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Wntb-catenin signaling induces proliferation survival and Interleukin-8 in human

Wntb-catenin signaling induces proliferation survival and Interleukin-8 in human
Wntb-catenin signaling induces proliferation survival and Interleukin-8 in human

Wnt/b -catenin signaling induces proliferation,survival and Interleukin-8in human endothelial cells

T.Ne ′stor H.Masckaucha ′n 1,Carrie J.Shawber 1,Yasuhiro Funahashi 1,Chi-Ming Li 2&Jan Kitajewski 1,2

1

Department of OB/GYN,Columbia University Medical Center,New York,USA;2Department of Pathology,Columbia University Medical Center,New York,USA

Received 11November 2004;accepted in revised form 23March 2005

Key words:b -catenin,endothelial cell,Interleukin-8,proliferation,Wnt

Abstract

Wnts are secreted signaling proteins able to control diverse biological processes such as cell di?erentiation and proliferation.Many Wnts act through a canonical,b -catenin signaling pathway.Here,we report that Wnt receptors and transcriptional e?ectors are expressed in primary human endothelial cells and that Wnt/b -catenin signaling promotes angiogenesis.Human umbilical vein and microvascular endothelial cells express Wnt receptors,Frizzled-4,-5,-6,and b -catenin-associated transcription factors,Tcf-1,-3,-4and Lef-1.In endothelial cells,ectopic expression of Wnt-1stabilized cytosolic b -catenin,demonstrating activation of the Wnt/b -catenin canonical sig-naling pathway.Expression of Wnt-1or a stabilized and active form of b -catenin,b -cateninS37A,promoted endothelial cell proliferation.Proliferation induced by Wnt/b -catenin signaling was optimal in the presence of bFGF.b -cateninS37A expression in endothelial cells promoted survival after growth factor https://www.wendangku.net/doc/e410733697.html,ing matrigel assays,Wnt-1or b -cateninS37A expression promoted the formation of capillary-like networks.To help de?ne the e?ectors of Wnt angiogenic function,microarray analysis was used to compare endothelial cells expressing Wnt-1to control cells.Interleukin-8,a known angiogenic factor,was identi?ed as a transcriptional target of Wnt/b -catenin signaling in endothelial cells.Expression of either Wnt-1or b -cateninS37A induced Interleukin-8transcripts and secreted protein.We thus conclude that Wnt/b -catenin signaling promotes angiogenesis possibly via the induction of known angiogenic regulators such as Interleukin-8.

Abbreviations:AoSMC --aortic smooth muscle cells;bFGF --basic ?broblast growth factor;EGF --epidermal growth factor;FEVR --familial exudative vitreoretinopathy;HA --hemagglutinin;HMVEC --human microvascular endothelial cells;HUAEC --human umbilical artery endothelial cells;HUVEC --human umbilical vein endothelial cells;IGF-1--insulin-like growth factor-1;IL-8--Interleukin-8;MOI --multiplicity of infection;sFRP --soluble frizzled-related protein;VEGF --vascular endothelial growth factor

Introduction

Wnt ligands are multifunctional secreted proteins able to regulate proliferation,apoptosis,branching mor-phogenesis,inductive processes,and cell polarity [1].Wnts also play important roles in maintaining stem cells in a progenitor/proliferative state [2,3].All of these processes are required for the development and maintenance of the vasculature.The roles of angio-genic factors,such as VEGF and Angiopoietins [4--9]

are well established in angiogenesis but several other growth factor families likely assist in orchestration of the angiogenic process.

Wnts,through their receptors,Frizzleds,can acti-vate multiple downstream signaling cascades.The canonical or Wnt/b -catenin signaling pathway utilizes b -catenin as an effector to transmit a receptor-medi-ated signal from the cytosol to the nucleus where it interacts with and activates the Tcf/Lef transcription factors [10].The non-canonical pathways function via either Ca ++or Rho/JNK signaling pathways [11].Studies have implicated the Wnt/b -catenin pathway as a regulator of endothelial cell growth.Expression of Wnts known to activate the Wnt/b -catenin

Correspondence to:Jan Kitajewski,Department of Pathology,Columbia University Medical Center,630West 168th Street --Room 16-417,New York,NY 10032,USA.E-mail:jkk9@https://www.wendangku.net/doc/e410733697.html,.

Angiogenesis (2005)8:43--51óSpringer 2005

DOI 10.1007/s10456-005-5612-9

pathway can promote endothelial cell proliferation [12].Wnt/b-catenin signaling activity has been detected in the vasculature in vivo,as measured using a b-catenin/Tcf responsive reporter mouse(BAT-gal) [13].A role for Wnt/b-catenin signaling in the vascula-ture is further supported by the identi?cation of Wnt target genes that encode angiogenic regulators.In one case,the Wnt/b-catenin pathway may indirectly regu-late angiogenesis via transcriptional activation of the VEGF-A gene in non-endothelial cells or tumors[14]. Seven b-catenin/Tcf binding sites are encoded in the upstream sequence of the VEGF-A promoter[15].In tumors where b-catenin/Tcf transcriptional activity was elevated,VEGF-A was overexpressed.

To elucidate the role of Wnt signaling in the endothelium,we identi?ed the endogenous Frizzleds and Tcf/Lefs expressed in primary human endothelial https://www.wendangku.net/doc/e410733697.html,ing cultured endothelial cells,we demonstrate that Wnt/b-catenin signaling promoted proliferation, survival,and capillary-like networks.We also screened endothelial cells for genes regulated by Wnt/b-catenin signaling using microarrays and identi?ed Interleukin-8, an angiogenic factor,as a target gene.

Materials and methods

Cells and reagents

HUVEC were isolated from human umbilical vein as previously described[16]and grown in EGM2 (BioWhittaker Inc.,USA)with VEGF,bFGF,EGF, IGF-1and2%FBS on dishes coated with Type I rat tail collagen(Upstate Biotechnology,USA).HMVEC and HUAEC and their media were obtained from BioWhittaker.Adenoviruses encoding for Wnts or mutant b-catenin were prepared as described[17]. Anti-b-catenin antibody was purchased from Upstate Biotechnology and pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs were generously pro-vided by Hans Clevers(University Hospital,Utrecht, The Netherlands).

Gene transfer into HUVEC

To infect cells,4·105passage3--5cells were trypsi-nized and resuspended in300l l full culture medium. Adenovirus stock was added at30MOI unless indi-cated otherwise for each experiment,and cells were incubated at37°C for1h with gentle shaking every 10min.Then,cells were seeded onto type I collagen-coated plates and harvested24--48h later depending on the assay.For retroviral gene transfer,the retrovi-ral vector pHyTCX[18]was used. 5.0·106GP293 packaging cells(Clontech)were seeded,transfected with10l g pHyTC-genes and pVSVG and retroviral-containing supernatants were collected48h later. Retroviruses were added to passage4HUVEC and 48h later cells were selected for4--5days with hygro-mycinB at300l g/ml and then maintained with hygromycinB at100l g/ml.An expression vector encoding the HA-tag(pHyTC-HA)was used as nega-tive controls.Protein expression in HUVEC was eval-uated by immunoblotting using antibodies to the HA epitope.Cells were lysed in TENT buffer(20mM Tris--HCl pH=8.0,2mM EDTA,150mM NaCl, 1%Triton X-100added with1tablet of Complete Mini protease inhibitors(Roche Diagnostics, Germany)per10ml of buffer),lysates were fraction-ated using SDS-PAGE,transferred to nitrocellulose membrane,and incubated with antibodies.Proteins were visualized by Enhanced Chemiluminescence (Amersham)in conjunction with goat antimouse IgG-HRP.Extracts from LacZ expressing cells were used as controls.

Cytosolic b-catenin evaluation

HUVECs were infected with adenoviruses encoding for Wnts or control gene(LacZ)as described above and seeded on10cm diameter dishes.Cytosols were isolated48hrs later by?rst washing the cells with ice cold PBS three times and then adding750ul of physi-ological buffer(10mM TRIS--HCl pH=7.4, 140mM NaCl,5mM EDTA,2mM DTT,0.5mM PMSF,0.2l g/ml aprotinin,0.1mg/ml leupeptin)to each dish.Cells were scraped with a rubber policeman and lysed in a chilled Dounce homogeneizer.Lysates were cleared from unbroken cells and nuclei by centri-fugation at500g for10min and the supernatants were then centrifuged at approx.100,000·g for 90min.Cytosolic fractions were frozen at)20°C until the protein content was evaluated and the immunoblot analysis was performed using a b-catenin antibody (Transduction Laboratories,USA).

Reporter assays

Cells were seeded on collagen-coated12-well plates (88,000cells per well).The next day,cells were transfected with Tcf/Lef transcriptional activation re-porter construct TopFlash containing Tcf responsive elements or FopFlash with mutated elements(control) and a b-galactosidase-encoding construct.Transfec-tions were performed using0.11l g of reporter plas-mid,0.1l g of b-galactosidase plasmid and0.11l g of inducer plasmid using 3.6l l of Effectene(Qiagen, USA)and 2.9l l of Effectene Enhancer per well according to manufacturer’s instructions.Cells were incubated at37°C for5h with transfection cocktail and then they were incubated overnight with fresh cul-ture media.Cells were harvested the next day and?re-?y luciferase activities were evaluated using the Enhanced Luciferase Assay Kit(BD Pharmingen, USA).

44T.N.H.Masckaucha′n et al.

Proliferation and survival assays in vitro

Low-passage primary cells were split and suspended in full endothelial culture media.Cells were infected by incubation with Wnt-adenoviruses as described above, using adenovirus encoding for the LacZ gene as a neg-ative control.Next,cells were seeded at10,000cells/ well on24-well plates that were previously collagen-coated containing full endothelial culture media.Cells were allowed to seed overnight and media was then re-placed with basal endothelial cell media EBM-2added with bFGF.Cell numbers at48h post-incubation with basal media were assessed with the Cell Counting Kit-8 assay(Dojindo,USA).All assays were performed in quadruplicate.

Endothelial network formation assays

Retroviral selected HUVEC expressing Wnt-1-HA,b-cateninS37A-HA or control gene(HA tag)were ana-lyzed by Western blot to verify the overexpression of the desired protein.Matrigels were prepared using24-well plates with0.3ml pure Growth Factor-reduced Matrigel(BD Biosciences)per well and incubating plates at37°C for1h.Retroviral selected HUVEC grown on collagen-coated plates were seeded at 100,000cells per well on top of Matrigel in the pres-ence of0.8ml of full EC culture medium.For testing the effect of IL-8on cells,recombinant human IL-8 (R&D Systems Inc.,USA)was used at12ng/ml per well with wild type HUVEC,as previously described [19].All assays were performed in duplicates.Pictures were taken with4·magni?cation after18--19h of incubation at37°C.

RT-PCR and DNA array analysis

Low passage human primary endothelial cells were cultured in EGM-2BulletKit(BioWhittaker,Inc.).To-tal RNA was isolated as described[20]and RT-PCR was performed using Omniscript Reverse Transcriptase (Qiagen,Inc.)and Platinum Taq DNA Polymerase (Invitrogen,Inc.).For Frizzled receptors,RT-PCR was performed with a Multigene-12RT-PCR Pro?ling Kit(SuperArray Bioscience Corp.)according to manu-facturer’s instructions for30cycles and results were then con?rmed with a separate set of primers for Friz-zled-4,-5,and-6:5¢CAACTTTCACACCGCTCA TCCAG3¢,upper primer,5¢AGCCAGCATCATAG CCACACTTG3¢,lower primer for Frizzled-4;5¢GAGGTCCTCTGCATGGATTACAAC3¢,upper primer,5¢CTGAAGGACGGCTGGTAGCAG3¢, lower primer for Frizzled-5;5¢CTACAGCAAATC ATGGCACTTC3¢,upper primer,5¢GAGAGTCT GGAG ATGGATGCTG3¢,lower primer for Friz-zled-6(yielding products of390,253and195bp, respectively).Primers for transcription factors Tcf-1, Lef-1,Tcf-3and Tcf-4were used as reported before [21]for30cycles,yielding products of240,421,178 and494bp,respectively.DNA array analysis was per-formed with cDNA obtained from HUVEC adeno-infected to express either Wnt-1or LacZ gene (control)at48h post-infection.Hu95Av2GeneChips, which query about10,000genes,were purchased from Affymetrix(Santa Clara,CA).Microarray and probes were prepared and used as described before[22].To analyze data,for each sample,the signals for each gene(probe set)were normalized to the values of the entire microarray and statistical analyses were done using GeneSpring TM Software(Silicon Genetics).For the IL-8RT-PCR,adeno-infected HUVEC to express either Wnt-1,Wnt-5A,b-cateninS37A or LacZ(con-trol)were used to isolate RNA at48hours postinfec-tion.Primers for IL-8were:5¢CATGACTTCC AAGCTGGCCG3¢upper primer,5¢AATTTTTTTA TGAATTCTCAGCCCTC3¢lower primer(30cycles). PCR products were separated on non-denaturing bis-acrylamide gels and stained with either ethidium bro-mide or Syber Green(Molecular Probes).To correct for sample variations in RT-PCR ef?ciency,b-actin expression was used to normalize the RNA samples using the following primers:5¢CGAGGCCCAGAG CAAGAGAG3¢upper primer;5¢CTCGTAGATG GGCACAGTGTG3¢lower primer.

ELISA assay for IL-8

An ELISA assay was used to evaluate the levels of se-creted IL-8from conditioned media of HUVEC and was performed as per manufacturers instructions using the human IL-8Quantikine ELISA(R&D Systems, USA).Samples consisted of collected cell culture media of adeno-infected endothelial cells diluted1:10 in1·calibrator diluent.All samples were run in tripli-cates and compared to a standard curve performed according to the manufacturers’instructions.

Results

Frizzled-4,-5,-6and Tcf/Lef genes are expressed

in human primary endothelial cells

The Frizzled gene family consists of ten genes encod-ing Wnt receptors.We sought to de?ne the expression of Frizzleds in cultured human endothelial cells as a means to understand the role of Wnt/Frizzled signal-ing in endothelium.We?rst evaluated for presence of transcripts for all Frizzleds and the Wnt antagonist, sFRP1in human primary endothelial and vascular smooth muscle cells.RNA was puri?ed from primary HUVEC,HMVEC,HUAEC and human AoSMC grown in culture,and RT-PCR was performed using speci?c primers for all human Frizzled receptors.This screening detected different levels of transcripts for Frizzled-4,-5and-6in the primary endothelial cells

Wnt signaling in primary endothelial cells45

examined (HUVEC,HMVEC,HUAEC)and smooth muscle cells (AoSMC)(Figure 1a).No message was detected for all other Frizzleds in any of these primary cells (data not shown).Transcript levels of Frizzled-4and -6were high in all primary cells analyzed.Friz-zled-5showed high levels only in HMVEC and AoSMC.In accordance to what has been reported [23],we observed expression of sFRP1in both HUVEC and AoSMC (data not shown).We were also able to analyze the levels of transcripts for Tcf/Lef transcription factors in these cells,?nding that all of them are expressed at high levels,except Lef-1which showed low levels of expression in HMVEC and was not detectable in HUVEC (Figure 1b).

Wnt-1and not Wnt-5a expression activates Wnt/b -catenin signaling in endothelial cells

In order to activate the Wnt/Frizzled signaling pathway in cultured endothelial cells,we used adenoviruses encoding for Wnt-1and Wnt-5a fused in frame with a single HA-epitope at the 3’end.We also used an adeno-virus encoding HA-tagged mutant form of b -catenin,b -cateninS37A,which is more resistant to proteolytic turnover and thus functions as a gain-of-function for Wnt/b -catenin signaling [17].Adenovirus infection of

endothelial cells led to expression of detectable Wnt-1,Wnt-5A,and b -cateninS37A proteins in lysates derived from infected cells compared to lysates infected with an adenovirus expressing LacZ (Figure 2a).We asked whether expression of Wnt-1or Wnt-5a in primary endothelial cells led to elevation of cytosolic b -catenin,a measure of Wnt/b -catenin signal activation [24].Cyto-sols from the Ad-infected primary endothelial cells were isolated and separated from cell membranes,which con-tain cadherin-bound b -catenin.Immunoblot analysis of cytosolic b -catenin demonstrated that Wnt-1

expression

Figure 1.Frizzled-4,-5,-6and Tcf/Lef genes are expressed in human

primary endothelial cells.RNA was puri?ed from human primary endo-thelial cells and aortic smooth muscle cells and RT-PCR analysis was done to estimate the transcript levels of (a)Frizzled receptors and (b)Tcf/Lef transcription

factors.

Figure 2.Activation of Wnt/b -catenin signaling in human endothelial cells.(a)Western blot analysis of lysates from adenovirus infected HUVEC expressing HA-tagged Wnt-1,Wnt-5A or b -cateninS37A,de-tected with an anti-HA antibody.(b)Western blot analysis of cytosolic fractions from human primary endothelial cells infected with adenovirus-es expressing Wnt-1,Wnt-5A or LacZ (control)detected with antibody anti-b -catenin.(c)Reporter luciferase assay using lysates from HMVEC transfected with plasmids to express either Wnt-1,Wnt-5A,b -cate-ninS37A or control (human Fc)and co-transfected with either reporter construct pTop?ash (black bars)or negative control construct pFop?ash (white bars)and a lacZ construct to allow for normalization of data.At 24h post-transfection,lysates were prepared and luciferase activity was measured.All samples were run in triplicates.

46T.N.H.Masckaucha ′n et al.

elevated b-catenin levels in HUVEC,HUAEC and HMVEC,relative to controls(Figure2b).Expression of Wnt-5a,that does not signal via the canonical pathway, did not signi?cantly alter cytosolic b-catenin levels relative to control.Another measure of Wnt/b-catenin signal activation is the increase in Tcf-mediated tran-scriptional activity.To measure b-catenin/Tcf mediated transcription,we performed luciferase-based reporter assays in conjunction with the TopFlash reporter plas-mid which contains four Tcf/Lef elements and responds to Wnt/b-catenin signal activation[17].HMVEC were transfected with plasmids encoding for either Wnt1, Wnt5a,b-catenin-S37A or LacZ(control)and co-trans-fected with pTopFlash or the control pFopFlash.We observed a signi?cant transcriptional activation in response to b-catenin-S37A expression(Figure2c).The transcriptional response of TopFlash after Wnt-1 expression was modest and Wnt-5a expression did not elicit any change in reporter activity(Figure2c).

Wnt-1/b-catenin signaling promotes endothelial

cell proliferation and survival

To analyze the effect of Wnt/b-catenin signaling on endothelial cell proliferation,adenovirus-infected HUVEC expressing either LacZ,VEGF-A,Wnt-1or b-cateninS37A were seeded on collagen-coated plates at low con?uency,and cell growth was evaluated at different time points.In this assay,the Ad-VEGF-A infected HUVEC functioned as a positive control for proliferation.When cultured in minimal endothelial media containing bFGF,both Wnt-1and b-cate-ninS37A were able to induce cell proliferation(62% and91.6%higher than the Ad-LacZ infected HU-VEC,respectively)(Figure3a).Ad-b-cateninS37A was more effective than Ad-Wnt-1in promoting prolifera-tion but less effective than Ad-VEGF.Greater prolif-eration of HUVEC was achieved by increasing the MOI of Ad-b-cateninS37A infection indicating a dose-response effect of b-cateninS37A(Figure3b).

Effects on endothelial cell survival were deter-mined by seeding Ad-infected HUVEC and plating at con?uent densities,so proliferation was inhibited by cell--cell contact,and determining if the cells can survive in the absence of VEGF.Extended cell sur-vival was observed in cells expressing b-cateninS37A

(60.4%higher)with a minimal increase(7.6%)in the Wnt-1expressing cells relative to the LacZ expressing HUVEC(Figure3c).

Wnt-1promotes network cord formation of HUVEC During angiogenesis,endothelial cells migrate together and form a network of tubes.We assessed the ability of Wnt-1to promote endothelial cell network like-structures in matrigel.This assay evaluates the ability of HUVEC to form a network on top of a gel con-taining extracellular matrix components overlayed with endothelial culture medium.To conduct such assays, we generated HUVEC cell lines which ectopically express Wnt-1HA,b-cateninS37A or the HA epitope alone(control)via retroviral infection and selection (Figure4).Retroviral selection allows for the ectopic expression of Wnt-1in primary endothelial and we found retroviral selected endothelial cells to be more optimal for this experiments than the adenoviral in-fected cells.After18--19h of culture on matrigel, Wnt-1and b-cateninS37A expressing HUVEC formed stable networks of cords compared to control HA expressing HUVEC.Experiments,described below,

Wnt signaling in primary endothelial cells47

identi?ed Interleukin-8(IL-8)as a gene regulated by the Wnt/b -catenin pathway in endothelial cells.Thus,we also demonstrated that IL-8was able to induce a sta-ble network in the Matrigel assay,similar to Wnt-1or b -cateninS37A,at the concentration tested compared to control cells.

Wnt-1and b -cateninS37A expression induce Interleukin-8expression in endothelial cells

To probe the mechanisms by which Wnt/b -catenin sig-naling promoted endothelial proliferation and network formation,we screened for genes regulated by Wnt signaling in endothelial cells.We conducted Af?matrix microarray analysis for genes induced by Wnt-1in HUVEC after infection with either Wnt-1or LacZ expressing adenoviruses.Results were compared using a value of P <0.05to select genes of interest (Ta-ble 1).The ectopic expression of Wnt-1by the Ad-Wnt-1was readily detected by the analysis.Among the

genes of interest whose expression was induced by Wnt-1expression was Interleukin-8(IL-8).IL-8is a known target of Wnt/b -catenin signaling in primary hepatocytes and can function as an angiogenic factor [19,25,25,26].We next con?rmed by RT-PCR that IL-8is expressed in endothelial cells and we deter-mined whether Wnt/b -catenin signal activation induced IL-8expression.Expression of Wnt-1or b -cateninS37A increased transcript levels of IL-8in HUVEC relative to control Ad-LacZ HUVEC (Fig-ure 5a).In HUVEC,Wnt-5A did not activate b -cate-nin signaling (Figures 2b--c)and did not alter the transcript levels of IL-8(Figure 5a).Thus,activation of the Wnt/b -catenin but not the Wnt-5A signaling pathway induced IL-8,consistent with IL-8being a b -catenin/Tcf target gene in endothelial cells.We next evaluated the level of IL-8protein secreted in the cul-ture media collected from Ad-infected HUVEC over-expressing Wnts or b -cateninS37A.An ELISA assay demonstrated a 6.7fold increase in secreted IL-8in HUVEC media as a consequence of b

-cateninS37A

Figure 4.Wnt/b -catenin signaling promotes capillary-like network formation in Matrigel.HUVEC lines expressing HA tag,Wnt-1or b -cateninS37A

were used for a network formation assay,where cells were seeded on top of Matrigel and photographed after 18--19h of culture.Also,wild type HUVEC were incubated in the presence of 12ng/ml IL-8for the same period of time.Assays were performed two times in duplicates,a representa-tive experiment is shown.

48T.N.H.Masckaucha ′n et al.

expression relative to control(Figure5b).Wnt-1 expression only moderately increased secreted IL-8in HUVEC while Wnt-5had no effect on secreted IL-8 levels compared to control Ad-LacZ infected HUVEC. Discussion

The role of Wnt/b-catenin signaling in angiogenesis is becoming apparent.Our work,and that of others,indicate that Frizzleds,are expressed in endothelial cells.Our analysis revealed that three Frizzled receptors,Frizzled-4,-5and-6,are expressed in three different types of human primary endothelial cells and in aortic smooth muscle cells(Figure1a).Previous reports indicated that Frizzled-3transcripts are de-tected in both HUVEC and smooth muscle cells by RT-PCR[12].The expression of Frizzled-1,-4,-6and -7as well as sFRP1in HUVEC was reported by an-other study[27].It is not clear why there are differ-ences in the identi?cation of Frizzleds expressed in these primary cells,as all studies used HUVEC and similar methodologies for analysis.Of these genes, Frizzled-4and Frizzled-5have been directly implicated in angiogenesis based on evaluation of mouse pheno-types after gene disruption.Frizzled-4mutant mice have recently been shown to have defective retinal angiogenesis[28].Frizzled-5mutant mouse embryos display vascular defects in yolk sac and placenta[29]. Both Frizzled-4and Frizzled-5have been shown to be capable of activating the Wnt/b-catenin signaling[28, 30].In addition,we show that endothelial cells express Tcf/Lef transcription factors that function in Wnt/b-catenin signaling;including Tcf1,Lef-1,Tcf3and Tcf4 (Figure1b).In light of this,human endothelial cells have receptors and transcriptional components to al-low for Wnt/b-catenin signaling.

To assess angiogenic activity of the Wnt/b-catenin pathway,we studied the consequences of activating this pathway in primary endothelial cell cultures by ectopically expressing Wnt-1or b-cateninS37A (Figure2a).Wnt-1is known to activate Wnt/b-cate-nin signaling and we found that Wnt-1expression in different primary endothelial cell types increased cyto-solic b-catenin levels,a measure of Wnt/b-catenin sig-nal activation(Figure2b).Furthermore,expression of a stabilized form of b-catenin,or to a lesser extent Wnt-1,activated Tcf-transcriptional activity in HMVEC(Figure2c).We found that the consequence of Wnt/b-catenin signal activation was the promotion

Table1.DNA array analysis of Wnt-1expressing endothelial cells.

Gene name Acronym Fold effect Wingless-type MMTV integration site family,member1WNT-11418

CDC6cell division cycle6homolog CDC615.4 Interleukin-8IL813.6 Peptidylprolyl isomerase D PPID12 Small inducible cytokine subfamily B member6SCYB6 6.96 Fyn-related kinase FRK 5.8 Asparagine synthetase ASNS 5.6 Sialophorin SPN 4.98 Ras protein-speci?c guanine nucleotide-releasing factor1RASGRF1 4.1 Zyxin ZYX 4.04 Tropomodulin TMOD0.18 Gap junction protein,alpha5(connexin40)GJA50.14 Fibulin2FBLN20.1 Stromal cell-derived factor1SDF10.09 Gap junction protein,alpha4(connexin37)GJA40.09 Adenovirus infected HUVEC expressing Wnt-1or LacZ were subjected to Affymetrix microarray analysis,performed in triplicate.Genes listed show

differences between Wnt-1compared to LacZ of either greater than a4-fold increase or greater than a5-fold

decrease.

Figure5.Wnt/b-catenin signaling induces IL-8expression in endothe-

lial cells.(a)Adenovirus infected HUVEC expressing LacZ,Wnt1,

Wnt5a or b-cateninS37A were used for RT-PCR analysis to measure

the transcript levels of Interleukin-8at48h postinfection.(b)ELISA

analysis of IL-8in culture media collected from adenovirus infected

HUVEC expressing LacZ,Wnt1,Wnt5a or b-cateninS37A.Assays

were performed in triplicate.

Wnt signaling in primary endothelial cells49

of endothelial proliferation and survival in culture (Figures3a--c).Although either Wnt-1or b-catenin expression promoted endothelial proliferation,the strongest response resulted from expression of a stabilized form of b-catenin,b-cateninS37A.Activa-tion of the Wnt/b-catenin pathway by overexpression of b-cateninS37A induced more robust Wnt/b-catenin signaling(Figure2c),as opposed to expression of ligand Wnt-1.It may also be possible that the Friz-zled receptors expressed in endothelial cells respond relatively poorly to Wnt-1and would respond more strongly to other Wnts that activate the canonical Wnt/b-catenin pathway.We found that expression of Wnt-2or Wnt-3,known to activate Wnt/b-catenin signaling,were also not as effective in inducing prolif-eration as b-cateninS37A(data not shown).

We noted that proliferative responses of endothelial cells to Wnt/b-catenin signal activation depended on the presence of another angiogenic factor.For instance, detection of Wnt-1induced HUVEC proliferation was optimal in minimal endothelial cell media supplemented with20ng/ml bFGF(Figure3a).The effect of Wnt/b-catenin expression on endothelial proliferation was less evident in complete endothelial media supplemented with VEGF and FGF or VEGF alone(data not shown). Thus,the activity of Wnts on HUVEC proliferation may not have been as apparent in the presence of VEGF and serum.Alternatively,Wnts may cooperate speci?-cally with bFGF in their angiogenic activities.Wnts are known to cooperate with FGFs in promotion of mam-mary tumors in mouse models[31].

Activation of the Wnt/b-catenin pathway by Wnt-1 expression increased HUVEC proliferation(Fig-ure3a),induced b-catenin stabilization(Figure2b) and activated TCF-responsive transcriptional reporter (Figure2c).Wnt signaling may not be the only means of regulation of cytosolic b-catenin levels and activity in endothelial cells.Stimulation of endothelial prolifer-ation by b-catenin can be mediated by PECAM signal-ing[32].Increased degradation of b-catenin via disintegrin has been correlated with increased endothe-lial apoptosis[33].In con?ict with our own?ndings,a recent report[27]found that Wnt-1signaling inhibits HUVEC proliferation in a co-culture of HUVEC and C57MG mammary epithelial cells expressing Wnt-1.A complication in this study lies in the fact the C57MG cells are transformed by Wnt-1and thus express many different gene products that may in?uence endothelial cell growth.Con?rming our?ndings,we have found that Wnt-3A,another Wnt known to activate the Wnt/b-catenin pathway,was also able to induce weak but detectable endothelial cell proliferation in vitro using conditioned medium from mouse L cells ectopi-cally expressing Wnt-3a(data not shown).

We also found that Wnt-1or b-cateninS37A expres-sion promoted in vitro angiogenesis using a matrigel assay,in a fashion similar to IL-8addition(Figure4). In this assay,endothelial cells can be induced to form a network of cord-like structures.In HUVEC,ectopic expression of either Wnt-1or b-cateninS37A led to a more extensive capillary-like network after about18h of incubation,when compared to control endothelial cells.This increase in network formation may re?ect a morphogenic function for Wnt/b-catenin signaling in endothelial https://www.wendangku.net/doc/e410733697.html,work formation may also be pro-moted by an increase in endothelial cell survival in matrigel,as a result of Wnt-1expression.However,we found that Wnt-1was not effective in promoting endothelial cell survival(Figure3c).

Using microarray analysis we identi?ed IL-8,an angiogenic factor,as a potential Wnt/b-catenin tran-scriptional target in endothelial cells(Table1).To val-idate the microarray results,we demonstrated that both IL-8message and protein are expressed at higher levels in human primary endothelial cells with acti-vated Wnt/b-catenin canonical signaling(Figures5a, b).Consistent with our?nding,IL-8was also trans-criptionally activated by b-catenin/Tcf-4signaling in primary hepatocytes[25].Using in vitro angiogenesis assays,IL-8was shown to induce endothelial cell pro-liferation and capillary tube organization as well as up-regulate matrix metalloproteinase production[34, 35].Neutralization of IL-8by anti-IL-8antibody suc-cessfully blocked IL-8-mediated capillary tube organi-zation[19,34,35].Together,these data suggest that Wnt/b-catenin canonical signaling functions to pro-mote endothelial cell differentiation.

Recent evidence further supports the notion that Wnt/b-catenin signaling can regulate several aspects of angiogenesis.In Norrie disease,a congenital degenerative condition which mimics the spectrum of phenotypes in the Frizzled-4knock-out mouse, impaired Wnt/b-catenin signaling has been implicated [36].The Norrin protein,product of the Norrie disease gene,is linked to several eye disorders that are mani-fested by abnormal retinal vascular development, including Norrie disease,FEVR,retinopathy of prematurity[37]and Coats’disease[38].Norrin can signal through Frizzled-4during retinal vascular devel-opment via activation of the Wnt/b-catenin pathway [28].Mutant forms of Frizzled-4,as found in FEVR, are defective in Norrin-dependent signaling.The impli-cation of Frizzled-4and Norrin in the vascularization of the human retina opens an exciting array of new possibilities for the research on the function of the Wnt/b-catenin pathway in endothelial cells.

This study offers insights into Wnt/b-catenin signal-ing in human endothelial cells,showing previously unreported angiogenic effects and a target gene in these cells.Continued exploration of more target genes regu-lated by this pathway and the role of non-canonical Wnt signaling need to be further explored.

50T.N.H.Masckaucha′n et al.

Acknowledgements

We thank Anshula Sharma,Arun Wiita and Liz Munoz for technical support.T.N.H.M.and C.S.are supported by DOD Breast Cancer Research Program fellowships DAMD17-03-1-0293and DAMD17-03-1-0218,respec-tively.T.N.H.M.was also supported by the Polycystic Kidney Research Foundation(PKRF),Postdoctoral Fel-lowship#00006/2000.This work was supported by Grant NIH RO1HL076411to J.K.

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