Molecular Medicine

YAP Controls Endothelial Activation and Vascular Inflammation Through TRAF6

Yang Lv, Kyungho Kim, Yue Sheng, Jaehyung Cho, Zhijian Qian, You-Yang Zhao, Gang Hu, Duojia Pan, Asrar B. Malik, Guochang Hu

Rationale: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. Objective: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. Methods and Results: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell–specific deletion of YAP. Conclusions: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced

Downloaded from http://ahajournals.org by on July 25, 2018 inflammation and organ failure. (Circ Res. 2018;123:43-56. DOI: 10.1161/CIRCRESAHA.118.313143.)

Key Words: acute lung injury ◼ E-selectin ◼ endothelial cells ◼ neutrophils ◼ sepsis ◼ ubiquitination

epsis is the leading cause of intensive care unit admissions, ultimately organ dysfunction.3 Therefore, maintenance or re- Swith high mortality and morbidity. In septic patients, wide- constitution of vascular endothelial homeostasis after a major spread vascular leakage and inflammation secondary to endo- systemic injury is a crucial determinant of outcome in sepsis.1,4 thelial activation and dysfunction is the primary mechanism of However, the underlying mechanisms limiting the severity of multiorgan failure.1 Endothelial cells are the key sentinel cells endothelial activation during sepsis remain to be identified. detecting microbial infection through the activation of pattern Meet the First Author, see p 3 recognition receptors to mount an inflammatory response. Endothelial activation during sepsis leads to the production of Activation of NF-κB (nuclear factor-κB) is critical for multiple proinflammatory cytokines and upregulation of adhe- regulating the expression of inflammatory genes and adhesion sion molecules E/P-selectins and ICAM (intercellular adhe- molecules.5 The NF-κB activation cascade is initiated by endo- sion molecule)-1 essential for polymorphonuclear neutrophil toxin lipopolysaccharide binding to TLR (Toll-like receptor)-4, (PMN) rolling, adhesion, and transendothelial PMN migra- which activates adaptor protein MyD88 (myeloid differen- tion.2 Sustained activation of endothelium results in massive tiation factor)-dependent and MyD88-independent signaling infiltration of PMNs, hyperinflammation, tissue damage, and pathways.6,7 MyD88 associates with IRAK (IL [interleukin]-1

Original received April 14, 2018; revision received May 16, 2018; accepted May 21, 2018. In April 2018, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.36 days. From the Department of Anesthesiology (Y.L., Guochang Hu) and Department of Pharmacology (K.K., J.C., A.B.M., Guochang Hu), University of Illinois College of Medicine, Chicago; Department of Pharmacology, Nanjing Medical University, Jiangsu, China (Y.L., Gang Hu); Department of Medicine and Cancer Research Center, The University of Illinois Hospital and Health Sciences System, Chicago (Y.S., Z.Q.); Program for Lung and Vascular Biology, Stanley Manne Children’s Research Institute, Ann & Robert H. Lurie Children’s Hospital of Chicago, IL (Y.-Y.Z.); Departments of Pediatrics and Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL (Y.-Y.Z.); and Howard Hughes Medical Institute and Department of Physiology, UT Southwestern Medical Center, Dallas, TX (D.P.). The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 118.313143/-/DC1. Correspondence to Guochang Hu, MD, PhD, Department of Anesthesiology, University of Illinois College of Medicine, 3200W UICH, MC 515, 1740 W Taylor St, Chicago, IL 60612-7239. E-mail [email protected] © 2018 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.118.313143 43 44 Circulation Research June 22, 2018

Novelty and Significance

What Is Known? Systemic endothelial activation and dysfunction induce vascu- lar inflammation and disruption of microvascular barrier integ- • Microvascular endothelial activation and dysfunction are critical deter- rity leading to microvascular leak, tissue edema, shock, organ minants in the progression from sepsis to organ failure. • YAP (Yes-associated protein) transcriptionally promotes cell survival, failure, and ultimate death. However, the molecular mechanisms chemotaxis, differentiation, and proliferation. responsible for controlling endothelial activation remains un- known. Herein, we identified a crucial role of cytoplasmic YAP What New Information Does This Article Contribute? as a checkpoint modulator of endothelial activation and vascular inflammation. YAP suppressed inflammatory signaling by inter- • Endothelial-specific deletion of YAP exaggerates sepsis-induced acting with TRAF6 and prevented the activation of NF-κB by en- vascular inflammation, lung inflammatory injury, and cardiovascular hancing degradation of TRAF6 via K48 ubiquitination as well as dysfunction. suppressing K63-linked ubiquitination. Our results suggest that • YAP limits NF-κB (nuclear factor-κB) activation via modification of the endothelial YAP may be a novel therapeutic target for prevention signaling adaptor TRAF6 (tumor necrosis factor receptor-associated and treatment of sepsis-induced tissue inflammatory injury and factor 6). organ failure.

TRAF6 leads to its degradation via the 26S proteasome, where- Nonstandard Abbreviations and Acronyms as K63-linked polyubiquitination is essential for activation of CKO conditional knockout TAK1 kinase and subsequent NF-κB signaling.13,14 CLP cecal ligation and puncture The Hippo signaling pathway consists of a serine kinase HA hemagglutinin cascade with associated regulatory and scaffolding proteins HEK human embryonic kidney that transcriptionally regulate the expression of genes control- HLMVECs human lung microvascular endothelial cells ling growth. The Ser/Thr kinase Hippo in Drosophila (Mst1 ICAM intercellular adhesion molecule [mammalian sterile 20-like kinase] and Mst2 in mammals) IκB inhibitor protein κB and Sav1 (salvador) form a complex that phosphorylates and activates LATS1/2 (large tumor suppressor) kinases, which IKK IκB kinase

Downloaded from http://ahajournals.org by on July 25, 2018 in turn phosphorylate YAP (Yes-associated protein) and TAZ IL interleukin (transcriptional coactivator with PDZ-binding motif), the 2 IRAK IL-1 receptor-associated kinase downstream effectors of the Hippo pathway, causing them to MyD88 myeloid differentiation factor be retained in the cytoplasm.15–17 Dephosphorylation of YAP NF-κB nuclear factor-κB and TAZ induces their nuclear translocation and activates ex- PMN polymorphonuclear neutrophil pression of genes that promote cell proliferation and inhibit sICAM soluble ICAM apoptosis.18,19 YAP is a potent oncogene first identified by its siRNA small interfering RNA ability to associate with Yes and Src tyrosine kinases.20 As a sVCAM soluble vascular cell adhesion molecule transcriptional coactivator, YAP activates the expression of TAK transforming growth factor-β–activated kinase target genes important for cell survival, chemotaxis, differen- TAZ transcriptional coactivator with PDZ-binding motif tiation, and proliferation.21,22 Activation of YAP or loss of its TLR Toll-like receptor upstream negative regulators leads to striking overgrowth and TNF tumor necrosis factor tumor phenotypes.23 Although Hippo/YAP signaling is impor- TRAF6 TNF receptor-associated factor 6 tant in oncogenesis, little is known about how YAP signals WT wild-type inflammation. We previously showed that Gram-positive bac- YAP Yes-associated protein teria activated Hippo signaling through Toll-dependent path- way in Drosophila.24 It is unknown if Hippo/YAP signaling also regulates inflammatory response induced by sepsis. receptor-associated kinase), which recruits E3 ligase TRAF6 We identified here a previously unrecognized YAP- (tumor necrosis factor [TNF] receptor-associated factor 6),6,8–10 dependent pathway regulating NF-κB activation in endothe- which in turn interacts with the TAK1 (transforming growth lial cells via a direct interaction of YAP with TRAF6. YAP factor-β–activated kinase 1) complex. TAK1 activates IKK induced the degradation of TRAF6 via K48 ubiquitination (IκB [inhibitor protein κB] kinase), leading to the phosphoryla- while simultaneously suppressing K63-linked autoubiquitina- tion and degradation of IκB, and consequent release of NF-κB tion to prevent downstream formation and activation of the and its nuclear translocation.11,12 TRAF6 lies downstream of TAK1 complex and NF-κB signaling. We found that deletion TNF receptor, IL-1 family and most TLR (TLR1-9) and func- of YAP expression in endothelial cells triggered inflammation tions as a node controlling the activation of NF-κB. The ubiqui- even in the absence of infection and caused hyperinflamma- tination of TRAF6 is a key regulatory event controlling the level tion in response to lipopolysaccharide or cecal ligation and of TRAF6 expression and TAK1 activation.13,14 TRAF6 can be puncture (CLP) challenge through unfettered activation of ubiquitinated through the E3 ubiquitin ligase in its RING (really NF-κB signaling. These results establish an important func- interesting new gene) domain.14 K48-linked ubiquitination of tional linkage between endothelial YAP and inhibition of Lv et al YAP Limits Endothelial Activation 45

NF-κB signaling through direct interaction of cytoplasmic The proportion of born WT (YAP+/+), heterozygous (Tie2-Cre; YAP with TRAF6 and point to a fundamental role of YAP in YAP f/+), and homozygous (Tie2-Cre; YAPf/f) mice complied limiting activation of endothelial cells. with Mendel’s laws, and their appearance was undistinguish- able from the littermates. Similarly, the male to female ratio Methods was also consistent with the law. No difference was observed The data that support the findings of this study are available from the with regard to body weight (Online Figure IIA) or organ size corresponding author on reasonable request. (Online Figure IIB and IIC) in YAP-CKO mice. The ratios of Details of materials and experimental procedures are available in organ to body weight were also not different in YAP-CKO mice the Online Data Supplement. except for the slightly higher ratio in spleen/body weight as Mice compared with WT mice (Online Figure IIC). YAP-CKO mice Endothelial-specific YAP-deficient mice were generated by crossing also showed normal fertility and the oldest mice survived for >2 YAP-floxed mice (YAPf/f) with Tie2-Cre transgenic mice. All mice years. Importantly, YAP-CKO mice displayed mild lung inflam- f/f used in the experiments were the littermates of genotypes of YAP mation and endothelial activation as evidenced by increased with Cre gene as endothelial YAP-conditional knockout (CKO) mice (these mice are hereafter referred to as YAP-CKO). Mice only carry numbers of total cells, PMNs, macrophages, and PMN/mac- YAP f/f as wild-type (WT) mice. rophage ratio in bronchoalveolar lavage fluid (Online Figures IID, IIE, and IIIA through IIIC) and increased expression of Mouse Models of Endotoxemia and Polymicrobial E-selectin and ICAM-1 in lung endothelial cells (Online Figure Sepsis IVA through IVC) as compared with WT mice. All experimental mice were sex- and age-matched. Endotoxemia was induced with injection of lipopolysaccharide (5 mg/kg, IP). Enhanced Inflammatory Response to Endotoxin Polymicrobial sepsis was created by CLP. and Polymicrobial Sepsis in YAP-CKO Mice Intravital Microscopy We next examined whether baseline increases in endothelial The cremaster muscle was exposed. PMNs and monocytes were vi- activation enhanced the susceptibility of YAP-CKO mice to sualized by infusion of Alexa Fluor 647-conjugated anti-Ly-6G and endotoxemia. In YAP-CKO mice challenged with lipopolysac- Alexa Fluor 647-conjugated anti-F4/80 antibody, respectively. The charide, the numbers of total cells (Online Figure IIIA), PMNs rolling influx and number of adherent PMNs or monocytes were (Figure 1A), macrophages (Online Figure IIIB), and the ratio determined. of PMN/macrophages (Online Figure IIIC) in bronchoalveolar Generation of Bone Marrow Chimeras lavage fluid as well as expressions of E-selectin and ICAM- The irradiated mice (CD45.2) were transplanted with bone marrow 1 in pulmonary endothelial cells (Online Figure IVA through Downloaded from http://ahajournals.org by on July 25, 2018 cells isolated from Ly5.1 (CD45.1). Six weeks after transplantation, IVC) dramatically increased compared with their WT coun- all the recipients were used for measurement of lung inflammation. terparts. The serum sICAM (soluble ICAM)-1 (Online Figure Depletion of TRAF6 in Mouse Pulmonary IVD) and sVCAM (soluble vascular cell adhesion molecule)-1 (Online Figure IVE) released by activated endothelium were Endothelium TRAF6 was depleted in mouse pulmonary vasculature by intrave- also elevated both basally and after lipopolysaccharide chal- nous injection of cationic liposome-TRAF6 small interfering RNA lenge. Lung tissue from WT counterparts after lipopolysac- (siRNA) complexes. charide challenge had severe histological changes, including alveolar congestion, exudates, and infiltration of inflammatory Statistics cells, whereas these alterations were markedly exacerbated in One-way ANOVA and Student Newman-Keuls test for post hoc com- parisons were used to test differences between the means of 3 or more lipopolysaccharide-challenged YAP-CKO mice, as evident by groups. Mann-Whitney U test was performed to detect significant dif- the significantly higher lung injury score (Figure 1B). To as- ferences between 2 experimental groups. Difference in the survival sess whether endothelial YAP knockout also augmented the between 2 groups was determined by Mantel-Cox test. All data are increase in lipopolysaccharide-induced pulmonary vascular expressed as mean±SD. Differences were considered significant permeability, we measured lung albumin-bound Evans blue when P<0.05. dye extravasation, an indicator of vascular leakage. We ob- served a striking aggravation of lipopolysaccharide-induced Results pulmonary vascular hyperpermeability in YAP-CKO mice Endothelial Cell–Specific Deletion of YAP Basally (Figure 1C). Production of the proinflammatory cytokines IL- Activates Inflammation 6, TNF-α, and IL-1β was greatly elevated in YAP-CKO mice To study the functional significance of YAP expression in en- after lipopolysaccharide challenge as compared with WT lit- dothelial cells, we generated YAP-CKO mice (Tie2-Cre; YAPf/f; termates (Figure 1D). Importantly, YAP-CKO mice had a sig- Online Figure IA). Cre and YAP-flox alleles in mice were iden- nificantly higher mortality rate after challenge with lethal dose tified by PCR (Online Figure IB). Specificity and efficiency of of lipopolysaccharide than WT counterparts (Figure 1E). In a YAP deletion were determined by real-time quantitative reverse polymicrobial sepsis model, deletion of YAP also enhanced transcription PCR and Western blotting. We found >85% re- CLP-induced increase in serum sICAM-1 (Online Figure duction in YAP mRNA (Online Figure IC) and protein levels IVF), sVCAM-1 (Online Figure IVG), IL-6 (Figure 1F), in isolated mouse lung endothelial cells but not in nonendo- and TNF-α (Figure 1G). In addition, mean blood pressure thelial cells (epithelial cells and fibroblasts; Online Figure ID). (Figure 1H), ejection fraction (Figure 1I and 1J), and frac- Immunoblotting assessment of YAP protein expression also ex- tional shortening (Figure 1I and 1K) dramatically decreased hibited 60% reduction in whole lung tissues (Online Figure IE). in YAP-CKO mice after CLP challenge compared with WT 46 Circulation Research June 22, 2018

Figure 1. YAP (Yes-associated protein) deletion in endothelial cells increases lung inflammatory injury, cardiovascular organ

Downloaded from http://ahajournals.org by on July 25, 2018 failure, and mortality in mice challenged by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Wild-type (WT) and YAP-conditional knockout (CKO) littermates were challenged with LPS (5 mg/kg, IP, A–D) or CLP (F–K). Bronchoalveolar lavage (BAL) was performed at 0, 6, and 24 h after LPS injection. A, Polymorphonuclear neutrophil (PMN) count in BAL fluid by cytospin analysis. n=6. **P<0.01, Mann-Whitney U test. B, Hematoxylin and eosin staining of sections of lungs. Left, Representative lung histology. Red arrow, PMNs; yellow arrow, erythrocyte. Magnification, 20×; inset, 40×.Right , quantification of histopathologic lung injury scores. n=5 to 7. **P<0.01, Mann-Whitney U test. C, Pulmonary vascular permeability measured by Evans blue dye extravasation. Left, Representative lung appearance after Evans blue dye administration. Right, Quantitative analysis of Evans blue-labeled albumin extravasation. n=6 to 8. ***P<0.001, Mann-Whitney U test. D, Levels of IL (interleukin)-6, TNF (tumor necrosis factor)-α, and IL-1β in the serum of WT and YAP- CKO mice challenged with LPS. n=6 to 7. *P<0.05, **P<0.01, Mann-Whitney U test. E, Survival of WT and YAP-CKO mice challenged with LPS (20 mg/kg, IP). n=15 mice/group. ***P<0.001, Mantel-Cox test. F and G, Levels of IL-6 (F) and TNF-α (G) in the serum of WT and YAP-CKO mice challenged with CLP. H, Mean blood pressure (MAP). Basal values (mm Hg): WT/YAP-CKO, systolic pressure, 142±22/128±19; diastolic pressure, 97±24/90±21; MAP, 112±23/102±20. Heart rate (beats/min), 508±77 (WT)/450±57 (YAP-CKO). n=6 to 7. I, Photographs of echocardiograms. Basal values (mm): WT/YAP-CKO, left ventricular anterior wall (systole), 1.18±0.21/1.06±0.11; left ventricular anterior wall (diastole), 0.81±0.19/0.73±0.12; left ventricular posterior wall thickness (systole), 1.19±0.32/0.98±0.13; left ventricular posterior wall thickness (diastole), 0.72±0.22/0.69±0.13. n=6 to 7. J, Ejection fractions (EF%). K, Fractional shortening (FS%). n=6 per group; **P<0.01, *** P<0.001, Mann-Whitney U test. L, Survival of WT and YAP-CKO mice subjected to CLP. Sham n=10, CLP n=15. *** P<0.001, Mantel-Cox test.

mice. Depletion of endothelial YAP resulted in 0% survival ≈95% reconstitution of WT and YAP-CKO bone marrow after CLP, whereas WT mice exhibited ≈70% survival within at 5 weeks after transplantation (Figure 2B and 2C; Online 96 hours (Figure 1L). Figure VB). Transplantation of WT bone marrow into YAP- Because Cre recombinase driven by the Tie2 promoter was CKO mice failed to prevent an increase in lung inflammation used to delete yap in the mouse studies, this promoter may also after lipopolysaccharide challenge (Figure 2D through 2F). induce YAP deletion in hematopoietic cells.25 To determine any Consistently, transplantation of YAP-CKO bone marrow into contribution of YAP deletion in myeloid cells to the enhanced WT mice did not change the phenotypes of lung inflamma- endothelial immune response, we generated chimeric mice tion after lipopolysaccharide challenge (Online Figure VC and (Figure 2A; Online Figure VA) to examine whether the hema- VD). These results indicate that deletion of YAP in hematopoi- topoietic cells favored lung inflammation after lipopolysaccha- etic cells per se did not contribute to augmented lung inflam- ride challenge. Bone marrow cells isolated from WT (Ly5.1) mation in YAP-CKO mice in response to lipopolysaccharide. were transplanted into lethally irradiated WT or YAP-CKO Because Cre is known to have nonspecific effects in mul- mice (Figure 2A). In other experiments, bone marrow cells tiple cell and tissues that could influence inflammation, we isolated from WT or YAP-CKO mice were transplanted into performed an additional experiment in Tie2-Cre mice to ad- lethally irradiated WT (Ly5.1) mice (Online Figure VA). FACS dress the potential role of Cre in a focused comparative in (fluorescence-activated cell sorting) analysis demonstrated vivo study with YAPf/f and YAP-CKO mice. Tie2 Cre mice Lv et al YAP Limits Endothelial Activation 47 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 2. Failure of wild-type (WT) bone marrow cells to rescue increased inflammatory response after lipopolysaccharide (LPS) stimulation in YAP (Yes-associated protein)-conditional knockout (CKO) mice. A, An experimental protocol showing bone marrow transplantation to lethally irradiated WT and YAP-CKO mice. B and C, FACS (fluorescence-activated cell sorting) analysis of the percentage of the CD45.1 marker-expressing in the reconstituted WT and YAP-CKO mice. ***P<0.001, 1-way ANOVA. D, Total cells, polymorphonuclear neutrophils (PMNs), macrophages (Mϕ), and PMN/Mϕ ratio in bronchoalveolar lavage (BAL) fluid were enumerated at 0 and 24 h after LPS challenge. The numbers of PMNs in BAL under basal condition are (0.82±0.42)×103 in WT mice and (7.36±1.78)×103 in YAP-CKO mice. n=5 to 7 per group. *P<0.05, **P<0.01, Mann-Whitney U test. E, Pulmonary vascular permeability measured by Evans blue dye extravasation. Quantitative analysis of Evans blue-labeled albumin extravasation was measured by spectrophotometry. n=5 to 7 per group. **P<0.01, Mann-Whitney U test. F, Levels of IL (interleukin)-6 and TNF-α in serum measured by ELISA. n=5 to 6 per group. **P<0.01, Mann-Whitney U test.

exhibited a similar pattern of inflammatory response with WT lipopolysaccharide challenge (Figure 3C; Online Movie I (YAPf/f) mice (Online Figure VIA and VIB). through IV). As measured by anti-F4/80 antibodies, monocyte adhesion to the inflamed endothelium was also enhanced in YAP Suppresses PMN and Monocyte Recruitment lipopolysaccharide-challenged YAP-CKO mice, compared in Microvessels with WT mice (Online Figure VII). Because of the small % in To investigate the role of YAP in systemic vascular inflam- leukocytes, no rolling and adherent monocytes were detected mation, we performed real-time fluorescence intravital mi- in unchallenged WT and CKO mice (data not shown). croscopy in a mouse model of lipopolysaccharide-induced We also performed immunohistochemistry in cremaster cremaster venular inflammation.26 YAP-CKO mice exhibited muscle vessels taken from the mice after intravital microscopy markedly increased number of adherent PMNs compared with to determine E-selectin and ICAM-1 expression, which medi- WT mice both basally and after lipopolysaccharide challenge ates PMN rolling and adhesion, respectively.2,27 Expressions (Figure 3A and 3B). PMN rolling was reduced in YAP-CKO of E-selectin and ICAM-1 in endothelial cells of YAP-CKO mice compared with that in WT mice both basally and after mice were significantly increased under basal conditions 48 Circulation Research June 22, 2018 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 3. Endothelial YAP (Yes-associated protein) deletion increases the number of adherent polymorphonuclear neutrophils (PMNs) in vivo. A, Representative images of cremaster muscle venule demonstrating the role of YAP in regulating PMN rolling and recruitment. PMN accumulation was visualized by infusion of Alexa Fluor 647-anti-Ly6G into wild-type (WT) or YAP-conditional knockout (CKO) mice challenged with lipopolysaccharide (LPS) for 0 and 24 h. Scale bar: 10 μm. Arrows show direction of blood flow and yellow dashed lines indicate the border of blood vessel. B and C, Numbers of adherent (B) and rolling (C) PMNs are shown. n=28 to 30 venules in 3 to 4 mice per group. D, Representative images of E-selectin and ICAM (intercellular adhesion molecule)-1 staining. Cremaster muscles from mice treated with LPS for 0 and 24 h were stained with PECAM-1 (platelet endothelial cell adhesion molecule-1) and E-selectin or ICAM-1 antibodies. Scale bar: 10 μm. E and F, Quantitative data showing changes in mean fluorescent intensity (MFI) of E-selectin (E) and ICAM-1 (F) expression. n=4 to 6. *P<0.05, **P<0.01, and ***P<0.001, 1-way ANOVA.

compared with control mice, and expressions of both adhesion time-dependent manner, peaked at 4 hours, and then gradually proteins were further increased after lipopolysaccharide chal- decreased to basal level at 24 hours (Figure 4A). A similar lenge (Figure 3D through 3F). result of YAP mRNA expression was seen in HUVECs af- ter lipopolysaccharide stimulation by real-time quantitative YAP Negatively Regulates Endothelial Activation reverse transcription PCR (Figure 4B). Expression of YAP To further determine the role of YAP in the endothelial activa- protein was also elevated at 4 hours after stimulation with

tion during sepsis, we first examined the expression of YAP other stimuli TNF-α or H2O2 in HUVECs (Figure 4C, left) in response to lipopolysaccharide in HUVECs (human um- and human lung microvascular endothelial cells (HLMVECs; bilical vein endothelial cells). We observed that YAP protein Figure 4C, right). To verify whether lipopolysaccharide regu- expression increased after lipopolysaccharide stimulation in a lates YAP protein expression in vivo, we challenged mice by Lv et al YAP Limits Endothelial Activation 49 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 4. Lipopolysaccharide (LPS) induces YAP (Yes-associated protein) expression in human endothelial cells and mouse lungs. A, Immunoblotting showing expression of YAP in the HUVECs (human umbilical vein endothelial cells) after LPS stimulation (1 µg/mL) at indicated times. Left, Representative Western blots for YAP and GAPDH (loading control); right, protein quantification by densitometry. n=3. **P<0.01 and ***P<0.001, 1-way ANOVA. B, Quantitative analysis of YAP mRNA levels in HUVECs by quantitative reverse transcription PCR (qRT-PCR). YAP mRNA levels were normalized to β-actin. n=3. *P<0.05, 1-way ANOVA. C, Immunoblot analysis of YAP expression in the HUVECs (left) and human lung microvascular endothelial cells (HLMVECs; right) after stimulation with different inflammatory mediators. The

cells were incubated with PBS, LPS (1 µg/mL), recombinant human TNF (tumor necrosis factor)-α (10 ng/mL) or H2O2 (500 µmol/L) for 4 h. Top, Representative Western blots for YAP and GAPDH (loading control); bottom, protein quantification by densitometry. n=3. *P<0.05, **P<0.01, and ***P<0.001, Mann-Whitney U test. D, YAP expression in wild-type (WT) mouse lungs after LPS challenge. Lung endothelial cells (EC) and nonendothelial cells (non-EC) were isolated from WT challenged with LPS (5 mg/kg) for 4 h. n=4. *P<0.05, Mann-Whitney U test.

IP administration with lipopolysaccharide to induce acute Figure VIIIC) or HLMVECs (Online Figure VIIID) inhibited lung injury. As shown in Figure 4D, YAP protein expression lipopolysaccharide-induced protein expression of ICAM-1 and in both endothelial and nonendothelial cells was increased in E-selectin. Ablation of YAP in HUVECs (Figure 5G and 5H) vivo after lipopolysaccharide challenge. or HLMVECs (Online Figure VIIIE) increased PMN adhesion We next deleted YAP protein in HUVECs with CRISPR/ (Figure 5G; Online Figure VIIIE, left) and transendothelial PMN Cas9 or overexpressed it with recombinant Flag-YAP cDNA migration (Figure 5H; Online Figure VIIIE, right) even in the and measured the production of proinflammatory cytokines absence of lipopolysaccharide stimulation. YAP deletion mark- on challenging the cells with lipopolysaccharide. YAP deletion edly augmented lipopolysaccharide-induced PMN adhesion and (Figure 5A) significantly increased the production of IL-6 and transendothelial PMN migration as compared with control cells IL-1β over time (Figure 5B). Conversely, overexpression of YAP (Figure 5G and 5H; Online Figure VIIIE). Consistently, overex- (Figure 5C) markedly attenuated the production of IL-6 and IL- pression of YAP in HUVECs (Figure 5I and 5J) or HLMVECs 1β (Figure 5D). Leukocyte adhesion via endothelial ICAM-1 (Online Figure VIIIF) inhibited lipopolysaccharide-induced and E-selectin and subsequent transendothelial migration are PMN adhesion and transendothelial PMN migration. Thus, YAP hallmarks of endothelial activation and inflammation and are modulated the state of endothelial activation basally as well as in central features of the innate immune function of endothelial response to lipopolysaccharide. cells.28 We found that depletion of YAP in HUVECs (Figure 5E; Online Figure VIIIA) or HLMVECs (Online Figure VIIIB) ba- YAP Interaction With TRAF6 Inhibits NF-κB sally upregulated protein expression of ICAM-1 and E-selectin Activation and their expression further enhanced by lipopolysaccharide. In We next addressed the mechanism by which YAP modu- contrast, overexpression of YAP in HUVECs (Figure 5F; Online lated lipopolysaccharide-induced endothelial activation. In 50 Circulation Research June 22, 2018 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 5. YAP (Yes-associated protein) limits lipopolysaccharide (LPS)-induced endothelial inflammation. HUVECs (human umbilical vein endothelial cells) were transfected with control CRISPR-Cas9 plasmid (Con), YAP CRISPR-Cas9 plasmid (YAP-KO), empty vector (EV), or recombinant Flag-YAP cDNA. At 48 h posttransfection, HUVECs were stimulated with LPS (1 µg/mL) for the indicated times. A and B, Effects of YAP deletion on the production of IL (interleukin)-6 and IL-1β. A, Representative Western blots for YAP (left) and protein quantification by densitometry right( ). B, The concentrations of IL-6 and IL-1β in HUVECs supernatants by ELISA. n=3. C and D, Effects of YAP overexpression on the production of IL-6 and IL-1β. C, Representative Western blots for YAP; D, the concentrations of IL-6 and IL-1β in HUVECs supernatants by ELISA. n=3 to 6. E and F, Representative immunoblots showing ICAM (intercellular adhesion molecule)-1 and E-selectin expression after YAP knockout (E) and YAP overexpression (F) in HUVECs. G and H, Effects of YAP depletion on polymorphonuclear neutrophil (PMN) adhesion to HUVECs (G) and PMN transendothelial migration (H). I and J, Effects of YAP overexpression on PMN adhesion to HUVECs (I) and PMN transendothelial migration (J). n=3. *P<0.05, **P<0.01, and ***P<0.001, Mann-Whitney U test.

endothelial cells, exposure to lipopolysaccharide induces en- XA). Stimulation of endothelial cells with lipopolysaccharide dothelial activation through TLR4 and triggers proinflamma- for 30 minutes significantly reduced the association of YAP tory cytokine secretion and expression of adhesion molecules with TRAF6 (Figure 6A; Online Figure XA). Confocal imag- via NF-κB signaling.28 Here, we first assessed whether YAP ing also showed a distinct colocalization of YAP with TRAF6 interacted with components of the TLR4 signaling pathway. in untreated cells and a decreased colocalization of YAP and We observed no association of YAP with many key compo- TRAF6 after lipopolysaccharide stimulation (Figure 6B). nents of the pathway TLR4, MyD88, IRAK-1, and IκBα To analyze the possibility of direct interaction of YAP (Online Figure IXA through IXD) by immunoprecipitation, and TRAF6, we searched TRAF-binding domains in YAP but only association with TRAF6 (Figure 6A; Online Figure and identified that human YAP has a major TRAF-binding Lv et al YAP Limits Endothelial Activation 51 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 6. YAP (Yes-associated protein) inhibits NF-κB (nuclear factor-κB) pathway secondary to direct interaction with TRAF6 (tumor necrosis factor receptor-associated factor 6). A, YAP associates with TRAF6 in HUVECs (human umbilical vein endothelial cells) in the absence and presence of lipopolysaccharide (LPS; 1 µg/mL) stimulation. Immunoblot analysis was assessed followed by coimmunoprecipitation with normal immunoprecipitation (IgG) or anti-TRAF6 antibody. B, YAP colocalizes with TRAF6 in HUVECs. Left, Representative confocal images showing colocalization of YAP and TRAF6. Scale bars, 10 µm. Right, Quantification of YAP and TRAF6 colocalization. ***P<0.001, Mann-Whitney U test. C, Major TRAF-binding site with YAP. Top, modular structure of TRAF6-binding motifs (red) in the PDZ-BM of YAP; Bottom, conservation across species of TRAF6-binding sites with YAP from the upper panel. PDZ-BM indicates PDZ domain-binding motif; SH3-BM, Src Homology 3 binding motif; TAD, transcription activation domain; and TID, TEAD transcription factor interacting domain. D, Direct interaction of YAP and TRAF6. Human embryonic kidney 293T (HEK293T) cells were transfected to overexpress Myc-his-TRAF6 and Flag-tagged wild-type (WT) YAP (Flag-YAP-WT) or Flag-tagged mutant YAP (Flag- YAP-mutant) cDNA. E, Exogenous expression of YAP-WT suppresses NF-κB activation. NF-κB activity was measured by luciferase assay. HEK293T cells transfected with NF-κB luciferase reporter, TRAF6 cDNA, plus empty vector (EV) or with increasing concentration (wedge) of Flag-YAP-WT cDNA. Results are presented relative to results obtained without Flag-YAP-WT expression. n=3. ***P<0.001, by 1-way ANOVA. F, Exogenous expression of YAP-mutant has no effect on NF-κB activation. HEK293T cells were transfected with NF-κB luciferase reporter, TRAF6 cDNA, empty vector (EV), or with increasing concentration (wedge) of Flag-YAP mutant vector. Results are presented relative to results obtained without Flag-YAP-mutant expression. n=4. ***P<0.001, by 1-way ANOVA. G, Effects of YAP deletion on accumulation of p65/p50 in nucleus, NF-κB p65 phosphorylation (p-p65), and IκB (inhibitor protein κB)-α degradation in endothelial cells in vivo. Total cell lysate, cytoplasmic, and nuclear fractions were obtained from lung endothelial cells isolated from WT and YAP-conditional knockout (CKO) mice challenged with LPS (5 mg/kg. IP) for 4 h. n=3 to 4.

motif ((P/S/A/T)-X-(Q/E)-E, where P/S/A/T indicates pro- 4 amino acids (Ser-Leu-Gln-Glu) in TRAF-binding domain. line, serine, alanine, or threonine, X indicates any amino We expressed Flag-tagged WT YAP (Flag-YAP-WT) or mu- acid, and Q/E indicates glutamine or glutamic acid29; site: tant YAP (Flag-YAP-mutant) together with Myc-his-tagged 471-MPSLQEALSS) in its PDZ domain-binding motif. The TRAF6 (Myc-his-TRAF6) in human embryonic kidney 293T region in YAP with the TRAF motif is well conserved across (HEK293T) cells and assessed the association of YAP and various species (Figure 6C). To identify whether YAP specifi- TRAF6 by immunoprecipitation of Myc-his-TRAF6 followed cally binds TRAF6 via this TRAF-binding motif, we generat- by immunoblot analysis of Flag-YAP. Our results demonstrat- ed YAP mutants in which alanine was substituted for all of the ed that expression of Flag-YAP-WT but not Flag-YAP-mutant 52 Circulation Research June 22, 2018

directly associated with Myc-his-TRAF6 (Figure 6D; Online Online Figure XIF) ubiquitination of TRAF6 in the presence Figure XB). or absence of Flag-YAP plasmid, respectively. These findings To determine whether YAP regulated NF-κB activation together indicate that YAP activates K48-linked ubiquitination through its binding to TRAF6, we next expressed NF-κB lu- to induce TRAF6 degradation and inhibits K63-linked ubiqui- ciferase reporter together with TRAF6 and increasing concen- tination of TRAF6, and thereby suppresses NF-κB signaling. trations of YAP-WT or YAP-mutant cDNA in HEK293T cells. YAP-WT strongly inhibited NF-κB activation in a concen- TRAF6 Depletion in Endothelial Cells Rescues tration-dependent manner (Figure 6E), whereas YAP-mutant Augmented Inflammatory Phenotype of YAP-CKO Mice failed (Figure 6F). To further elucidate the effects of YAP on To address whether TRAF6 expression was required and suffi- NF-κB signaling in endothelial cells in vivo, we isolated lung cient for lipopolysaccharide-induced increase in lung inflam- endothelial cells from WT and YAP-CKO mice challenged mation in YAP-CKO mice, we depleted TRAF6 in YAP-CKO with lipopolysaccharide and observed that YAP deletion lung vascular endothelium using liposomes incorporating significantly increased NF-κB p65 phosphorylation (activa- TRAF6 siRNA. Injection of liposomes containing scrambled tion), IκBα degradation, and accumulation of p65 and p50 in siRNA was used as control. Western blot analysis of pulmo- nucleus both basally and after lipopolysaccharide challenge nary endothelial cells isolated from YAP-CKO mice trans- compared with control WT cells (Figure 6G; Online Figure fected with siRNAs confirmed efficiency of TRAF6 depletion XC through XE). (Figure 8A; Online Figure XIIA). TRAF6 protein expression YAP Modulates Ubiquitination and Degradation of in bone marrow cells was not altered by injection of liposome- TRAF6 in Endothelial Cells TRAF6 siRNA mixture (Online Figure XIIB). Depletion of We next determined the mechanism of YAP-mediated TRAF6 TRAF6 protein expression in YAP-CKO lung endothelial cells suppression of NF-κB activation in endothelial cells. We ob- completely rescued lipopolysaccharide-induced increase in served that CRISPR/Cas9 knockout of YAP in endothelial the numbers of total cells, PMNs (Figure 8B), macrophages, cells basally upregulated TRAF6 protein expression and also PMN/macrophage ratio (Figure 8C) in bronchoalveolar la- enhanced lipopolysaccharide-induced TRAF6 protein expres- vage fluid, and the release of IL-6 and TNF-α (Figure 8D), sion as compared with control endothelial cells stimulated suggesting that YAP suppressed endothelial activation and with lipopolysaccharide (Figure 7A; Online Figure XIA). We lung inflammation in vivo via TRAF6. also observed in HEK293T cells transiently transfected with Myc-his-TRAF6 and Flag-YAP that YAP inhibited TRAF6 Discussion Downloaded from http://ahajournals.org by on July 25, 2018 protein expression in a concentration-dependent manner YAP, a transcriptional coactivator, serves as a key regulator of (Figure 7B; Online Figure XIB). Based on these findings, tissue growth and organ size by promoting cell proliferation we addressed the possibility that YAP functions by promot- and suppressing apoptosis.31 In cardiac and vascular smooth ing TRAF6 degradation through ubiquitination. Proteins are muscle cells, YAP is an important regulator of cardiovascular targeted for degradation by the proteasome with covalent at- development.32 Here, we identified a crucial role of cytoplas- tachment of K48-linked polyubiquitin chains, whereas K63- mic YAP as a checkpoint modulator of endothelial activation linked ubiquitin activates the downstream TAK1 activation and vascular inflammation. Specific deletion of YAP in endo- important for NF-κB activation.13,30 We found that deletion thelial cells did not alter the size of organs; instead, it induced of YAP in HUVECs significantly decreased total and K48- vascular inflammation in the absence of inflammatory stimuli linked ubiquitination of TRAF6 but increased K63-linked and greatly augmented inflammation during endotoxemia and ubiquitination of TRAF6 (Figure 7C; Online Figure XIC). To polymicrobial sepsis. YAP negatively regulated inflammatory validate the role of YAP in the mechanism of ubiquitination signaling by directly interacting with the E3 ligase TRAF6 of TRAF6, we transfected HEK293T cells with Flag-YAP, which induced degradation of TRAF6 through K48-linked Myc-his-TRAF6, and HA (hemagglutinin)-tagged ubiqui- TRAF6 ubiquitination and simultaneously inhibited K63- tin (HA-Ub) and performed the in vivo ubiquitination assay. linked autoubiquitination of TRAF6. Expression of Flag-YAP increased total Myc-his-TRAF6 Our study clearly demonstrated the requisite role of YAP ubiquitination (Figure 7D; Online Figure XID). To identify in limiting endothelial activation, and thereby excessive vas- the type of TRAF6 ubiquitination regulated by YAP, we trans- cular inflammation, and organ dysfunction. This notion was fected HEK293T cells with Flag-YAP and Myc-his-TRAF6 strongly supported by studies in 2 murine experimental models together with plasmids encoding K48-linked (HA-K48) of sepsis in which YAP was conditionally deleted in endothe- (Figure 7E; Online Figure XIE) or K63-linked ubiquitin lial cells (YAP-CKO mice). Although mice with endothelial (HA-K63; Figure 7F; Online Figure XIF). Ubiquitin mutants cell–specific deficiency of YAP did not display any gross phe- containing single lysine-to-arginine mutation at positions notypic alteration up to adulthood, PMN infiltration into the 48 (HA-K48R) and 63 (HA-K63R) were individually trans- lung and enhanced expression of adhesion molecules suggest fected into HEK293T cells. Expression of YAP resulted in a mild lung endothelial inflammatory activation in YAP-CKO more K48-linked ubiquitination of TRAF6 (Figure 7E; Online mice. The inflammatory response to lipopolysaccharide and Figure XIE) but less K63-linked ubiquitination of TRAF6 polymicrobial sepsis in YAP-CKO mice was markedly en- (Figure 7F; Online Figure XIF). Coexpression of either HA- hanced as evident by higher tissue PMN infiltration, severe K48R or HA-K63R prevented these changes in K48-linked lung histopathologic changes, and edema formation. Intravital (Figure 7E; Online Figure XIE) and K63-linked (Figure 7F; microscopy studies in the cremaster muscle microcirculation Lv et al YAP Limits Endothelial Activation 53 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 7. YAP (Yes-associated protein) activates K48-linked ubiquitination to induce TRAF6 (tumor necrosis factor receptor- associated factor 6) degradation and inhibits K63-linked TRAF6 ubiquitination. A, Effects of YAP depletion on TRAF6 protein expression in HUVECs (human umbilical vein endothelial cells) stimulated with lipopolysaccharide (LPS). HUVECs were transfected with control CRISPR-Cas9 plasmid (Con) or YAP CRISPR-Cas9 plasmid (YAP-KO). At 48 h posttransfection, cells were incubated with LPS (1 µg/mL). B, YAP decreased TRAF6 protein expression. Human embryonic kidney 293T (HEK293T) cells transfected Myc-tagged TRAF6 cDNA with increasing concentration of Flag-YAP-wild-type (WT) cDNA. C, Effects of YAP depletion on ubiquitination. Endogenous TRAF6 autoubiquitination was performed by immunoblot analysis of total ubiquitin (Ub), K48-linked ubiquitin (K48-Ub), or K63-linked ubiquitin (K63-Ub) in HUVECs transfected with control CRISPR-Cas9 plasmid or YAP CRISPR-Cas9 plasmid, assessed after immunoprecipitation with anti-TRAF6 antibody. D, Effects of exogenous YAP expression on total ubiquitination of exogenous TRAF6 expression. HEK293T cells were transfected to express Myc-his-TRAF6 and HA-Ub (hemagglutinin-tagged total ubiquitin) in the presence (+) or absence (−) Flag-YAP vector. TRAF6 ubiquitination was assessed by immunoprecipitation with anti-Myc antibody. E and F, Immunoblot analysis of K48-linked ubiquitin (E) and K63-linked ubiquitin (F) of TRAF6 in HEK293T cells transfected to express Myc-his-TRAF6, K48-linked ubiquitin (HA-K48-Ub), K48-linked ubiquitin mutant (HA-K48R-Ub), K63-linked ubiquitin (HA-K63-Ub), K63-linked ubiquitin mutant (HA- K63R-Ub), with or without Flag-YAP expression. TRAF6 ubiquitination was assessed by immunoprecipitation with anti-Myc antibody.

also showed marked PMN adhesion to cremasteric vessel en- cells, as opposed to hematopoietic cells, was responsible for dothelial cells of YAP-CKO mice both in the absence of in- endothelial cell activation and inflammation seen in the YAP- flammatory stimulus and after lipopolysaccharide exposure. CKO mice. Because Tie2 promoter/enhancer-driven Cre expression was TLR4/NF-κB signaling is essential for endothelial activa- used to deplete the yap gene in endothelial cells in our study, a tion and the consequent inflammation during sepsis.5,28 Our possible concern is that augmented endothelial activation and findings indicated that YAP prevented NF-κB activation by di- vascular inflammation may also result from deletion of the rect interaction with TRAF6 in endothelial cells. We observed yap gene in hematopoietic cells. To circumvent this concern, that coexpression of YAP-WT cDNA and TRAF6 cDNA we transplanted either WT or YAP-CKO bone marrow cells inhibited NF-κB activation. Furthermore, amino acid sub- into lethally irradiated YAP-CKO or WT mice and observed stitution mutations within the TRAF-binding site (Ser-Leu- similar levels of endothelial activation and inflammatory re- Gln-Glu) of YAP abolished the interaction of YAP and TRAF6. sponses in these mice. Thus, deletion of YAP in endothelial Interestingly, coexpression of YAP-mutant and TRAF6 cDNA 54 Circulation Research June 22, 2018 Downloaded from http://ahajournals.org by on July 25, 2018

Figure 8. Deletion of TRAF6 (tumor necrosis factor receptor [TNF]-associated factor 6) in vascular endothelial cells prevents lipopolysaccharide (LPS)-induced lung inflammation in YAP (Yes-associated protein)-conditional knockout (CKO) mice. Mice were transfected with scrambled (Sc) or TRAF6 siRNA (si)-liposome complex through tail vein. At 48 h posttransfection, mice were challenged with LPS for 24 h. A, Representative Western blot analysis showing TRAF6 knockdown in primary lung endothelial cells isolated from wild-type (WT) and YAP-CKO mice. B and C, Total cells (B, left), polymorphonuclear neutrophils (PMNs; B, right), macrophages (Mϕ; C, left), and PMN/Mϕ ratio (C, right) in bronchoalveolar lavage (BAL) fluid were enumerated at 24 h after LPS challenge. n=4.D , Levels of IL (interleukin)-6 (left) and TNF-α (right) in serum measured by ELISA. n=4 to 6. **P<0.01 and ***P<0.001, 1-way ANOVA. E, Model of YAP in regulating TLR (Toll-like receptor)-4/NF-κB (nuclear factor-κB) signaling via modulation of TRAF6.

failed to affect NF-κB activation. Thus, it seems that specific K63-linked nondegradative chains at multiple locations. In the binding of YAP to TRAF6 leading to inhibiting NF-κB activa- latter case, the N-terminal RING domain of TRAF6 functions tion plays a crucial role in the immunomodulatory function as an E3 ubiquitin ligase that facilitates its own site-specific of YAP in endothelial cells. TRAF6 on the basis of its ligase ubiquitination through the generation of K63-linked ubiquitin function is a key downstream regulator of multiple immuno- chains.36 K63-linked autoubiquitination of TRAF6 is essential regulatory receptors, including members of the TNF recep- for the formation and activation of TAK1 complex and NF- tor superfamily, TLR family, transforming growth factor-β κB signaling.38 In this study, deletion of YAP in endothelial receptors, and T cell receptor.33 CD40–TRAF6 interactions cells upregulated TRAF6 expression, prevented K48 ubiqui- are known to play an essential role in the development of ath- tination of TRAF6, and significantly enhanced K63 ubiqui- erosclerosis and restenosis.34,35 The ubiquitination of TRAF6 tination of TRAF6. These results suggest that YAP prevented is a crucial mechanism for NF-κB activation.36,37 TRAF6 the activation of NF-κB by both enhancing the degradation of can be ubiquitinated with both K48-linked degradative and TRAF6 via K48 ubiquitination and blocking TAK1 activation Lv et al YAP Limits Endothelial Activation 55

via suppression of K63-linked ubiquitination. Taken together, (Figure 8E). In this context, upregulation and stabilization of YAP expression controls endothelial activation and thereby YAP expression in endothelial cells may be a novel pharmaco- prevents hyperinflammatory response to endotoxin through logical approach for preventing sepsis-induced tissue inflam- restraining TRAF6-mediated NF-κB activation. matory injury and organ failure. In the present study, YAP expression in human and mouse endothelial cells was upregulated by lipopolysaccharide, but Acknowledgments then the expression recovered toward baseline after the in- We thank Yongkang Zhang (Department of Anesthesiology, flammatory response exaggerated. In tumor-initiating stem- University of Illinois at Chicago) for technical assistance. like cells39 and adipose tissue-derived mesenchymal stem cells,40 TLR ligands also induced the expression of YAP1 Sources of Funding and TAZ. These findings suggest a negative feedback loop This study was supported by National Institutes of Health Grant between YAP and TLRs, whereby TLR signaling results in R01HL104092 (G. Hu), PPG1–P01 HL060678 (A.B. Malik), PPG2– increased expression of YAP, which limits activation of NF- P01 HL077806 (A.B. Malik), and HL130028 (J. Cho). κB to proceed and holds the inflammatory response in check. Thus, YAP signaling may act as a molecular brake to restrict Disclosures endothelial activation and prevent excessive inflammation None. and organ failure. YAP is known to play a key role in cell proliferation, References survival, differentiation, tissue regeneration, and organ size 1. Lee WL, Slutsky AS. Sepsis and endothelial permeability. N Engl J Med. 2010;363:689–691. doi: 10.1056/NEJMcibr1007320. 41 determination. Global YAP knockout mice died at embry- 2. Andonegui G, Goyert SM, Kubes P. Lipopolysaccharide-induced leuko- onic day 8.5.42 Kidney-specific CKO of YAP in mice resulted cyte-endothelial cell interactions: a role for CD14 versus Toll-like receptor in defective kidneys with abnormal glomeruli, ducts with- 4 within microvessels. J Immunol. 2002;169:2111–2119. out lumen.43 Ectopic expression of YAP in the liver induced 3. Goldenberg NM, Steinberg BE, Slutsky AS, Lee WL. Broken barriers: a new take on sepsis pathogenesis. Sci Transl Med. 2011;3:88ps25. doi: liver enlargement and the liver reverted to a normal size on 10.1126/scitranslmed.3002011. switching-off YAP overexpression.44,45 Liver enlargement was 4. Deutschman CS, Tracey KJ. Sepsis: current dogma and new perspectives. also observed with knockout of Mst1/2 (molecules upstream Immunity. 2014;40:463–475. doi: 10.1016/j.immuni.2014.04.001. 5. Lawrence T. The nuclear factor NF-kappaB pathway in inflam- of YAP). However, there was no discernable change in the mation. Cold Spring Harb Perspect Biol. 2009;1:a001651. doi: size of the kidney, intestines, and lung in Mst1/2 knockout 10.1101/cshperspect.a001651. 46

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