Vascular Endothelial Growth Factor(VEGF) Induced Airway Epithilium Barrier Disruption By PI3K And JNK Pathway

BACKGROUND AND AIM:Asthma,as a most common chronic respiratory disease,has caused lots of trouble for human being. Statistic suggests that there are300million people,10percent are Chinese,are suffering from asthma.The number is increasing.Thanks to the standardized therapeutic schedule given by GINA,lots of symptoms about asthmahas been controled to certain level. However, the therapy can’t inhibit the pote ntial developping trend of the disease. So the pathogenesis of asthma is still indistinct, which need more researchs to study the pathogenesis of asthma and find new therapeutic targets for it.AECs,as the first gate to against outside stimuli, plays an essential role in keeping the balance and stability of airway internal environment. AECs plays a central role in the onset and development of asthma. Although asthma is a TH-2type-dominant chronic respiratory diseases,the TH-2immune cells mediated-inflammation donot completely explain the hypersensitive of patients on the allergen and the pathophysiologic process of asthma.In recent years,increasing researches shows that airway structural cells feel the harmful irritation,further plays an important role in the onset and development of inflammation. Researchs showed that there exits airway epithelia barrier dysfunction among asthma patients, and the distribution or expression of junction proteins was abnormal compared with that of non-asthma people.After epithelium barrier was damaged by allergens such as HDM, pollen or fungus,epithelial cells secreted various inflammatory cytokines and allergens contacted with subepithelial dendritic cells.Finally immune cells like DC cell were activated and caspase inflammatory reaction was promoted.Meanwhile,airway epithelial cells produced numerous cytokines to facilitate mucus secretion,basement membrane thickness or smooth muscle cells proliferation,and induced airway remodeling.Further a more powerful and long-lasting inflammatory reaction was caused,ultimately the asthma became irreversibly.Family of vascular endothelial growth factor(VEGF) majorly includes VEGF-A、 B、C、D、E、F,most of studies are about VEGF-A,gene of human VEGF-A consists of eight exon and seven intron,mRNA of VEGF-A is cut for five isomerides(VEGF121、VEGF145、VEGF165、VEGF189. VEGF206) in differ modes.In most cases,expression of VEGF165dominates others,and it mainly plays biological effect of VEGF.Therefore VEGF in domestic and foreign researches is VEGF165.As a multifunctional factor,VEGF is potent stimulator of vascular angiogenesis,permeability and remodeling.And it also plays important roles in wound healing and tissue cytoprotection.A number of domestic and foreign researches demonstrated that compared with the normal, the levels of VEGF were increased in biologic fluids from patients with asthma. Laterly,the animal studies show that the symptom and airway inflammation of TDI-asthma mices were obviously relieved.In other study, VEGF transgenic mices arised asthma-like symptom such as angiogenesis,mucus metaplasia,airway hyperresponsiveness. Further studies showed that VEGF activated subepithelial DCs leading to heightened adaptive Th2immunity,it also caused mucus metaplasia,airway remodeling and airway hyperresponsiveness(AHR),and so on. These discoveries all confirmed that VEGF contributed to the pathogenesis of asthma, and the effect was mediated by its regulating airway inflammation.In addition, there are researches showed that the common allergen such as CrAg、pollen etc, induced VEGF release in BAECs and alterd bronchial airway permeability.Our research team have conformed that TDI(or HDM) increased the permeability of HBE cell monolayers(or airway primary epithelium),partly through a VEGF-mediated pathway in vitro.However,it doesn’t show whether triggers the airway inflammation or not through directly destructing barrier function of airway epithelium.The therapeutic role of glucocorticoids(GCs) in human allergic asthma is very immportant,which is mainly bacause of its powerful anti-inflammatory characteristic.The most inmmportant mechanism of the anti-inflammatory function of GCs is their ability to inhibit the infiltration of inflammaory cells into affected tissues,human airway epithelia cells also express functional GC receptors. A recent report demonstrated GCs enhance airway barrier intergrity through re-distribution of TJ proteins(ZO-1and OCLN) and that EGFR signaling could be involved in this process. However, the protective effects of Glucocorticoids on the distribution and expression of E-cadherin under allergic conditions have not been clarified.Accordingly, the purpose of the present study was further to assess the influence of Recombinant Human VEGF165on the barrier function of respiratory tract epithelium.And additional aim of this study was that discuss the intervention of dexamethasone (DEX) on the effect of VEGF165and define the mechanism involved.Section one:Effect of VEGF on the barrier function of respiratory tract epithelium(human bronchial epithelium16HBE-14o and human lung adenocarcinoma cell A549)METHODS1.Methyltetrazolium (MTT) assay was used to assess the16HBE(or A549) cell viability under different concentrations of VEGF, groups as follows:The normal control group、VEGF10ng/ml group、VEGF50ng/ml group、VEGF100ng/ml group、VEGF150ng/ml group (n=8), stimulated cell for24hours, then MTT colorimetric assay detected cell viability. 2.Transepithelial Electrical Resistance(TEER) and fluorescein isothiocyanate(FITC) assessed paracullular permeability of16HBE(or A549) under differ time of VEGF50ng/ml, groups as follows:The normal control group、VEGF30min group、VEGF2h group、VEGF6h group、VEGF12h group、VEGF24h group (n=6).After stimulated,the permeability of cell monolayar was measuremented by TEER and FITC.3.Western blot was used to detect the expression of adherent junction proteins(E-Cadherin、beta-catenin) and tight junction proteins(ZO-1、Occludin、 claudin-2) in16HBE(or A549) under differ time of VEGF50ng/ml, groups as follows:The normal control group、VEGF30min group、VEGF2h group、VEGF6h group、VEGF12h group、VEGF24h group (n=6).After stimulated, the cells were collected.4.Immunofluorescence staining was used to detect the distribution of E-Cadherin and beta-catenin in16HBE(or A549) under differ time of VEGF50ng/ml, groups as follows:The normal control group、VEGF6h group (n=4).After stimulated, the cells were collected.Statistical analysisSPSS13.0analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Tamhanes was used for Multiple comparisons among the groups. Significance was accepted when P <0.05.Results:l.The effect of different concentrations of VEGF on cell viability of16HBE and A549:VEGF with the concentrations of10ng/ml,50ng/ml,100ng/ml, for16HBE (or A549) did not significantly affect cell viability(P=0.23);but VEGF150ng/ml significantly inhibited cell viability(P<0.05). 2.Changes in paracullular permeability of16HBE(or A549) by50ng/ml VEGF under differ time.TEER and FITC showed:In comparison with normal control group,50ng/ml under differ time VEGF significantly increased permeability of16HBE(P<0.05),and the most significant was50ng/ml VEGF exposure for6h(P<0.01); In comparison with normal control group,50ng/ml under differ time VEGF significantly increased permeability of A549(P<0.05),and the effect appeared a time-dependent.3.The expression of junction proteins in16HBE(or A549) by50ng/ml VEGF under differ time. Western blot showed:expression of adherent junction proteins and tight junction proteins in16HBE(or A549) were not signifantly altered after treatment with VEGF50ng/ml.4.Changes in the distribution of E-cadherin and beta-catenin induced by50ng/ml VEGF. Confocal microscopy showed that strong E-cadherin(or beta-catenin) staining in16HBE(or A549) located at the plasma membrane level at sites of cell-cell contact in untreated cells.50ng/ml VEGF exposure for6h induced disassembly of E-cadherin(or beta-catenin).Conclusion1.VEGF significantly destructed the barrier function of16HBE and A549.2.VEGF caused redistribution of E-cadherin(or beta-catenin) in I6HBE(or A549).But it did not alter the expression of adherent junction proteins and tight junction proteins in16HBE(or A549).Section two:VEGF destructed the barrier function of16HBE by PI3K and JNK signaling pathwayMETHODS1. Western blot detected the phosphorylated level of signaling molecule by VEGF50ng/ml under differ time in16HBE. groups as follows:The normal control group, VEGF5min group, VEGF10min group, VEGF15min group, VEGF30min group, VEGF1h group, VEGF2h group, VEGF6h group(n=6). After stimulated, the cells were collected.2. Western blot detected the inhibitory effect of LY294002(or SP600125or U0126) on the phosphorylated level of signaling molecule when16HBE were treated with50ng/ml VEGF. groups as follows:The normal control group, VEGF30min group, LY294002(or SP600125or U0126) pretreated group, LY294002(or SP600125or U0126) treated group(n=6). After stimulated, the cells were collected.3.Transepithelial Electrical Resistance(TEER) and fluorescein isothiocyanate(FITC) assessed the inhibitory effect of LY294002(or SP600125or U0126) on the destruction of50ng/ml VEGF in16HBE. groups as follows:The normal control group、VEGF6h group、LY294002(or SP600125or U0126) pretreated group, LY294002(or SP600125or U0126) treated group (n=6).After stimulated,the permeability of cell monolayar was measuremented by TEER and FITC.4.1mmunofluorescence staining detected the inhibitory effect of LY294002(or SP600125or U0126) on the redistribution of E-Cadherin and beta-catenin induced by VEGF in16HBE, groups as follows:The normal control group、VEGF6h group、 LY294002(or SP600125or U0126) pretreated group, LY294002(or SP600125or U0126) treated group (n=4).After stimulated, the cells were collected.Statistical analysisSPSS13.0analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Tamhanes was used for Multiple comparisons among the groups. Significance was accepted when P <0.05.Results:1. signaling pathway(PI3K、JNK and Erk)were activated by VEGF under differ time in16HBE. Western blot showed that VEGF exposure increased threonine/tyrosine phosphorylation of Akt、JNK and Erk after15min and30min stimulation (P<0.05).2. Pretreatment with LY294002(or SP600125or U0126) could inhibit PI3K(or JNK or Erk)activation by VEGF. Western blot showed that the PI3K(or JNK or Erk) activation of was inhibited by LY294002(or SP600125or U0126).3.Effect of LY294002(or SP600125or U0126) on the VEGF-destructed barrier function of16HBE. TEER and FITC showed:In comparison with normal control group, LY294002(or SP600125) could partly recover the destructed barrier function(P<0.05);but U0126could not do that.4. Effect of LY294002(or SP600125or U0126) on the VEGF-induced E-cadherin (or beta-catenin) aberrant of16HBE. In comparison with VEGF treated group, pretreatment with LY294002(or SP600125) for1h, the immunoreactivity of E-cadherin(or beta-catenin) was almost recovered to control levels, but the immunoreactivity of proteins did not be recovered with U0126treated group.ConclusionVEGF can activate the signaling pathway including PI3K、JNK and Erk in16HBE.The inhibition of PI3K(or JNK) can attenuate the VEGF-induced destruction of epithelium barrier and redistribution of E-cadherin(or beta-catenin).The result hint VEGF redistribute of E-cadherin(or beta-catenin),and then destruct of epithelium barrier partly by PI3K and JNK.Section three:DEX attenuated the VEGF-induced destruction of epithelium barrier by PI3K in16HBEMETHODS1. Transepithelial Electrical Resistance(TEER) and fluorescein isothiocyanate(FITC) assessed Protection role of DEX on the VEGF-induced destruction of epithelium barrier in16HBE. groups as follows:The normal control group、VEGF6h group、10-4M DEX pretreated group,10-4M DEX treated group (n=5).After stimulated,the permeability of cell monolayer was measuremented by TEER and FITC.2.Immunofluorescence staining detected the inhibitory effect of DEX on the VEGF-induced redistribution of E-Cadherin and beta-catenin in16HBE, groups as follows:The normal control group、VEGF6h group、10-4M DEX pretreated group,10-4M DEX treated group (n=4).After stimulated, the cells were collected.3. Western blot detected the inhibitory effect of DEX on the phosphorylated level of signaling molecule when16HBE were treated with50ng/ml VEGF. groups as follows:The normal control group, VEGF30min group,10-4M DEX pretreated group,10-4M DEX treated group(n=7). After stimulated, the cells were collected.Statistical analysisSPSS13.0analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Tamhanes was used for Multiple comparisons among the groups. Significance was accepted when P <0.05.Results:1.Effect of DEX on the VEGF-destructed barrier function of16HBE. TEER and FITC showed:In comparison with normal control group, DEX could partly recover the destructed barrier function(P<0.05); but barrier function of DEX treated group did not change compared with control group.2. Effect of DEX on the VEGF-induced E-cadherin (or beta-catenin) aberrance in16HBE. In comparison with VEGF treated group, pretreatment with DEX for1h, the immunoreactivity of E-cadherin(or beta-catenin) was almost recovered to control levels, protein distribution of DEX treated group did not change compared with control group.3. Pretreatment with DEX could inhibit PI3K activation by VEGF.Western blot showed that the VEGF-incuced threonine/tyrosine phosphorylation of Akt was inhibited by DEX, but the VEGF-incuced phosphorylation of JNK did not be inhibited by DEX.Conclusion1.DEX can attenuate the VEGF-induced destruction of epithelium barrier redistribution.2.DEX can attenuate the VEGF-induced of E-cadherin(or beta-catenin) aberrance in16HBE.3. DEX donot protect the epithelium barrier in a short period of time.