffer was added on ice for 30 min. After centrifugation at 10,000g for 10 min at 4 °C, the supernatant was collected. NADPH activity was measured by colorimetric method using a commercial kit. The readings were normalized to total protein level. The final results were expressed as relative NOX activity normalized to vehicle controls.

Detection of ROS by dihydroethidium and 2′,7′-dichlorodihydrofluorescein diacetate staining

Thoracic aortic segments were fresh-frozen in OCT and sectioned (1 × 10−7 M) using a Leica CM 1000 cryostat. The aortic section or MAECs were washed thrice with phosphate buffer saline (PBS), loaded with 5 × 10−6 M dihydroethidium (DHE) or 10−5 M 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), respectively, and incubated for 30 min at 37 °C in darkness. Differential interference contrast images were obtained using confocal microscopy (Nikon C2, Japan) and Image J was used to analysis fluorescence intensity.

Measurement of NO production in MAECs

As similar to the DCFH-DA staining, intracellular NO level was determined by using a NO-sensitive dye (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-DA). The amount of NO was evaluated by measuring the fluorescence intensity with confocal microscopy (Nikon C2, Japan) and Image J.

Western blotting analysis

Protein samples were prepared from thoracic aortic tissue, mesenteric arterial tissue or endothelial cells, respectively, in cold RIPA lysis buffer including protease inhibitor and phosSTOP phosphatase inhibitor (Roche). Nuclear and cytoplasmic protein samples were collected by Nuclear and Cytoplasmic Protein Extraction Kit. The protein samples were separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane, as previously described [6]. The membranes were immunoblotted with primary antibodies against test proteins (see Supplemental Table 2), at 4 °C overnight, and then incubated with secondary antibodies for 1 h at room temperature. The bound antibodies were detected with an enhanced chemiluminescence detection system (ECL, GE Biotech, USA) and quantified by densitometry using the Chemc-Genius Bio Imaging System (Syngene, Cambridge, UK). To ensure equal sample loading, the ratio of band intensity to β-actin, GAPDH or lamin B for total, nuclear and cytoplasmic protein, respectively, was obtained to quantify the relative protein expression level.

Co-immunoprecipitation (Co-IP)

After nuclear protein collection, the concentration of the sample was adjusted to 1 μg/μl. Then 1 ml of sample was added with 10 μl agarose beads and incubated at 4 °C for 1 h. After centrifugation, the supernatant was incubated with anti-YAP antibody or IgG, respectively, at 4 °C overnight, and then mixed with 50 μl protein A/G PLUS-agarose beads at 4 °C for 3 h. The immunoblotting was performed as described in the Western blotting analysis.

Real-time PCR analysis

Total RNA was extracted from the HUVECs by using TRIzol Reagent. RNA sample was reverse-transcribed into cDNA with the RT kit. Real-time PCR amplification and detection were performed using the SYBR Premix Ex TaqTM according to the manufacturer’s protocol. Amplification was carried out with the following specific primers (Sangon biotech, Shanghai, China): Gal-3, forward primer: 5′-ATGGCAGACAATTTTTCGCT-3′, reverse primer: 5′-GCCTGTCCAGGATAAGCC-3′; β-actin, forward primer: 5′-ATCATGTTTGAGACCTTCAACA-3′, reverse primer: 5′-CATCTCTTGCTCGAAGTCCA-3′. Relative mRNA expression in each sample was determined by comparison with a control group. The expression of target genes was normalized to that of β-actin using the method of 2−ΔΔct.

RNA interference

Specific siRNA molecules targeted to human YAP were synthesized by Gene Pharma Company (Shanghai, China). The siRNA and non-targeting control siRNA molecules at 5 × 10−8 M were transfected into HUVECs using Lipofectamine 2000 reagent for 6 h according to the manufacturer’s instructions. The transfected HUVECs were harvested for analysis. The sequences of YAP siRNA were shown as follows: Sense: 5′-CCUUAACAGUGGCACCUAUTT-3′, Anti-Sense: 5′-AUAGGUGCCACUGUUAAGGTT-3′.

Chromatin immunoprecipitation (ChIP) assay

We performed ChIP assays to evaluate the Tead1 and Gal-3 gene binding activities in the nucleus using Simple ChIP Enzymatic Chromatin IP Kit. The potential binding sites of Tead1 on human Gal-3 gene promoter region were predicted by the UCSC genome website and JASPAR database. According to the manufacturer’s instructions, ChIP assays were performed using an anti-Tead1 antibody. The promoter regions were defined as −2000 to 500 bp from the transcriptional start site of the Gal-3 gene. Immunoprecipitated DNA fragments were reverse cross-linked and DNA binding was quantified as the percentage of input using qPCR. The Gal-3 specific primers (Sangon biotech, Shanghai, China) were used in Supplemental Table 3.

Immunofluorescence staining

HUVECs were fixed with 4% paraformaldehyde for 10 min at room temperature. HUVECs were blocking with 1% BSA for 1 h, follow by permeabilization in 0.05% Triton X-100 (in PBS). Then cells were incubated with the primary anti-YAP antibody at 4 °C overnight. After 3 washes in PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:200) for 1 h at room temperature. Immunofluorescence staining for nuclei was performed with DAPI (1:1000). The fluorescence signal was visualized by confocal microscopy and quantified (Nikon C2, Japan).

Statistical analysis

All data were expressed as mean ± SEM and n represented the number of animals or the number of independent assays. Differences between two groups were assessed using the unpaired two-tailed Student’s t-test, or Mann–Whitney U test for non-normal distribution. Correlations between the nitric oxide and Gal-3 levels were assessed using the unpaired two-tailed Pearson correlation analysis. To analyze data sets involving longitudinal observations or over two groups, two-way ANOVA with repeated measures followed by Tukey’s post-hoc test using Prism GraphPad version 6 (GraphPad Software, Inc., San Diego, California, USA) was used. Multivariable logistic regression analysis was performed to explore the relationship between Gal-3 and hypertension, and adjusted for sex, age, body mass index, nitric oxide, fasting blood glucose, low-density lipoproteins and renal or hepatic function as covariates in the analyses. P < 0.05 was considered statistically significant.

Results

Increased Gal-3 levels in hypertensive patients or in Ang II-treated mice or endothelial cells

Clinical characteristics of hypertensive patients are detailed in Supplemental Table 4. There was no significant difference between hypertensive (n = 51) and healthy control (n = 34) groups in age, body mass index, levels of blood glucose and lipids, as well as renal or hepatic function. The serum level of Gal-3 was significantly higher in hypertensive patients compared with healthy controls (Fig. 1a). The serum NO level was lower in the hypertension group (3.49 ± 0.23 μmol/l vs 4.47 ± 0.40 μmol/l in control), and there was a negative correlation between serum levels of Gal-3 and NO in hypertensive patients (Fig. 1b, n = 32, R2 = 0.2574, P = 0.003). The multivariable logistic regression analyses revealed that Gal-3 increased the odds of hypertension (OR: 2.303, 95% CI: 1.147–4.621, Supplemental Table 5). Ang II (0.75 mg/kg/day, 2 weeks) infusion significantly increased Gal-3 levels in serum, thoracic aorta and mesenteric artery tissue of WT mice, whilst Gal-3 was undetectable in serum, aortic and mesenteric artery tissues of Gal-3−/− mice (Fig. 1c, d). In cultured HUVECs, Ang II (10−6 M) significantly increased mRNA level of Gal-3 at 24 h and protein level at 48 h (Fig. 1e, f). The results indicated that vascular endothelial cells confer higher Gal-3 levels in serum and vascular tissues.

Fig. 1.

Fig. 1

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Increased Gal-3 level in hypertensive patients and Ang II-treated mice or endothelial cells. a Serum Gal-3 level in hypertensive patients (n = 51) and health control (n = 34) measured by ELISA. b Correlation between nitric oxide and Gal-3 levels in the serum of hypertensive patients (n = 32). c, d Gal-3 levels in serum (n = 6/group), isolated thoracic aorta (TA) and mesenteric artery (MA) tissue (n = 4/group) measured by ELISA or Western blotting analysis, respectively, in WT and Gal-3−/− mice infused with normal saline (control group) or Ang II (0.75 mg/kg/day) for 2 weeks via osmotic minipump. e Relative level of Gal-3 mRNA detected by real-time PCR in human umbilical vein endothelial cells (HUVECs) treated with Ang II (10−6 M) for 24 h (n = 6/group). f Western blotting images and quantitative analysis of Gal-3 protein levels in HUVECs treated with Ang II (10−6 M) for 0–48 h (n = 4/group). All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ang II

Ang II upregulates Gal-3 expression through promoting YAP nuclear translocation and YAP-Tead1 interaction in HUVECs

To explore the mechanism of Ang II-induced Gal-3 upregulation in vascular endothelium, we investigated the role of Src-YAP signaling in aortic tissue and HUVECs. In aortic tissues from WT mice treated with Ang II for 2 weeks, abundance of p-SrcY418, p-YAPY357 and total YAP were significantly elevated versus control (Fig. 2a–c). In cultured HUVECs incubated with Ang II (10−6 M) for various time points, the level of p-YAPY357 was increased by Ang II stimulation from 1 to 24 h (Fig. 2d), which was associated with an increased abundance of p-SrcY418 following Ang II treatment for 1 h (Fig. 2e). By contrast, pre-incubation of HUVECs with the Src inhibitor Saracatinib (SA, 1.25 × 10−4 M) reversed Ang II-induced increment in the abundance of p-SrcY418 and p-YAPY357 (Fig. 2e, f). Thus, Ang II-activated Src signaling mediates the phosphorylation of YAPY357 in endothelial cells.

Fig. 2.

Fig. 2

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Increased phosphorylation of Src and YAP in thoracic aorta from Ang II-treated mice and HUVECs. a–c Western blotting of total Src and YAP as well as p-SrcY418 and p-YAPY357 in isolated thoracic aorta tissue from mice infused with Ang II (0.75 mg/kg/day) or vehicle for 2 weeks. d Western blotting of phosphorylated YAPY357 in HUVECs treated with Ang II (10−6 M) for 0–24 h (n = 5/group). e, f Western blotting of phosphorylated SrcY418 and YAPY357 in HUVECs with or without (Control) treatment of Ang II (10−6 M), and the Src inhibitor Saracatinib (SA, 1.25 × 10−4 M) plus Ang II for 1 h. n = 4/group, All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control; #P < 0.05 vs Ang II

It has been shown that phosphorylation of YAPY357 enhances YAP nuclear translocation thereby promoting endothelial cell activation in response to blood flow [15]. We then assessed the effect of Ang II treatment on YAP nuclear translocation in HUVECs. Following exposure to Ang II (10−6 M) for 6 h, there was increased nuclei with YAP-positive stain compared with control indicating increased YAP nuclear localization (Fig. 3a). Similarly, Western blotting of nuclear and cytoplasmic protein fractions from HUVECs revealed that Ang II stimulation for 6 h markedly increased YAP nuclear abundance together with reduced cytoplasmic YAP abundance (Fig. 3b). In HUVECs with YAP gene knockdown using specific YAP-siRNA, upregulation of Gal-3 by Ang II was abolished (Fig. 3c). Co-immunoprecipitation of nuclear protein in HUVECs treated with Ang II (10−6 M) for 6 h showed that increased interaction of YAP and the transcription factor Tead1 (Fig. 3d). Using ChIP assay, we further found that in HUVECs Ang II (10−6 M) treatment for 6 h increased enrichments of Tead1 binding to site-1(−1878/−1868 bp from the transcriptional start site) of the Gal-3 promoter region, other putative sites (site-2: −1523/−1511, site-3: −1286/−1276, site-4: −318/−308 bp from the transcriptional start site) of Gal-3 gene promoter region in HUVECs showed no significant binding by Tead1 (Fig. 3e). Furthermore, PY-60 (5 × 10−6 M and 10−5 M, 48 h), a small molecular and specific activator for YAP transcriptional activity [18], significantly increased Gal-3 protein expression in HUVECs (Fig. 3f). Collectively, these results demonstrate that Ang II increases nuclear translocation of YAP and its interaction with Tead1, a prerequisite for Gal-3 upregulation.

Fig. 3.

Fig. 3

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Ang II increases YAP nuclear translocation in HUVECs together with enhanced Gal-3 expression. a Confocal microscopic detection of YAP (green) and nuclear stain with DAPI (blue) in HUVECs treated with Ang II (10−6 M) for 0–24 h. Scale bar corresponds to 20 µm. b YAP abundance detected by Western blotting in nuclear (Lamin B as maker) and cytoplasmic (GAPDH as marker) protein fractions of HUVECs treated with Ang II (10−6 M) for 6 h. c Protein levels of Gal-3 in HUVECs transfected with control-siRNA and YAP-siRNA followed by treatment with Ang II (10−6 M) for 48 h. d Co-immunoprecipitation of YAP in nuclear proteins to determine molecular interactions in HUVECs treated with Ang II (10−6 M) for 6 h. The lysate was immunoprecipitated with anti-YAP antibody and then immunoblotted with anti-Tead1 antibody. The protein expression was detected by Western blotting analysis. The experiments were repeated three times. e HUVECs treated with or without Ang II (10−6 M) for 6 h were analyzed by ChIP assay with the use of anti-Tead1 antibody. IgG was used as an isotype control. Tead1 promoter enrichment was quantified by qPCR. f Effect of YAP activation on protein expression of Gal-3. Western blotting of Gal-3 in HUVECs treated with PY-60 (5 × 10−6 and 10−5 M, 48 h), a specific activator of YAP transcriptional activity, n = 4/group, All data are mean ± SEM, *P < 0.05, **P < 0.01 vs Control, ###P < 0.001 vs Ang II

Gal-3 mediates Ang II-induced impairment of NO-dependent relaxation in conduit and resistance arteries

SBP was similar between WT and Gal-3−/− mice at baseline or with vehicle infusion (Fig. 4a). In WT mice, infusion of Ang II caused a time-dependent increase in SBP, which was abolished in Gal-3−/− mice (Fig. 4a). In the aorta from WT or Gal-3−/− mice, endothelium-dependent relaxation in response to ACh was evident and was blocked by incubation with eNOS inhibitor l-NAME (10−4 M) (Fig. 4b, c, Supplemental Fig. 1a, b). Ang II infusion or pre-incubation (10−9 M, 24 h) in wild-type but not Gal-3−/− mice significantly blunted such endothelium-dependent relaxation in response to ACh (Fig. 4b, Supplemental Fig. 1a). In contrast, endothelium-independent relaxations of aortas in response to SNP (Fig. 4d and Supplemental Fig. 1c) or vasoconstriction in response to phenylephrine (10−10–10−6 M) (Supplemental Fig. 1d) were comparable between WT and Gal-3−/− mice without or with Ang II treatment. These results indicate that Gal-3 mediates Ang II-induced impairment of NO-mediated relaxation in the thoracic aorta in vivo and in vitro.

Fig. 4.

Fig. 4

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Gal-3 deletion restores NO-mediated endothelium-dependent relaxation and eNOS phosphorylation in thoracic aorta from Ang II-infused mice. a Systolic blood pressure measured by tail-cuff system in WT and Gal-3−/− mice treated for 14 days with Ang II (0.75 mg/kg/day) or vehicle (n = 10/group). Cumulative concentration–response curves to ACh of aortas in the absence b or presence of L-NAME (10−4 M) c in endothelium-intact and phenylephrine (Phe) pre-contracted thoracic aortas from WT and Gal-3−/− mice treated with Ang II (0.75 mg/kg/day) or vehicle for 2 weeks (n = 5 mice/group). d SNP-induced endothelium-independent relaxation of thoracic aortas from WT and Gal-3−/− mice treated with Ang II or vehicle (n = 5 mice /group). e, f Protein level of phosphorylated eNOS (p-eNOSS1176 and p-eNOST494) in isolated aorta tissue of WT and Gal-3−/− mice with Ang II or vehicle (n = 4/group). All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control, #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ang II

Phosphorylation of eNOSS1177 in human (eNOSS1176 in mouse) is known to promotes NO production [19]. We investigated, in isolated aortic tissue, alterations in the level of p-eNOSS1176 as the potential mechanism by which Gal-3 affected NO-mediated relaxation. In comparison with vehicle control, Ang II infusion significantly decreased the level of p-eNOSS1176 in aortas from WT mice (Fig. 4e). Interestingly, Gal-3 deletion per se enhanced eNOSS1176 phosphorylation in aortas relative to that of WT control, and blunted the decline of p-eNOSS1176 induced by Ang II (Fig. 4e). In addition, the level of p-eNOST494 in aortas from Ang II-treated WT mice was increased and the effect of Ang II was attenuated in aortas from Gal-3−/− mice (Fig. 4f).

In view of the more important role of resistance arterioles in regulating blood pressure, we examined whether NO-mediated endothelium-dependent relaxation in mesenteric arteries from Ang II-infused mice was affected by Gal-3 deletion. Ang II infusion attenuated NO-mediated relaxation in response to ACh in mesenteric arteries from WT mice (Fig. 5a, b), which was not obvious in Gal-3−/− mice infused with Ang II (Fig. 5c, d). Gal-3−/− restored the change of NO-mediated endothelium-dependent relaxation by Ang II (Fig. 5e, f).

Fig. 5.

Fig. 5

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Gal-3 deletion restores NO-mediated endothelium-dependent relaxation in mesenteric arteries from Ang II-infused mice. a, c, e Cumulative concentration–response curves to ACh of mesenteric arteries in the absence or presence of l-NAME (10−4 M) in endothelium-intact and phenylephrine (Phe) pre-contracted mesenteric arteries from WT and Gal-3−/− mice treated with Ang II (0.75 mg/kg/day) or vehicle for 2 weeks (n = 5 mice/group). The area under the endothelium-dependent relaxation curve for NO-mediated endothelium-dependent relaxation as indicated in panel b, d, f. All data are mean ± SEM, *P < 0.05, **P < 0.01 vs Control, #P < 0.05, ##P < 0.01 vs Ang II

Gal-3 deletion improves Ang II-induced eNOS uncoupling and NO decrease in MAECs

Endothelial dihydrofolate reductase (DHFR) catalyzes the regeneration of tetrahydrobiopterin (BH4) from its oxidized form dihydrobiopterin (BH2) and is critical for preserving eNOS coupling and NO bioavailability [20]. In MAECs isolated from WT mice, we observed that Ang II (10−6 M, 48 h) significantly reduced protein expression of DHFR (Fig. 6a) and eNOS dimer-to-monomer ratio, an indicator of the uncoupling state of eNOS (Fig. 6b). The effects of Ang II on DHFR and eNOS uncoupling in MAECs was prevented by Gal-3 deletion. Similarly, NO production was blunted in MAECs by Ang II (Fig. 6c). Gal-3 deletion restored the decreased NO production in MAECs.

Fig. 6.

Fig. 6

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Gal-3 deletion reverts Ang II-induced changes in eNOS uncoupling and NO production in mouse aorta endothelial cells (MAECs). a Representative Western blotting and grouped densitometric data of DHFR expression in MAECs isolated from WT and Gal-3−/− mice and treated with Ang II (10−6 M, 48 h) (n = 5/group). b Protein levels of eNOS dimer and monomer detected by using low-temperature SDS-PAGE in MAECs treated with Ang II for 48 h (n = 5/group). c Representative images and summarized data showing NO production (DAF-DA fluorescence) of MAECs isolated from WT and Gal-3−/− mice and treated with Ang II (10−6 M, 48 h) (n = 6/group). Scale bar corresponds to 100 µm. All data are mean ± SEM, *P < 0.05, **P < 0.01 vs Control, #P < 0.05, ##P < 0.01 vs Ang II

Gal-3 deletion inhibits Ang II-induced oxidative stress

We then explored the expression in thoracic aortas of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), which interferes eNOS coupling and NO bioavailability. Ang II infusion in WT mice significantly increased aortic expression of both NOX2 and its subunit p47phox relative to control, and such effect of Ang II was remarkably blunted in aortic tissue from Gal-3−/− mice (Fig. 7a–c). In addition, in WT mice the aortic activity of NADPH oxidase was enhanced by Ang II infusion, which was attenuated by Gal-3 deletion (Fig. 7d), a finding consistent with the unregulated NOX expression. Superoxide production was determined in the aorta using dihydroethidium (DHE) as a probe. In the aortas of Ang II-infused wild-type mice, the generation of superoxide was significantly enhanced as indicated by fluorescence intensity (Fig. 7e, f), and Ang II-evoked increment of ROS in the aorta was prevented in Gal-3−/− mice.

Fig. 7.

Fig. 7

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Gal-3 deletion reverts Ang II-induced changes of NOX2, and superoxide in isolated aorta tissue. a–c Western blotting for protein expression of NOX2 and its subunit p47phox in isolated thoracic aorta tissue of WT and Gal-3−/− mice treated with Ang II (0.75 mg/kg/day) or vehicle for 2 weeks. d NADPH activity measured by a colorimetric method in thoracic aorta tissue from WT and Gal-3−/− mice treated with Ang II or vehicle. The NOX activities in different groups were normalized to controls. e, f Confocal microscopic images of thoracic aorta stained with DHE for detection of superoxide and quantitative analysis of the density of DHE stains in aortas from WT and Gal-3−/− mice treated with Ang II or vehicle. Scale bar corresponds to 200 µm. n = 4/group, All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control, #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ang II

To explore endothelial dysfunction contributing to the changes observed in thoracic aortas, we studied in mouse aortic endothelial cells (MAECs) prepared from Gal-3−/− mice and WT littermates. As shown in Fig. 8a, b, Ang II (10−6 M, 48 h) significantly increased the expression of NOX2 and p47phox in MAECs from WT mice, and this effect was attenuated in MAECs from Gal-3−/− mice. Consistently, immunofluorescence staining showed that Gal-3 deletion reversed Ang II-induced generation of ROS using probes of DHE and DCFH-DA in MAECs (Fig. 8c, f).

Fig. 8.

Fig. 8

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Gal-3 deletion reverts Ang II-induced changes of NOX2 and ROS in MAECs. a, b Western blotting and relative level of NOX2 and its subunit p47phox in MAECs isolated from WT and Gal-3−/− mice and treated with Ang II (10−6 M, 48 h) (n = 4/group). c–f Images of DHE and DCF staining and density levels in MAECs isolated from WT and Gal-3−/− mice and treated with Ang II (10−6 M, 48 h). Scale bar corresponds to 100 µm. All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control, #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ang II

Gal-3 deletion alleviates Ang II-evoked vascular inflammation

We then determined the role of Gal-3 in aortic inflammation induced by Ang II. In isolated thoracic aortas from Ang II-infused WT mice, expressions of macrophage marker CD68, inflammatory cytokine IL-6 and adhesion molecule VCAM-1 were significantly increased relative to control values. These effects of Ang II were abolished in Gal-3−/− mice (Fig. 9a–d). Enhanced protein expression of IL-6 and VCAM-1 by treatment with Ang II (10−6 M, 48 h) was evident in MAECs isolated from WT mice but was absent in MAECs from Gal-3−/− mice (Fig. 9e, f). Similarly, Ang II-infusion increased serum levels of IL-6 and MCP-1 in WT mice, which was also partially or totally reversed by Gal-3 deletion (Fig. 9g, h). Thus, in Ang II-induced hypertensive model, Gal-3 deletion alleviated vascular inflammatory response.

Fig. 9.

Fig. 9

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Gal-3 deletion alleviates Ang II-induced inflammation. a–d Protein expression of CD68, IL-6 and VCAM-1 in aortic tissue from WT and Gal-3−/− mice treated with Ang II (0.75 mg/kg/day) or vehicle for 2 weeks (n = 4/group). e, f Protein expression of IL-6 and VCAM-1 in MAECs isolated from WT and Gal-3−/− mice and treated with Ang II (10−6 M, 48 h) (n = 4/group). g, h Serum levels of IL-6 and MCP-1 measured by ELISA in WT and Gal-3−/− mice treated with Ang II or vehicle (n = 5/group). All data are mean ± SEM, *P < 0.05, **P < 0.01 vs Control, #P < 0.05, ##P < 0.01 vs Ang II

Discussion

In the present study, we demonstrated for the first time that (1) in Ang II-induced hypertension model or Ang II-pretreated endothelial cells, expression of Gal-3 in the vascular endothelium was increased; (2) Ang II-induced Gal-3 upregulation in endothelial cells was mediated by tyrosine kinase Src-driven phosphorylation of YAPY357 with enhanced nuclear localization and transcriptional activity through increased YAP-Tead1 interaction; (3) Genetic Gal-3 deletion abolished rise in SBP in response to Ang II infusion, preserved NO-mediated endothelium-dependent relaxation of conduit and resistance arteries of Ang II-infused mice, and increased NO bioavailability in MAECs; and (4) Gal-3 deletion suppressed oxidative stress and alleviated inflammation in Ang II-treated thoracic aorta and endothelial cells. These findings highlight the role of Gal-3 in Ang II-induced endothelial dysfunction and hypertension.

Ang II level is well known to be elevated in patients with essential or secondary hypertension. Ang II, the key effector of the renin–angiotensin–aldosterone system, plays a central role in the initiation and development of hypertension through vasoconstriction, activation of the sympathetic nervous system and secretion of aldosterone leading to cardiovascular and renal complications as well as uncontrolled hypertension. Using Ang II or aldosterone-induced hypertension model, previous studies showed that either Gal-3−/− or Gal-3 inhibitors are protective against cardiovascular fibrosis and remodeling [6, 7]. However, the role and mechanism of Gal-3 in relating Ang II-induced vascular endothelial dysfunction have not previously been addressed. Our results show that the serum level of Gal-3 was higher in hypertensive patients as well as in Ang II-induced hypertensive mice and that Gal-3 levels correlated negatively with NO levels in hypertensive patients, indicating a causal relationship. As far as we are aware, Gal-3 can be secreted by activated macrophage, cardiac myocytes and fibroblasts, as well as vascular smooth muscle cells and endothelial cells as shown in our study. We recently reported that the elevated blood level of Gal-3 is associated with inflammatory status or activation of the sympathetic nervous system [2]. In the setting of hypertension, systemic inflammation with activation of circulating inflammatory cells contributes to the elevation of Gal-3 in serum, and increased sympathetic activity plays a key role in the release of Gal-3 from the above multiple cells. Moreover, Gal-3 protein expression in aortic and mesenteric artery tissue was significantly increased following Ang II-infusion. Similarly, in endothelial cells Gal-3 mRNA and protein levels were increased by Ang II in a time-dependent manner. The nuclear localization of YAP, which permits its transcriptional activity, is regulated by its phosphorylation status following signals including stimulation of GPCRs [10–14]. Whereas phosphorylation of YAPS127 or YAPS397 reduces its nuclear localization together with enhanced cytoplasmic retention and degradation, phosphorylation of YAPY357 leads to increased stability and nuclear translocation [15, 21]. YAP as a transcription coactivator binds to numerous transcription factors and epigenetic factors to regulate the expression of a range of target genes thereby promoting cell proliferation and survival [14, 22]. Recent studies have provided evidence for regulation by YAP of Gal-3 expression. For example, interfering YAP reduced Gal-3 mediated proliferation of pulmonary arterial smooth muscle cells or aggressive phenotypes of gastric adenocarcinoma cells [23, 24]. In the current study on vascular endothelial cells, Ang II stimulation upregulated Gal-3 expression via Src-mediated YAPY357 phosphorylation with enhanced nuclear translocation and interaction with Tead1, and increased enrichments of Tead1 binding to site-1 of the Gal-3 promoter region. Importantly, ablation of YAP abolished the endothelial Gal-3 expression stimulated by Ang II, and YAP activation by PY-60 significantly increased Gal-3 protein expression. Collectively, these findings establish the notion that Ang II upregulates endothelial Gal-3 expression via Gq-protein coupled AT1R-Src-YAP signaling.

Additional evidence for the role of Gal-3 in mediating endothelial dysfunction comes from the impairment of endothelium-dependent relaxation by myograph in Ang II-treated mice. Importantly, Gal-3 deletion similarly restored NO-mediated endothelium-dependent relaxation in aortas and mesenteric arteries after Ang II infusion in vivo or Ang II pre-incubated aortas in vitro. All these findings demonstrate that Gal-3 is involved in Ang II-induced vascular endothelial dysfunction, which is known to contribute to the onset of hypertension [25].

Our finding of a blunted hypertensive response induced by Ang II suggests that Gal-3 as a potential mediator is involved in blood pressure variation. González et al. previously reported that in Ang II-induced hypertension, Gal-3 deletion prevented ventricular dysfunction without affecting blood pressure or left ventricular hypertrophy [6]. The difference between the current and González’s studies might be partly due to a much lower concentration of Ang II used in our study (0.75 vs 4.32 mg/kg/day) in addition to a short duration (2 vs 8 weeks). In addition, Calvier et al. reported that treatment with Gal-3 inhibitor MCP (modified citrus pectin) for 3 weeks blunted aldosterone-induced hypertension and vascular fibrosis [7]. However, in 30-week-old spontaneously hypertensive rats Gal-3 inhibition did not modify SBP level albeit renal abnormalities were blunted [26]. These findings together with ours supported the potential therapeutic benefit of Gal-3 inhibition in early hypertension.

NO produced in vascular endothelium via eNOS is a key factor for vascular homeostasis [27]. While phosphorylation of Ser1176 in the mouse results in the activation of eNOS, the phosphorylation of Thr494 reduces the eNOS function [28, 29]. Here we identified that p-eNOSS1176 level was significantly decreased while p-eNOST494 level was increased in aorta tissue of Ang II-infused mice, both of which were better preserved in Gal-3−/− mice treated with Ang II. Moreover, Decreased NO bioavailability with elevated production of superoxide characterizes endothelial dysfunction in the setting of hypertension. Superoxide anion can directly bind to NO to generate peroxynitrite (ONOO−), which induces eNOS uncoupling through oxidation of eNOS cofactor BH4, and ultimately endothelial dysfunction [30]. Various in vivo studies on Ang II-induced hypertensive models have found that the uncoupled eNOS as a source of superoxide that is pro-hypertensive [31, 32]. We showed that Ang II decreased DHFR expression and eNOS dimer-to-monomer ratio with decreased NO production in MAECs of WT mice, and Gal-3 deletion restored these changes induced by Ang II. As much as we are aware, this is the first study demonstrating that Ang II regulates NO bioavailability partially via Gal-3 upregulation in vascular endothelium.

Oxidative stress plays a pivotal role in mediating the production and secretion of cytokines, thus linking ROS to inflammation signal and endothelial dysfunction [33, 34]. Ang II is known to increase NOX activity and ROS production in endothelial cells. In endothelial cells, NOXs, specifically NOX2 isoform, are enzyme complexes that catalyze the reduction of molecular oxygen to superoxide anion, thus producing a burst of ROS [35, 36]. NOX2 is able to promote endothelial dysfunction and inflammation in experimental hypertension models [37]. Fleming et al. found that in Ang II-induced hypertension models, knockout of either NOX2 or the subunit p47phox gene effectively ameliorates blood pressure elevation [38, 39]. In the present study, we found that enhanced levels of expression of NOX2 and p47phox and NOX activity as well as ROS production, seen in the aorta of WT mice with Ang II infusion and MAECs isolated from WT and treated with Ang II, were blunted in Gal-3−/− mice. Therefore, it is reasonable to postulate that Ang II regulates oxidative stress through increasing Gal-3 protein in vascular endothelial cells, thereby promoting endothelial dysfunction and the occurrence of hypertension.

Inflammation is involved in vascular injury and remodeling in settings of hypertension and diabetes. There has been accumulating evidence that Ang II is able to induce the expression of several proinflammatory mediators, such as adhesion molecules (ICAM-1 and VCAM-1) and chemokines or cytokines (MCP-1, IL-8, IL-6 and TNF-α) that are involved in vascular inflammation [40]. However, the regulation by Gal-3 of this process is incompletely understood. We found that Ang II resulted in a significantly higher level of IL-6 in either aorta tissue or serum as well as MAECs from WT mice, and the effect was almost abolished in Gal-3–/– mice. In WT mice, Ang II enhanced expression of VCAM-1 in aortas and MAECs as well as MCP-1 level in serum, factors known to promote monocyte recruitment and migration. Indeed, there was a significant increase in CD68 as a macrophage marker in the aortas of WT mice treated with Ang II. All these in vivo and in vitro changes were found to be largely blunted by Gal-3 deletion. Therefore, our results strongly suggest that Gal-3 mediates Ang II-induced vascular inflammation by repressing the expression of inflammatory mediators in the endothelium.

Gal-3 is broadly expressed in inflammatory cells to induce the functions of macrophage chemotaxis and inflammation responding to biochemical and biophysical stimuli. In the present study, we found that Gal-3 deletion per se caused a decrease of CD68 expression in aorta tissue and blunted the increment of CD68 induced by Ang II. Therefore, systemic knockout of Gal-3 inhibits macrophage infiltration into the vascular wall and vascular inflammation, which likely confers protection against Ang II-induced hypertension.

Conclusions

This study for the first time provides direct evidence that Ang II upregulates Gal-3 expression via YAP nuclear translocation in endothelial cells, and that enhanced Gal-3 level impairs arterial endothelial function in a mouse model of early-phase hypertension. Thus, Ang II/YAP/Gal-3 signaling pathway would be a therapeutic target for the treatment of impaired vasodilatation associated with hypertension.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 247 KB) (246.6KB, docx)

Acknowledgements

None.

Abbreviations

Ang II

Angiotensin II

DHFR

Dihydrofolate reductase

eNOS

Endothelial nitric oxide synthase

Gal-3

Galectin-3

GPCRs

G protein-coupled receptors

HUVECs

Human umbilical vein endothelial cells

IL-6

Interleukin-6

MAECs

Mouse thoracic aorta endothelial cells

MCP-1

Monocyte chemoattractant protein-1

NO

Nitric oxide

NOX2

Nicotinamide adenine dinucleotide phosphate oxidase 2

ROS

Reactive oxygen species

Tead1

TEA domain transcription factor 1

VCAM-1

Vascular cell adhesion molecule-1

YAP

Yes-associated protein

Author contributions

ZDP, XS, XJD and XLD contributed to the study conception and design. Material preparation, data collection and analysis were performed by ZDP, XS, RYB, MZH, YJZ, WW, YZ, BCL, YIZ, YW, XJD and XLD. The first draft of the manuscript was written by ZDP, XJD and XLD. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers 81870223 and 82170298), Natural Science Foundation of Shannxi province (grant number 2022JM-557). Yulin Science and Technology Project (grant number CXY-2020-048).

Data availability

Enquiries about data availability should be directed to the authors.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

The animal study was approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. The human study was performed in accordance with the guidelines in the Declaration of Helsinki and was approved by the Ethics Committee of Xi’an Jiaotong University Health Science Center (No. 2020-607).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zheng-Da Pang and Xia Sun contributed equally to this work.

Contributor Information

Xiao-Jun Du, Email: xiao-jun.du@baker.edu.au.

Xiu-Ling Deng, Email: dengxl@mail.xjtu.edu.cn.

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Supplementary Materials

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Data Availability Statement

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