VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

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  • VEGF Increases ParacellularPermeability in Brain Endothelial Cells

    via Upregulation of EphA2ZIWEI MIAO, YANBIN DONG, WENGANG FANG, DESHU SHANG, DONGXIN LIU,

    KE ZHANG, BO LI, AND YU-HUA CHEN*Department of Developmental Cell Biology, Key Laboratory of Cell Biology, Ministry of

    Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, ChinaMedical University, Heping District, Shenyang, China

    ABSTRACTNeurological disorders are associated with an increase in the perme-

    ability of human brain microvascular endothelial cells (HBMEC). Our pre-vious findings have indicated that EphA2 could increase the permeabilityof HBMEC. Recent evidence has linked EphA2 and vascular endothelialgrowth factor (VEGF) to abnormalities in the vascular response. However,it is unclear whether EphA2 is involved in the VEGF-induced changes inthe permeability of HBMEC. Here, changes in permeability were deter-mined by measuring transendothelial electrical resistance (TEER) and theflux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abol-ished the VEGF-induced reduction in TEER and increase in flux of fluores-cent dextran. Moreover, VEGF-induced redistribution of ZO-1 and therecruitment of detergent-soluble occludin and claudin-5 were also pre-vented. Further results showed that VEGF increased EphA2 expression ina time- and dose-dependent manner, which was inhibited by a neutralizingantibody against VEGFR2 or SU1498. VEGF-induced EphA2 expressionwas suppressed in the brain endothelium following treatments with thePI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Dp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Ourdata suggest that VEGF upregulates the expression of EphA2 in HBMECthrough binding to VEGFR2 and subsequently activating the intracellularPI3K/Akt and ERK1/2 signaling pathways, which contribute to an increasein paracellular permeability. These data reveal a novel role for VEGF as aregulator of EphA2 expression in the brain endothelial cells and provideinsights into the molecular mechanisms of VEGF-mediated changes in par-acellular permeability. Anat Rec, 297:964972, 2014. VC 2014 WileyPeriodicals, Inc.

    Key words: EphA2; VEGF; human brain microvascular endo-thelial cell; PI3K/Akt; ERK1/2

    Abnormalities in the bloodbrain barrier (BBB) per-meability are associated with serious pathological eventsin a variety of neurological disorders, including stroke,bacterial meningitis, Alzheimers disease, multiple scle-rosis, and brain tumors (Zlokovic, 2008). It is well knownthat vascular endothelial growth factor (VEGF), alsoknown as the vascular permeability factor, plays a pri-mary role in disrupting the BBB, which consists ofmicrovascular endothelial cells lining the brain micro-

    Grant sponsor: National Natural Science Foundation of China;Grant numbers: 30970120, 31171291, 81171537, 81101225.

    *Correspondence to: Yu-Hua Chen, Department of Develop-mental Cell Biology, Key Lab of Cell Biology, Ministry of PublicHealth of China, China Medical University, Shenyang 110001,China. Fax: 186-24-23260246. E-mail: yhchen@mail.cmu.edu.cn

    Received 16 October 2013; Accepted 13 December 2013.

    DOI 10.1002/ar.22878Published online 24 January 2014 in Wiley Online Library(wileyonlinelibrary.com).

    THE ANATOMICAL RECORD 297:964972 (2014)

    VVC 2014 WILEY PERIODICALS, INC.

  • vessels (Ma et al., 2012). VEGF has been demonstratedto increase paracellular permeability by downregulatingthe expression of tight junction (TJ) proteins, such asclaudin-5, occludin, and zonula occludens-1 (ZO-1), inthe brain endothelial cells (Argaw et al., 2009; Morin-Brureau et al., 2011). Conversely, VEGF has been shownto have a negative influence on BBB permeability afterintracerebral hemorrhage, which is closely related to anincrease in AQP4 expression induced by VEGF (Chuet al., 2013). Thus, it is possible that VEGF may triggerthe expression of different molecules in the brain endo-thelial cells to regulate paracellular permeability.

    Recent evidence has linked VEGF and EphA2 to neo-vascularization and vasopermeability abnormalities.EphA2, a member of the largest known family of proteintyrosine kinase receptors, has been implicated in theendothelial cells migration and vascular assembly(Brantley-Sieders et al., 2004). In bovine retinal endo-thelial cells, EphA2 receptor stimulation by its ligandephrinA1 has been shown to inhibit VEGF-induced phos-phorylation of VEGF receptor 2 (VEGFR2), leading to areduction in the VEGF-induced angiogenic cell activity(Ojima et al., 2006). The alterations in paracellular per-meability induced by EphA2-ephrinA1 have been shownto be similar to the changes induced by VEGF in thelung vascular endothelial cells (Larson et al. 2008). Ourprevious findings indicated that EphA2 was involved inmodulating paracellular permeability in human brainmicrovascular endothelial cells (HBMEC) (Zhou et al.,2011). However, it is still unclear whether EphA2 areinvolved in VEGF-induced paracellular permeability inthe brain endothelial cells.

    In the present study, we provide evidence to demon-strate that VEGF upregulates the expression of EphA2,leading to an increase in the permeability of HBMEC.Furthermore, VEGFR2, the principal VEGF receptor, andits associated downstream signaling cascades, includingphosphoinositide 3-kinase (PI3K)/Akt and ERK1/2, con-tribute to VEGF-induced EphA2 expression.

    MATERIALS AND METHODS

    Chemicals and Antibodies

    Recombinant human VEGF and neutralizing antibodiesagainst VEGFR1 and VEGFR2 were purchased from R&DSystems (Minneapolis, MN). The inhibitors, PD98059,SP600125, LY294002, wortmannin, Y27632, Go6976,Go6983, PP1, PP2, PP3, and SU1498 were obtained fromCalbiochem (San Diego, CA). Akt1/2 kinase inhibitor andFITC-dextran were obtained from Sigma-Aldrich (St.Louis, MO). Antibodies against VEGFR2, occludin, andFITC-conjugated ZO-1 were purchased from Invitrogen(Grand Island, NY). The anti-claudin-5 antibody was pur-chased from Abcam (Cambridge, MA). The anti-VEGFR1antibody was obtained from Santa Cruz Biotechnology(Santa Cruz, CA). The anti-EphA2 antibody was pur-chased from Millipore (Billerica, MA). Antibodies againstERK, phospho-ERK, Akt, and phospho-Akt were obtainedfrom Cell Signaling Technology (Danvers, MA).

    Cell Culture

    The HBMEC were a generous gift from Dr K.S. Kim(Johns Hopkins University School of Medicine). HBMECwere cultured in RPMI 1640 medium supplemented with

    10% fetal bovine serum (Life Technologies, Grand Island,NY), 10% Nu-serum (BD Biosciences, Franklin Lakes,NJ), 2 mM glutamine, 1 mM sodium pyruvate, 13 non-essential amino acids and 13 MEM vitamin. A stableHBMEC cell line transfected with a dominant-negativePI3K (p110gD948981) was constructed as described pre-viously (Li et al., 2009).

    RNA Interference

    The small interfering RNA (siRNA) sequences targetingEphA2, VEGFR2, and nonsilencing control were synthe-sized and purified by GenePharma (Shanghai, China).The siRNA sequences are listed in Table 1. The siRNAsequences for VEGFR1 and ERK1/2 were used asdescribed previously (Li et al., 2013). Transient transfec-tion procedures were performed using Lipofectamine2000reagent according to the manufacturers instructions(Invitrogen Corporation, Carlsbad, CA). The transfectedcells were harvested for experiments 4872 hr aftertransfection.

    Real-Time Reverse Transcription (RT)-PCR

    Total RNA from cells was extracted with TRIzol Rea-gent (Sigma-Aldrich, St. Louis, MO) and then wasreverse-transcribed using M-MLV reverse transcriptase(Promega, Madison, WI). Real-time PCR was performedon the ABI 7500 real-time PCR system (Applied Biosys-tems, Foster City, CA) using a SYBR green premix ExTaq kit (Takara Biotechnology, Tokyo, Japan), accordingto the manufacturers instructions. The relative quantifi-cation was calculated by the 2-DDCT method and wasnormalized against the internal control (GAPDH). Theprimer sequences for human EphA2 were 50-CCAACCAGGCTGTGTTGAAGT-30 and 50-CCCTTGTACACCTCCCCAAA-30. The primer sequences for human GAPDHwere 50-GAAGGTGAAGGTCGGAGTC-30 and 50-GAAGATGGTGATGGGATTTC-30.

    Cell Fractionation and Western Blot

    Cell fractionation experiments were performed asdescribed previously (Li et al., 2006). The protein con-centrations for each sample were quantified by using aBCA protein assay reagent kit (Pierce, Rockford, IL).Following protein quantification, the fractions were ana-lyzed by Western blot. Equal amounts of samples wereseparated by SDS-PAGE and transferred to PVDF mem-branes (Millipore, Billerica, MA). The membrane wasthen blocked and incubated with primary antibodiesovernight at 4C. The blots were incubated with anHRP-conjugated secondary antibody (Santa Cruz Bio-technology, Santa Cruz, CA) and visualized by SuperSig-nal West Pico Chemiluminescent Substrate (Pierce,Rockford, IL) using a LAS 3000 mini (Fuji Film, Tokyo,Japan). Quantification of band density was performedusing Image J software.

    TABLE 1. The siRNA sequences for RNA interference

    Target gene SiRNA sequence

    EphA2 50-CCAUCAAGAUGCAGCAGUAUA-30

    VEGFR2 50-CCGGGAUAUUUAUAAAGAUTT-30

    Nonsilencing control 50-UUCUCCGAACGUGUCACGUTT-30

    VEGF INDUCES EphA2 EXPRESSION FOR PERMEABILITY 965

  • Transendothelial Electrical ResistanceMeasurement

    The HBMEC transfected with EphA2 siRNA or nonsilenc-ing control siRNA were seeded onto 0.4-lm pore size Trans-wells (Corning, Lowell, MA) in 24-well plates. Experimentswere conducted when transendothelial electrical resistance(TEER) was >200 Ohmcm2. The cells were stimulated with100 ng/mLVEGF for 24 hr and TEER was measured using aMillicell-ERS (Milipore, Billerica, MA) according to the man-ufacturers protocol. The final TEER values were calculatedas Ohmcm2 by

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