AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK–YAP–JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.


Introduction
Obesity is an independent risk factor for cardiovascular disease [1].Increased blood glucose (hyperglycemia) is associated with endothelial dysfunction in obesity [2,3].Endothelial inflammation is closely associated with obesity and diabetes [2,4].The overexpression of pro-inflammatory cytokines in obesity is considered to be a link between obesityassociated inflammation and endothelial dysfunction [3].Hyperglycemia, insulin resistance, oxidative stress, etc., play critical roles in endothelial dysfunction in obesity [5].However, the exact mechanisms have not been fully elucidated.Recent studies have revealed that the diminished activity of AMPK plays a significant role in hyperglycemia-associated endothelial dysfunction [6,7], although the detailed mechanisms are not fully understood.
YAP is the effector of the Hippo pathway, a highly conserved kinase cascade that controls organ size, cell contact, cancer metastasis, and tissue homeostasis [8,9].Previous studies showed that YAP activation in the vascular endothelium plays a critical role in disturbed flow-induced atherogenesis through JNK activation [10,11].We hypothesize that YAP activation participates in endothelial dysfunction.YAP was reportedly activated in multiple organs under diabetic situations.For example, in patients with diabetic foot, neutrophil extracellular traps suppressed angiogenesis and delayed diabetic wound healing by inducing endothelial-to-mesenchymal transition via activating the YAP pathway [12].YAP activation in diabetic nephropathy was associated with renal fibrosis [13].A recent study showed that high glucose promoted vascular inflammation by activating YAP in diabetic mice [14].However, this study only examined the effect of shear forces on glucosetreated endothelial cells without exploring upstream regulators of YAP.Meanwhile, the activation of YAP and TAZ was observed in white adipocytes from obese humans and mice [15], and YAP activation in adipose tissue induced adipocyte hypertrophy and insulin resistance [16].However, the involvement of YAP in endothelial dysfunction under obese conditions remains unknown.
AMPK was found to act as a negative regulator of YAP activity through both the direct and indirect phosphorylation of YAP at S127 in human embryonic kidney 293 cells (HEK293) and cervical cancer HeLa cell lines [17].AMPK phosphorylates angiomotin-like 1 (AMOTL1) and promotes LATS1 to phosphorylate YAP in HEK293A cells and HaCaT cells [18].Xiong et al. reported that decreased phosphorylation of AMPK resulted in increased YAP expression, leading to aggravated myocardial fibrosis and inflammation in rats with diabetic cardiomyopathy [19].However, the role of the AMPK/YAP signaling pathway in endothelial dysfunction has not been explored.
Metformin, a first-line medication for type 2 diabetes associated with obesity, lowers blood sugar levels, reduces inflammation, improves insulin sensitivity, and improves endothelial function in rodents and humans [20][21][22][23][24][25][26].Clinical studies have shown that metformin treatment promotes weight loss, improves the metabolic profile, and increases insulin sensitivity in obese adolescents [27,28].In vitro studies have shown that metformin increased nitric oxide production in insulin-resistant endothelial cells and suppressed vascular endoplasmic reticulum stress in diet-induced obese mice [29,30].Metformin has recently been described to suppress YAP in multiple cancer cells through activating AMPK [31][32][33].However, whether the AMPK/YAP axis is involved in any beneficial effects of metformin in the vascular endothelium is basically unclear.

Animals
The animal protocols were approved by the Animal Research Ethical Committee of the Chinese University of Hong Kong (CUHK), and the mice were handled according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.Male C57BL/6 mice at 8 weeks old were kept in the CUHK Laboratory Animal Service Center with free access to food and water in a temperature-controlled environment (23 ± 1 • C) and humidity (55 ± 5%) on a 12 h light-dark cycle.Some C57BL/6 mice were fed a high-fat diet (a rodent diet with 60 kcal% fat; D12492; Research Diets, Inc., New Brunswick, NJ, USA) for 8 weeks to induce high-fat diet-induced obesity.

Metformin Treatment
The mice fed with a normal diet and a high-fat diet were randomly divided into 2 groups, respectively, and were administered orally with metformin (200 mg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) [34] suspended in distilled water or a solvent control for 3 weeks.

RNA-seq and Microarray Analyses
Sequencing data are available from NCBI's Gene Expression Omnibus (GEO).Human aortic endothelial cells (HAECs) were obtained from diabetic and non-diabetic patients (n = 3 per group) at autopsy (GSE77108).Fibrovascular membranes (FVMs) were surgically removed from normal humans (n = 3) and patients with diabetic retinopathy (n = 6) (GSE60436).Coronary artery segments of atherosclerotic lesions were from diabetichypercholesterolemic (n = 10 lesions) and hypercholesterolemic (n = 13 lesions) swine (GSE162391).For RNA-seq, Fastqc and trim galore were used to perform quality control.The sequences were mapped to the human genome (GRCh38 release 109) for the human samples and to the Sus Scrofa reference genome (V.11.1) for the pig samples using Hisat2 [35].The tool featureCounts [36] was used to generate a matrix of read counts per gene, and DESeq2 (v1.38.1) software [37] was then used to determine differential gene expression.For the microarrays, the raw gene expression data were normalized, and differential gene expression was selected using the limma package [38].The significance was defined as p < 0.05.Additionally, gene set enrichment analysis (GSEA) was performed on the gene expression profiles using the Molecular Signatures Database (MsigDB, v2023.1).|NES score| > 1 and p < 0.05 were used as the cutoff values for significantly enriched gene sets.The Benjamini-Hochberg procedure was used as the p adjustment method for the analyses above.

Functional Assay by Wire Myograph
The C57BL/6 mice were sacrificed by CO 2 anesthesia, and the mouse aortas or mesenteric resistance arteries were dissected out and placed in oxygenated ice-cold Krebs solution (NaCl: 119 mM, KCl: 4.7 mM, CaCl 2 : 2.5 mM, MgCl 2 : 1 mM, NaHCO 3 : 25 mM, KH 2 PO 4 : 1.2 mM, and D-glucose: 11 mM).Ring segments of the thoracic aortas or mesenteric resistance arteries (~2 mm in length) were suspended between two tungsten wires (40 µm in diameter) in organ chambers (Multi Myograph System) filled with 5 mL oxygenated Krebs solution at 37 °C.The changes in the isometric tone of the rings were recorded by wire myograph (Danish Myo Technology, Aarhus, Denmark).The rings were stretched to an optimal baseline tension of 3 mN (thoracic aortas) and 2 mN (mesenteric resistance arteries) and kept for equilibration for 1 h.The rings were contracted with a 60 mM KCl solution and rinsed in Krebs solution.Endothelium-dependent relaxations induced by acetylcholine (Ach, 10 nM to 10 µM, Sigma-Aldrich, St. Louis, MO, USA) or insulin (0.1 µM to 5 µM, Sigma-Aldrich, St. Louis, MO, USA) were determined in phenylephrine (Phe, 3 µM; Sigma-Aldrich, St. Louis, MO, USA) -precontracted rings.The relaxation was presented as a percentage reduction of phenylephrine-induced contraction [39,40].

Ex Vivo Culture of Mouse Arteries
For the ex vivo studies, thoracic aortas or mesenteric resistance arteries were cut into ring segments (~2 mm in length).All the segments were cultured in a 24-well plate with Dulbecco's Modified Eagle's Media (DMEM; 12320032; Gibco, Carlsbad, CA, USA) containing 1% penicillin/streptomycin and 10% FBS and kept at 37 • C and 5% CO 2 in an incubator.To explore whether metformin ameliorates YAP activation-induced endothelial dysfunction, 1 µL of YAP adenovirus (10 10 plaque-forming units) and GFP control adenovirus were added to the corresponding wells.After 12 h of incubation, metformin was added and the wells were incubated for another 24 h.To study whether metformin ameliorates insulin resistance, metformin and/or high glucose were added to the culture medium for 24 h.After incubation, the arterial rings were transferred to oxygenated Krebs solution, suspended in myograph for functional studies or kept for western blotting (4 aortic rings were merged into one sample).

ROS Determination by Dihydroethidium (DHE) Staining
Intracellular ROS generation was measured by DHE fluorescence staining.Briefly, HUVECs were washed in phosphate-buffered saline and incubated with DHE (5 µM; Invitrogen, Carlsbad, CA, USA) at 37 • C for 15 min.Hoechst 33324 was used to stain the nucleus.The DHE fluorescence was recorded by a fluorescence microscope [39].

Data Analysis and Statistics
The results were analyzed using GraphPad Prism 8 software (8.0.1) and are presented as the means ± SD.The statistical significance was determined using the unpaired t-test or non-parametric Mann-Whitney test for two groups comparison.Multiple comparisons were analyzed by one-way analysis of variance, followed by the Bonferroni multiple comparison test.A value of p < 0.05 was taken as statistically significant.

High Glucose-Induced YAP Activation Accelerates Vascular Injury
To study the role of YAP signaling in high glucose-induced vascular injury, we analyzed the RNA-seq and microarray data of diabetic human aortic endothelial cells (HAECs) and fibrovascular membranes (FVMs) from the GEO database.We found significant activation of the YAP pathway and increased expression of YAP and its target genes, CTGF and CYR61, in the HAECs of diabetic patients compared with healthy subjects (Figure 1A,B).Similarly, in the FVMs of patients with diabetic retinopathy, the expression of the YAP target genes, CTGF and CYR61, also increased (Figure 1C,D).In addition, we analyzed coronary artery data from diabetic-hypercholesterolemic pigs.The expression of CYR61 and inflammatory factors was increased in the coronary artery lesions of diabetic pigs compared with non-diabetic pigs (Supplementary Materials, Figure S1A,B), while the AMPK pathway was significantly inhibited (Supplementary Materials, Figure S1C).These results show that YAP signaling is activated under hyperglycemic or diabetic conditions.

Data Analysis and Statistics
The results were analyzed using GraphPad Prism 8 software (8.0.1) and are presented as the means ± SD.The statistical significance was determined using the unpaired t-test or non-parametric Mann-Whitney test for two groups comparison.Multiple comparisons were analyzed by one-way analysis of variance, followed by the Bonferroni multiple comparison test.A value of p < 0.05 was taken as statistically significant.

High Glucose-Induced YAP Activation Accelerates Vascular Injury
To study the role of YAP signaling in high glucose-induced vascular injury, we analyzed the RNA-seq and microarray data of diabetic human aortic endothelial cells (HAECs) and fibrovascular membranes (FVMs) from the GEO database.We found significant activation of the YAP pathway and increased expression of YAP and its target genes, CTGF and CYR61, in the HAECs of diabetic patients compared with healthy subjects (Figure 1A,B).Similarly, in the FVMs of patients with diabetic retinopathy, the expression of the YAP target genes, CTGF and CYR61, also increased (Figure 1C,D).In addition, we analyzed coronary artery data from diabetic-hypercholesterolemic pigs.The expression of CYR61 and inflammatory factors was increased in the coronary artery lesions of diabetic pigs compared with non-diabetic pigs (Supplementary Materials, Figure S1A,B), while the AMPK pathway was significantly inhibited (Supplementary Materials, Figure S1C).These results show that YAP signaling is activated under hyperglycemic or diabetic conditions.

Metformin Improves Endothelial Function through Inhibiting YAP Signaling
To investigate if metformin protects vascular function through YAP inhibition, we first examined the effect of metformin on YAP phosphorylation and showed that YAP S127 phosphorylation was increased by metformin in a time-dependent fashion (Figure 2A,B).The expression of the YAP target protein CTGF was suppressed by 24 h treatment of metformin (Figure 2C).To verify whether metformin ameliorates YAP activation-induced endothelial dysfunction, we infected isolated mouse aortas with adenovirus for 12h to overexpress YAP and then treated them with metformin for 24 h.The results showed that the expression of YAP in aortic rings infected by YAP adenovirus significantly increased, as well as the YAP target genes CTGF and ANKRD1 (Supplementary Materials, Figure S2A).YAP overexpression impaired acetylcholine-induced endothelium-dependent relaxations (EDRs), which can be rescued by metformin treatment (Figure 2D,E).Correspondingly, YAP overexpression increased the expression of the YAP target genes CTGF, ANKRD1, and CYR61, which were reversed by metformin (Figure 2F).Taken together, these results show that metformin protects endothelial function against YAP overexpression in mouse aortas.

Metformin Improves Endothelial Function through Inhibiting YAP Signaling
To investigate if metformin protects vascular function through YAP inhibition, first examined the effect of metformin on YAP phosphorylation and showed that YAP S phosphorylation was increased by metformin in a time-dependent fashion (Figure 2A The expression of the YAP target protein CTGF was suppressed by 24 h treatment of m formin (Figure 2C).To verify whether metformin ameliorates YAP activation-induced dothelial dysfunction, we infected isolated mouse aortas with adenovirus for 12h to ov express YAP and then treated them with metformin for 24 h.The results showed that expression of YAP in aortic rings infected by YAP adenovirus significantly increased, as well as YAP target genes CTGF and ANKRD1 (Supplementary Materials, Figure S2A).YAP overexpr sion impaired acetylcholine-induced endothelium-dependent relaxations (EDRs), wh can be rescued by metformin treatment (Figure 2D,E).Correspondingly, YAP overexpr sion increased the expression of the YAP target genes CTGF, ANKRD1, and CYR61, wh were reversed by metformin (Figure 2F).Taken together, these results show that metf min protects endothelial function against YAP overexpression in mouse aortas.

Metformin Reverses High Glucose-Induced YAP Activation
To investigate whether metformin protects endothelial cells under hyperglycem conditions through inhibiting YAP signaling, we examined the effect of 30 mM glucose YAP phosphorylation in HUVECs and found that high glucose suppressed YAP ph phorylation and increased the expression of the YAP target protein CYR61.These chan were reversed by metformin (Figure 3A-C).The phosphorylation of endothelial nitric ide synthase (eNOS) on Ser 1177 augments endothelial function [41].We next determin eNOS phosphorylation in HUVECs treated with high glucose and metformin.The resu

Metformin Reverses High Glucose-Induced YAP Activation
To investigate whether metformin protects endothelial cells under hyperglycemic conditions through inhibiting YAP signaling, we examined the effect of 30 mM glucose on YAP phosphorylation in HUVECs and found that high glucose suppressed YAP phosphorylation and increased the expression of the YAP target protein CYR61.These changes were reversed by metformin (Figure 3A-C).The phosphorylation of endothelial nitric oxide synthase (eNOS) on Ser 1177 augments endothelial function [41].We next determined eNOS phosphorylation in HUVECs treated with high glucose and metformin.The results showed that reduced eNOS phosphorylation at Ser 1177 in high glucose-treated HUVECs was reversed by metformin (Figure 3A,D).The increased production of endothelial reactive oxygen species (ROS) participates in endothelial dysfunction associated with hyperglycemia [42].We found that high glucose-increased endothelial ROS production was suppressed by metformin (Figure 3E,F), as also reported in [43].
oxidants 2023, 12, x FOR PEER REVIEW 8 of showed that reduced eNOS phosphorylation at Ser 1177 in high glucose-treated HUVE was reversed by metformin (Figure 3A,D).The increased production of endothelial re tive oxygen species (ROS) participates in endothelial dysfunction associated with hyp glycemia [42].We found that high glucose-increased endothelial ROS production was su pressed by metformin (Figure 3E,F), as also reported in [43].To further evaluate the vasoprotective effect of metformin, mouse aortas were in bated with high glucose and metformin.As insulin resistance plays a critical role in pathogenesis of obesity and diabetes-related vascular diseases, we also explored the eff of metformin on insulin sensitivity in mouse aortic endothelial cells.Consistent with results in the HUVECs, high glucose inhibited YAP phosphorylation, which was revers by metformin in mouse aortas (Figure 4A,B).Since Akt is the upstream regulator for eN phosphorylation [44], we measured the effect of metformin on Akt phosphorylation a found that metformin treatment restored high glucose-induced reduction in basal and sulin-induced Akt phosphorylation (Figure 4A,C).High glucose exposure decreased bo basal and insulin-induced eNOS phosphorylation in mouse aortas, which was revers by co-treatment with metformin (Figure 4A,D).To further show that metformin amel rates insulin resistance, we found that the high glucose-induced impairment of insul induced relaxations in mouse mesenteric arteries was reversed by metformin (Figu 4E,F).
Taken together, these results demonstrate that metformin is effective at inhibiti high glucose-induced YAP activation and the associated endothelial dysfunction.To further evaluate the vasoprotective effect of metformin, mouse aortas were incubated with high glucose and metformin.As insulin resistance plays a critical role in the pathogenesis of obesity and diabetes-related vascular diseases, we also explored the effect of metformin on insulin sensitivity in mouse aortic endothelial cells.Consistent with the results in the HUVECs, high glucose inhibited YAP phosphorylation, which was reversed by metformin in mouse aortas (Figure 4A,B).Since Akt is the upstream regulator for eNOS phosphorylation [44], we measured the effect of metformin on Akt phosphorylation and found that metformin treatment restored high glucose-induced reduction in basal and insulin-induced Akt phosphorylation (Figure 4A,C).High glucose exposure decreased both basal and insulin-induced eNOS phosphorylation in mouse aortas, which was reversed by co-treatment with metformin (Figure 4A,D).To further show that metformin ameliorates insulin resistance, we found that the high glucose-induced impairment of insulin-induced relaxations in mouse mesenteric arteries was reversed by metformin (Figure 4E,F).
Taken together, these results demonstrate that metformin is effective at inhibiting high glucose-induced YAP activation and the associated endothelial dysfunction.

Metformin Improves Endothelial Function through AMPK-Mediated YAP Inhibition
Our previous study reported that the beneficial effects of metformin in the aortas of diet-induced obese mice can be blunted by an AMPK inhibitor compound C [30].We found a marked increase in the level of AMPK phosphorylation in HUVECs after 12 h and 24 h of treatment with metformin (Figure 5A,B).Metformin-induced AMPK phosphorylation is in consistence with metformin-increased YAP phosphorylation, suggesting that metformin increases YAP phosphorylation, probably through AMPK activation.In addition, met-formin reversed the high glucose-induced inhibition of AMPK phosphorylation in isolated mouse aortas (Figure 5C,D) and HUVECs (Supplementary Materials, Figure S3A,B).Further support comes from the observation that compound C reversed metformin-induced AMPK phosphorylation in high glucose-treated HUVECs (Figure 5E,F) and also suppressed metformin-induced YAP phosphorylation (Figure 5E,G), thus indicating that metformin improves endothelial function through promoting AMPK-mediated YAP phosphorylation.Furthermore, compound C inhibited the metformin-increased phosphorylation of Akt and eNOS in high glucose-exposed HUVECs (Figure 5E,H,I).These results show that metformin ameliorates endothelial dysfunction through AMPK-mediated YAP inhibition.
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Metformin Improves Endothelial Function through AMPK-Mediated YAP Inhibition
Our previous study reported that the beneficial effects of metformin in the aortas of diet-induced obese mice can be blunted by an AMPK inhibitor compound C [30].We found a marked increase in the level of AMPK phosphorylation in HUVECs after 12 h and 24 h of treatment with metformin (Figure 5A,B).Metformin-induced AMPK phosphorylation is in consistence with metformin-increased YAP phosphorylation, suggesting that metformin increases YAP phosphorylation, probably through AMPK activation.In addition, metformin reversed the high glucose-induced inhibition of AMPK phosphorylation in isolated mouse aortas (Figure 5C,D) and HUVECs (Supplementary Materials, Figure S3A,B).Further support comes from the observation that compound C reversed metformin-induced AMPK phosphorylation in high glucose-treated HUVECs (Figure 5E,F) and also suppressed metformin-induced YAP phosphorylation (Figure 5E,G), thus indicating that metformin improves endothelial function through promoting AMPK-mediated YAP phosphorylation.Furthermore, compound C inhibited the metformin-increased phosphorylation of Akt and eNOS in high glucose-exposed HUVECs (Figure 5E,H,I).These

Metformin Reduces Vascular Inflammation through Inhibiting the YAP-JNK Pathway
To confirm whether metformin protects endothelial function also through inhibiting the YAP-JNK-inflammation cascade, we treated HUVECs with metformin and determined the mRNA levels of pro-inflammatory genes by real-time PCR.The results showed that metformin suppressed the expression of pro-inflammatory genes, E-selectin, VCAM1, CCL2, and TNFα (Figure 6A).YAP overexpression increased the expression of E-selectin, ICAM1, VCAM1, CCL2, and TNFα, which was reversed by metformin treatment in HUVECs (Figure 6B).JNK is the main activated MAPK in response to inflammatory stress, which is associated with glucose intolerance in obese mice [45].Metformin inhibited high glucoseinduced JNK phosphorylation (Supplementary Materials, Figure S4A,B; Figure 6C,D).To test the role of JNK activation in YAP-induced inflammation, HUVECs were first infected with a plasmid overexpressing YAP and then treated with a JNK inhibitor, SP600125, for 4 h.The results showed that the JNK inhibitor reversed the YAP overexpression-induced expression of E-selectin and ICAM1 (Figure 6E).To substantiate that JNK is the downstream effector of YAP, we treated HUVECs with different concentrations of a JNK stimulator, phorbol 12-myristate 13-acetate (PMA), in the absence or presence of metformin and found that metformin failed to suppress PMA-induced expression of inflammation in HUVECs (Supplementary Materials, Figure S4C,D), thus suggesting that metformin reduces vascular inflammation through the inhibition of YAP without a direct effect on the JNK activity.

Metformin Reduces Vascular Inflammation through Inhibiting the YAP-JNK Pathway
To confirm whether metformin protects endothelial function also through inhibiting the YAP-JNK-inflammation cascade, we treated HUVECs with metformin and determined the mRNA levels of pro-inflammatory genes by real-time PCR.The results showed that metformin suppressed the expression of pro-inflammatory genes, E-selectin, VCAM1, CCL2, and TNFα (Figure 6A).YAP overexpression increased the expression of E-selectin, ICAM1, VCAM1, CCL2, and TNFα, which was reversed by metformin treat- overexpression-induced expression of E-selectin and ICAM1 (Figure 6E).To substantiate that JNK is the downstream effector of YAP, we treated HUVECs with different concentrations of a JNK stimulator, phorbol 12-myristate 13-acetate (PMA), in the absence or presence of metformin and found that metformin failed to suppress PMA-induced expression of inflammation in HUVECs (Supplementary Materials, Figure S4C,D), thus suggesting that metformin reduces vascular inflammation through the inhibition of YAP without a direct effect on the JNK activity.

Metformin Reverses HFD-Induced YAP Activation and Vascular Inflammation
C57BL/6 mice were fed a high-fat diet for eight weeks to induce obesity.The mice were treated with metformin (200 mg/kg/day) or a vehicle for three weeks.After that, the mouse aortas were isolated for western blotting and real-time PCR.Metformin treatment reversed the HFD-induced reduction of AMPK phosphorylation (Figure 7A,B).

Metformin Reverses HFD-Induced YAP Activation and Vascular Inflammation
C57BL/6 mice were fed a high-fat diet for eight weeks to induce obesity.The mice were treated with metformin (200 mg/kg/day) or a vehicle for three weeks.After that, the mouse aortas were isolated for western blotting and real-time PCR.Metformin treatment reversed the HFD-induced reduction of AMPK phosphorylation (Figure 7A,B).Importantly, YAP signaling was activated by HFD feeding, which was reversed by metformin administration (Figure 7A,C).In addition, metformin suppressed the mRNA expression of the YAP target genes CTGF, ANKRD1, and CYR61 (Figure 7D).Metformin treatment also reversed the HFD-induced reduction of eNOS phosphorylation (Figure 7A,E).Furthermore, HFD increased JNK phosphorylation and VCAM1 protein expression, which was reversed by metformin (Figure 7A,F,G).In addition, the HFD-induced mRNA expression of VCAM1 and TNFα was also inhibited by metformin (Figure 7H,I).
formin administration (Figure 7A,C).In addition, metformin suppressed the mRNA expression of the YAP target genes CTGF, ANKRD1, and CYR61 (Figure 7D).Metformin treatment also reversed the HFD-induced reduction of eNOS phosphorylation (Figure 7A,E).Furthermore, HFD increased JNK phosphorylation and VCAM1 protein expression, which was reversed by metformin (Figure 7A,F,G).In addition, the HFD-induced mRNA expression of VCAM1 and TNFα was also inhibited by metformin (Figure 7H,I).

Discussion
Our previous report showed that the effector of the Hippo pathway, YAP, is a mechanosensory gene responsible for atherosclerotic plaque formation upon its activation [10].However, clinical endothelial dysfunction, or atherosclerosis, rarely occurs alone and is often secondary to metabolic disorders such as obesity and diabetes.The key signaling pathways underlying hyperglycemic inflammation have not been fully elucidated.
YAP signaling is vital for vascular dysfunction.A recent study has shown that high glucose accelerates atherosclerosis by SNO-GNAI2 coupling with CXCR5, activating the HIPPO/YAP pathway [46].YAP activation is also related to pathological mechanisms in non-vascular disorders, such as diabetic nephropathy [47,48], tumors [49], and renal fibrosis [50].YAP signaling in Hippo-dependent and -independent pathways in non-vascular models indicates that unknown mechanisms of YAP regulation may exist in hyperglycemia-associated vascular injury.Further research on the vascular YAP pathway could provide a better understanding of the role of hyperglycemia or other metabolic disorders in vascular injury.Based on our previous study, metformin treatment reversed the obesity-

Discussion
Our previous report showed that the effector of the Hippo pathway, YAP, is a mechanosensory gene responsible for atherosclerotic plaque formation upon its activation [10].However, clinical endothelial dysfunction, or atherosclerosis, rarely occurs alone and is often secondary to metabolic disorders such as obesity and diabetes.The key signaling pathways underlying hyperglycemic inflammation have not been fully elucidated.
YAP signaling is vital for vascular dysfunction.A recent study has shown that high glucose accelerates atherosclerosis by SNO-GNAI2 coupling with CXCR5, activating the HIPPO/YAP pathway [46].YAP activation is also related to pathological mechanisms in non-vascular disorders, such as diabetic nephropathy [47,48], tumors [49], and renal fibrosis [50].YAP signaling in Hippo-dependent and -independent pathways in nonvascular models indicates that unknown mechanisms of YAP regulation may exist in hyperglycemia-associated vascular injury.Further research on the vascular YAP pathway could provide a better understanding of the role of hyperglycemia or other metabolic disorders in vascular injury.Based on our previous study, metformin treatment reversed the obesity-associated impairment of endothelium-dependent relaxations [30], and we further explored new mechanisms underlying metformin-induced vascular benefits.
In this study, through mining and analyzing human vascular transcriptome data, we found the high glucose-induced activation of YAP in vascular endothelial cells and the increased expression of YAP target genes.The expression of the YAP target gene CTGF was significantly elevated, and the AMPK pathway was inhibited in diabetic pig aortic tissues, which was consistent with our findings in both the cell and animal experiments.Next, we examined whether metformin, as an activator of AMPK, can reverse the pathological process of high glucose-treated endothelial cells.Our results show that metformin-induced the phosphorylation of both AMPK and YAP in human endothelial cells, suggesting that YAP inhibition may be responsible for metformin-induced vascular beneficial effects.To reveal the role of YAP in endothelial function, we performed a functional assay on a wire myograph and showed that YAP overexpression impairs endothelial function reflected by diminished acetylcholine-induced endothelium-dependent relaxations.Metformin treatment ameliorated YAP overexpression-induced endothelial dysfunction and reversed the YAP overexpression-induced expression of YAP target genes.Metformin treatment decreased the high glucose-induced inhibition of AMPK phosphorylation, thus reversing high glucose-induced YAP activation.Furthermore, this effect of metformin was abolished by the AMPK inhibitor compound C.These new findings suggest that metformin inhibits YAP activity, likely through an AMPK-dependent mechanism, to protect endothelial function.The YAP-JNK cascade contributes to the pro-inflammatory action of YAP activation.We treated HUVECs with high glucose and found that the increased JNK phosphorylation was reversed by metformin.Meanwhile, metformin did not affect the expression of inflammatory genes induced by a JNK inducer PMA, thus suggesting that JNK is likely a downstream effector of YAP-induced inflammation.These results imply that metformin protects endothelial function by inducing YAP phosphorylation and subsequently inhibiting JNK-mediated inflammation via AMPK activation (Figure 8).associated impairment of endothelium-dependent relaxations [30], and we further explored new mechanisms underlying metformin-induced vascular benefits.
In this study, through mining and analyzing human vascular transcriptome data, we found the high glucose-induced activation of YAP in vascular endothelial cells and the increased expression of YAP target genes.The expression of the YAP target gene CTGF was significantly elevated, and the AMPK pathway was inhibited in diabetic pig aortic tissues, which was consistent with our findings in both the cell and animal experiments.Next, we examined whether metformin, as an activator of AMPK, can reverse the pathological process of high glucose-treated endothelial cells.Our results show that metformininduced the phosphorylation of both AMPK and YAP in human endothelial cells, suggesting that YAP inhibition may be responsible for metformin-induced vascular beneficial effects.To reveal the role of YAP in endothelial function, we performed a functional assay on a wire myograph and showed that YAP overexpression impairs endothelial function reflected by diminished acetylcholine-induced endothelium-dependent relaxations.Metformin treatment ameliorated YAP overexpression-induced endothelial dysfunction and reversed the YAP overexpression-induced expression of YAP target genes.Metformin treatment decreased the high glucose-induced inhibition of AMPK phosphorylation, thus reversing high glucose-induced YAP activation.Furthermore, this effect of metformin was abolished by the AMPK inhibitor compound C.These new findings suggest that metformin inhibits YAP activity, likely through an AMPK-dependent mechanism, to protect endothelial function.The YAP-JNK cascade contributes to the pro-inflammatory action of YAP activation.We treated HUVECs with high glucose and found that the increased JNK phosphorylation was reversed by metformin.Meanwhile, metformin did not affect the expression of inflammatory genes induced by a JNK inducer PMA, thus suggesting that JNK is likely a downstream effector of YAP-induced inflammation.These results imply that metformin protects endothelial function by inducing YAP phosphorylation and subsequently inhibiting JNK-mediated inflammation via AMPK activation (Figure 8).Although this study reveals a relatively complete pathway leading to vascular inflammation associated with high glucose-induced changes along the AMPK-YAP-JNK Although this study reveals a relatively complete pathway leading to vascular inflammation associated with high glucose-induced changes along the AMPK-YAP-JNK cascade, we do not rule out that metformin may also phosphorylate other sites of YAP in addition to S127, which deserves future investigation.Actually, except for the phosphorylating YAP127 site, AMPK can directly bind to and phosphorylate YAP at S61, S94, and T119 [51,52], where the S94 residue is critical for YAP-TEAD interaction in HEK293 cells [17].

Figure 1 .
Figure 1.The expression of YAP signaling in diabetic HAECs and FVMs.(A) Normalized enrichment scores (NES) of the YAP-related pathway gene sets (|NES| > 1, p < 0.05) from Molecular Signatures Database (MsigDB) significantly enriched in HAECs of diabetic patients.(B) The relative expression levels of YAP target genes (CTGF and CYR61) in diabetic HAECs.(C) NES of the YAP-

Figure 8 .
Figure 8. Schematic diagram of AMPK-dependent YAP inhibition mediates the protective effect of metformin against obesity-associated endothelial dysfunction.

Figure 8 .
Figure 8. Schematic diagram of AMPK-dependent YAP inhibition mediates the protective effect of metformin against obesity-associated endothelial dysfunction.