| Sepsis is a common complication after severe trauma,surgery,shock and infection,and is one of the leading causes of intensive care unit admissions,with high mortality and morbidity.The diagnostic criteria for sepsis include multiple indicators and parameters such as infection,organ dysfunction,hemodynamics,and inflammatory response.However,due to its excessively complicated standards,it is still difficult to diagnose patients in clinic.Currently,according to the evaluation of sepsi-related organ failure assessment(SOFA),the index includes the number of respiration rate,the status of the patient’s consciousness and systolic pressure.Although there are some treatment options for sepsis patients,most of them are mainly through anti-infection,organ protection and maintenance and fluid resuscitation.Therefore,to strengthen the pathogenesis of sepsis and to find new drug targets,the establishment of an effective clinical treatment program is an important objective of the current study on sepsis.In septic patients,widespread vascular leakage and inflammation secondary to endothelial activation and dysfunction is the primary mechanism of multi-organ failure.Endothelial cells are the key sentinel cells detecting microbial infection through the activation of pattern recognition receptors to mount an inflammatory response.Endothelial activation during sepsis leads to the production of multiple pro-inflammatory cytokines and up-regulation of adhesion molecules E/P-selectins and intercellular adhesion molecule-1(ICAM-1)essential for polymorphonuclear neutrophil(PMN)rolling,adhesion and transendothelial PMN migration.Sustained activation of endothelium results in massive infiltration of PMNs,hyperinflammation,tissue damage,and organ dysfunction.Therefore,maintenance or reconstitution of vascular endothelial homeostasis after a major systemic injury is a crucial determinant of outcome in sepsis.However,the underlying mechanisms limiting endothelial activation during sepsis remain to be identified.Activation of nuclear factor-B(NF-B)is critical for regulating the expression of inflammatory genes and adhesion molecules.The NF-B activation cascade is initiated by endotoxin lipopolysaccharide(LPS)binding to Toll-like receptor(TLR)4,which activates adaptor protein myeloid differentiation factor(My D88)-dependent and-independent signaling pathways.My D88 associates with IL-1 receptor-associated kinase(IRAK),which recruits E3 ligase TNF-associated factor 6(TRAF6),which in turn interacts with TGF-β-activated kinase 1(TAK1)complex.TAK1 thereby activates I B kinase(IKK),leading to the phosphorylation and degradation of I B,and consequent release of NF-B and its nuclear translocation.TRAF6 mediates the signaling from TNF receptor,IL-1 family and most TLR(TLR1-9)and functions as a node controlling NF-B activation.The ubiquitination of TRAF6 is a key regulatory event controlling TRAF6 expression and TAK1 activation.TRAF6 can be ubiquitinated through the E3 ubiquitin ligase in its RING domain.K48-linked ubiquitination of TRAF6 leads to its degradation via the 26 S proteasome whereas K63-linked polyubiquitination is essential for activation of TAK1 kinase and subsequent NF-B signaling.The Hippo signaling pathway consists of a serine kinase cascade with associated regulatory and scaffolding proteins that transcriptionally regulate the expression of genes controlling growth.The Ser/Thr kinase Hippo in Drosophila(Mst1 and Mst2 in mammals)and Sav1 form a complex that phosphorylates and activates LATS1/2 kinases,which phosphorylate Yes-associated protein(YAP)and transcriptional coactivator with PDZ-binding motif(TAZ),the two downstream effectors of the Hippo pathway,causing them to be retained in the cytoplasm.Dephosphorylation of YAP and TAZ induces their nuclear translocation and activates expression of genes that promote cell proliferation and inhibit apoptosis.YAP is a potent oncogene first identified by its ability to associate with Yes and Src tyrosine kinases.As a transcriptional coactivator,YAP activates the expression of target genes important for cell survival,chemotaxis,differentiation,and proliferation.Activation of YAP or loss of upstream negative regulators leads to striking overgrowth and tumor phenotypes.While Hippo/YAP signaling is important in regulating tissue growth,little is known about its role in signaling inflammation.The previous study showed that Gram-positive bacteria activated Hippo signaling in fat bodies through Toll-dependent pathway in Drosophila.It is unknown if Hippo/YAP signaling also regulates inflammatory response induced by stimuli such as sepsis.Part Ⅰ The correlation between YAP and inflammationOBJECTIVE: To investigate the role of YAP in the endothelial activation-induced by stimuli.METHOD: The changes in the expression of YAP in protein or m RNA levels in endothelial cells were detected by Western blotting and real-time quantitative PCR after the stimulation of inflammatory mediators LPS,TNF-alpha or H2O2.8 to 12 weeks of wild type mice(wild type,WT,YAPf/f)were administrated 5 mg/kg of LPS(i.p.)to establish sepsis model.After 4 hours,sacrifice the mice and isolate lung endothelial and non-endothelial cells to determin the changes of YAP expression when compared to the control group.Using the CRISPR/Cas9 system to knock out YAP gene in the endothelial cells,then the cells were stimulated by LPS,and the secretion levels of IL-6 and IL-β were detected by using ELISA kit.The effect of YAP deficiency in endothelial cells on inflammatory response induced by LPS was observed and compared with the control group.At the same time,we transfected the Flag-YAP plasmid into the endothelial cells and observed the effect of YAP overexpression in endothelial cells on the release level of inflammatory cytokines IL-6 and IL-1β.The human neutrophil in the blood were also isolated and performed the neutrophil-endothelium adhesion and migration assay.To observe the effect of neutrophil adhesion or transmigration when YAP deletion or overexpression in the endothelial in the basal level or after LPS stilulation.RESULTS:(1)YAP protein expression increased following LPS stimulation in a time-dependent manner,peaked at 4 h,and then gradually decreased to basal level at 24 h.(2)A similar result of YAP m RNA expression was seen in HUVECs after LPS stimulation by real-time quantitative reverse transcription PCR(q RT-PCR).(3)The expression of YAP protein was elevated at 4 h following stimulation with other stimuli TNF-α or H2O2.(4)WT mice were challenged by i.p.administration with LPS to induce acute lung injury.YAP protein expression was also increased both in lung endothelial cells and lung non-endothelial cells,but it has a more significant increase in the lung endothelial cells after LPS challenge.(5)YAP deletion by CRISPR/Cas 9 significantly increased the production of IL-6 and IL-1β over time.Conversely,overexpression of YAP markedly attenuated the production of IL-6 and IL-1β.(6)Depletion of YAP in HUVECs basally upregulated protein expression of ICAM-1 and E-selectin and their expression further enhanced by LPS stimulation.Ablation of YAP in endothelial cells increased PMN adhesion and transendothelial PMN migration even in the absence of LPS stimulation.Depletion of YAP markedly augmented LPS-induced PMN adhesion and transendothelial PMN migration as compared to control cells.(7)In contrast,overexpression of YAP inhibited LPS-induced protein expression of ICAM-1 and E-selectin.And inhibit PMN adhesion and transendothelial migration compared to control cells.CONCLUTION:(1)LPS induces YAP expression in human endothelial cells and mouse lung ECs and non-ECs.(2)YAP inhibits endothelial cells cytokine release induced by LPS.(3)YAP negatively regulates PMN adhesion and transendothelial migration after endothelial antivation.PartⅡ The effects of YAP deletion in endothelial cells on mice sepsis modelOBJECTIVE: To investigate the functional significance of YAP in LPS-indeced mice sepsis model.METHOD: The Cre-Loxp system was used to establish YAP specific knockout in mice endothelial cells(YAP-CKO mice)and the DNA from mice tail was extracted to detect the expression of YAPf/f and Cre genes.Pulmonary endothelial and non-endothelial cells were isolated from mice and YAP expression in lung ECs or lung non-ECs were detected by using Western Blotting and real-time quantitative PCR,to make sure the deletion of YAP was only happened in mouse endothelial cells.The body weight of WT(YAPf/f)and YAP-CKO mice were monitored at 4,8 and 40 weeks,and the fertility and survival of mice were also recorded.The morphological,size,visceral coefficient and of the multi-organ tissues and the cell tytes and contents in BAL were determined between 8 and 12 weeks old in WT and YAP-CKO mice.WT and YAP-CKO mice were injected with 5 mg/kg LPS to prepare sepsis model.BAL from WT and YAP-CKO mice were collected and the cell type and numbers were counted at 6 and 24 h after LPS administration.At the same time,the serum was extracted from the venous blood of the mice,and the levels of IL-6,TNF-α and IL-1β in serum of WT and YAP-CKO mice were detected by ELISA kit.After 24 hours of LPS stimulation,the lung tissue was flushed to remove red blood cells in the blood vessels and H&E staining was performed.The changes in lung tissue were observed under the microscope and the lung injury score was observed.After 24 hours of LPS stimulation of mice,Evan’s Blue solution was injected intravenously.After 40 minutes,the lung tissue was taken out and the blood was removed,the content of Evans Blue in the lung tissue was extracted and calculated.The survival rate of WT and YAP-CKO mice was observed and compared after intraperitoneal injection of lethal dose of LPS(20 mg/kg).After the mice challenged by CLP model,blood pressure and ejection fraction and fractional shortening were measured to evaluate heart failure.To generate chimeric mice,WT and YAP-CKO mice were performed bone marrow transplantation(bone marrow transplantation,BMT)experiment.Ly5.1 mice bone marrow cells(bone marrow,BM)were transplanted into WT and YAP-CKO mice which were lethally irradiated.5 five weeks after bone marrow transplantation,the peripheral blood of mice(BMT-WT and BMT-YAP-CKO)were collected to detect bone marrow transplantation efficiency using FACS.6 weeks after bone marrow transplantation,BMT-WT and BMT-YAP-CKO mice were injected 5 mg/kg LPS and after stimulation for 6 and 24 h,the number of macrophages and neutrophils in BAL,the level of serum inflammatory cytokines(IL-6,TNF-α and IL-1β)in mice,and also the content of Evan’s blue in the lung were detected.WT and YAP-CKO(6-8 weeks old)mice were challenged with LPS(5 mg/kg,i.p.)or saline.At 24 h following LPS administration,the mouse was anesthetized and the cremaster muscle was exposed.PMNs were visualized by infusion of Alexa Fluor 647-conjugated anti-Ly-6G through a jugular cannula.PMNs rolling and adhesion were observed and compared between WT and YAP-CKO mice.In addition,the expression of ICAM-1 and E-selectin were detected by immunofluorescence assay in the cremaster muscle vessel endothelial cells.RESULTS:(1)A greater than 95% reduction in YAP m RNA and protein levels in isolated mouse lung endothelial cells but not in non-endothelial cells(epithelial cells and fibroblasts).(2)Immunoblotting assessment of YAP protein expression also exhibited 60% reduction in whole lung tissues.(3)The proportion of born wild-type(YAP+/+),heterozygous(Tie2-Cre;YAP floxf/+)and homozygous(Tie2-Cre;YAPf/f)mice complied with the Mendel’s laws and their appearance was undistinguishable from the littermates.Similarly,the male to female ratio was also consistent with the law.YAP-CKO mice also showed normal fertility and the oldest mice survived for more than 2 years.(4)No difference was observed with regard to body weight or organ size in YAP-CKO mice.The ratios of organ to body weight were also not different in YAP-CKO mice except for the slightly higher ratio in spleen/body weight as compared with wild-type mice.(5)Importantly,YAP-CKO mice displayed mild lung inflammation endothelial activation as evidenced by increased PMN counts in bronchoalveolar lavage(BAL)fluid and increased expression of E-selectin and ICAM-1 in endothelial cells as compared to WT mice.(6)In YAP-CKO mice challenged with LPS,the number of PMNs in BAL fluid(7)as well as expressions of E-selectin and ICAM-1 in pulmonary endothelial cells dramatically increased compared to their WT counterparts.(8)Lung tissue from WT counterparts following LPS challenge had severe histological changes,including alveolar congestion,exudates,and infiltration of inflammatory cells,while these alterations were markedly exacerbated in LPS-challenged YAP-CKO mice,as evident by the significantly higher lung injury score.(9)A striking aggravation of LPS-induced pulmonary vascular hyperpermeability was observed in YAP-CKO mice by measuring measured lung albumin-bound Evans blue dye extravasation,an indicator of vascular leakage after LPS stimulation for 24 h.(10)The production of the proinflammatory cytokines IL-6,TNF-α,and IL-1β were greatly elevated in YAP-CKO mice following LPS challenge as compared to WT littermates.(11)Importantly,YAP-CKO mice had a significantly higher mortality rate after challenge with lethal dose of LPS than WT counterparts.(12)After CLP challenge,YAP-CKO mice exhibited more severe heart failure than WT mice.(13)Bone marrow cells isolated from WT(Ly5.1)mice were transplanted into lethally irradiated WT(Ly5.2)and YAP-CKO(Ly5.2)mice.FACS analysis demonstrated 90% reconstitution of WT and YAP-CKO bone marrow at 5 weeks after transplantation.(14)Transplantation of WT bone marrow into YAP-CKO mice failed to prevent the increase of lung inflammation after LPS challenge,the number of PMNs in the BAL,the cytokine IL-6 and TNF-α release level and pulmonary vascular permeability in BMT-YAP-CKO mice were still much higher than those in BMT-WT mice.(15)YAP-CKO mice exhibited markedly increased number of adherent PMNs compared with WT mice both basally and after LPS challenge.PMN rolling was reduced in YAP-CKO mice compared to that in WT mice both basally and after LPS challenge.(16)Expressions of E-selectin and ICAM-1 in endothelial cells of YAP-CKO mice were significantly increased under basal conditions compared with control mice,and expressions of both adhesion proteins were further increased following LPS challenge.CONCLUTION:(1)Specific deletion of YAP in endothelial cells did not alter the size of organs;instead it induced vascular inflammation in the absence of inflammatory stimuli.(2)Enhanced inflammatory response and heart failure to endotoxin in YAP-CKO mice.(3)YAP suppresses PMN recruitment in microvessels.Part Ⅲ The impact role and mechanism of targeting YAP in inflammationOBJECTIVE: To address the mechanism by which YAP modulated LPS-induced endothelial activation.METHOD: To assess whether YAP interacted with components of the TLR4 signaling pathway by immunoprecipitation.The co-localiztion of endogenous YAP and TRAF6 in endothelial cells were detected by cell immunofluorescence.The possible binding sites between YAP and TRAF6 were predicted by software,and then generated Flag-YAP-WT,Flag-YAP-Mutant(binding site mutation)and Myc-his-TRAF6 plasmid,co-transfected them into HEK-293 T cells,the interaction of exogenous Flag-YAP-WT and Myc-his-TRAF6 or Flag-YAP-Mutant and Myc-his-TRAF6 were detected by using immunoprecipitation.To determine whether YAP regulated NF-B activation through its binding to TRAF6,NF-B luciferase reporter together with TRAF6 and increasing concentrations of YAP-WT or YAP-mutant c DNA were transfected in HEK293 T cells.Western Blotting was used to study the role of YAP in regulating NF-B activation in vivo and the expression of TRAF6,both in endogenous and exogenous level.The effects of endogenous YAP deletion on total ubiquitination,K48 and K63 linked ubiquitination of endogenous TRAF6 were detected by immunoprecipitation.Conversely,the effects of exogenous YAP overexpression on the total ubiquitination,K48 and K63 linked ubiquitination of exogenous TRAF6 in HEK-293 cells were also detected.TRAF6 small interfering RNA(si TRAF6)was transfected into mice by liposomes.The knockdown efficiency of si TRAF6 in mouse lung endothelial cells was detected by Western Blotting.At 48 h posttransfection,the mice were stimulated by LPS,number of PMNs in BAL and inflammatory cytokines release were determed beteen WT and YAP-CKO mice.RESULTS:(1)YAP has the interaction with TRAF6.(2)Confocal imaging also showed a distinct co-localization of YAP with TRAF6.(3)YAP directly associated with TRAF6 via its PDZ-BD(site: 471-MPSLQEALSS).(4)The expression of Flag-YAP-WT but not Flag-YAP-mutant directly associated with Myc-his-TRAF6.(5)YAP-WT strongly inhibited NF-B activation in a concentration-dependent manner whereas YAP-mutant failed.(6)YAP deficiency in endothelial cells increased NF-B activation both basally and after LPS challenge.(7)CRISPR/Cas9 knockout of YAP in endothelial cells basally upregulated TRAF6 protein expression and also enhanced LPS-induced TRAF6 protein expression as compared to control endothelial cells stimulated with LPS.(8)In HEK293 T cells transiently transfected with Myc-his-TRAF6 and Flag-YAP that YAP inhibited TRAF6 protein expression in a concentration-dependent manner.(9)Deletion of YAP in HUVECs significantly decreased total and K48-linked ubiquitination of TRAF6 but increased K63-linked ubiquitination of TRAF6.(10)The overexpression of Flag-YAP increased total Myc-his-TRAF6 ubiquitination.Expression of YAP resulted in more K48-linked ubiquitination of TRAF6 but less K63-linked ubiquitination of TRAF6.Co-expression of either HA-K48 R or HA-K63 R prevented these changes in K48-linked and K63-linked ubiquitination of TRAF6 in the presence or absence of Flag-YAP plasmid,respectively.(11)Western blot analysis of pulmonary endothelial cells isolated from YAP-CKO mice transfected with si RNAs confirmed efficiency of TRAF6 depletion was about 90%.(12)Depletion of TRAF6 protein expression in YAP-CKO lung endothelial cells completely rescued LPS-induced increase in total cell count and PMN in BAL fluid,and the release of IL-6 and TNF-α.CONCLUTION:(1)YAP interaction with TRAF6 inhibits NF-кB activation.25(2)YAP functions by modulating the ubiquitination and degradation of TRAF6.(3)Knockdown of TRAF6 in endothelial cells rescues augmented inflammatory phenotype of YAP-CKO mice.The major contributions of the present study:1.YAP was involved in the process of endothelial activation.The expression of YAP protein was elevated following stimulation with different stimuli.Deletion of YAP protein in HUVECs with CRISPR/Cas9 increased cytokines release by endothelial cells and augmented PMN adhesion and PMN transendothelial migration in the absence or presence LPS.These findings provide the academic foundation for targeting YAP in regulating endothelial activation.2.Clearly demonstrate the essential role of YAP in limiting endothelial activation and excessive vascular inflammation.This notion was strongly supported by studies in a mouse model in which YAP was conditionally deleted only in endothelial cells(YAP-CKO mice).Although mice with endothelial cell-specific deficiency of YAP did not display any gross phenotypic alteration up to adulthood,PMN infiltration into the lung and enhanced expression of adhesion molecules suggest a mild,lung endothelial inflammatory activation in YAP-CKO mice.Robust lung inflammation in YAP-CKO mice following LPS challenge was evident by higher tissue PMN infiltration,severe lung histopathological changes,and edema formation.Intravital microscopy studies in the cremaster muscle microcirculation also showed marked PMN adhesion to cremasteric vessel endothelial cells of YAP-CKO mice both in the absence of inflammatory stimulus and following LPS exposure.Promisingly,YAP is likely to reveal a novel therapeutic strategy by inhibiting endothelial activation and vascular inflammation.TRAF6 in endothelial cells.Co-expression of YAP-WT c DNA and TRAF6 c DNA inhibited NF-B activation in a concentration-dependent manner.Deletion of YAP in endothelial cells up-regulated TRAF6 expression,prevented K48 ubiquitination of TRAF6,and significantly enhanced K63 ubiquitination of TRAF6.These findings provide a new target and strategy for the treatment of sepsis patients.3.Clarify YAP prevented NF-κB activation by direct interaction with TRAF6 in endothelial cells.Co-expression of YAP-WT c DNA and TRAF6 c DNA inhibited NF-B activation in a concentration-dependent manner.Deletion of YAP in endothelial cells up-regulated TRAF6 expression,prevented K48 ubiquitination of TRAF6,and significantly enhanced K63 ubiquitination of TRAF6.These findings provide a new target and strategy for the treatment of sepsis patients. |
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