Antiangiogenic Effects of VH02, a Novel Urea Derivative

17 Pages·2016·12.87 MB·English

molecules
Article
Antiangiogenic Effects of VH02, a Novel Urea
Derivative: In Vitro and in Vivo Studies
SuwadeePhowichit1,MihoKobayashi2,YurikoFujinoya2,YasufumiSato2,
KingkarnSanphanya3,OpaVajragupta3,LindaChularojmontri4
andSuvaraK.Wattanapitayakul1,*
1 DepartmentofPharmacology,FacultyofMedicine,SrinakharinwirotUniversity,114Sukhumvit23,
Bangkok10110,Thailand;[email protected]
2 DepartmentofVascularBiology,InstituteofDevelopment,AgingandCancer,TohokuUniversity,
4-1Seiryo-machi,Aoba-ku,Sendai980-8575,Japan;[email protected](M.K.);
[email protected](Y.F.);[email protected](Y.S.)
3 DepartmentofPharmaceuticalChemistry,FacultyofPharmacy,MahidolUniversity,447SriAyudhyaRoad,
Bangkok10400,Thailand;[email protected](K.S.);[email protected](O.V.)
4 DepartmentofPreclinicalSciences,FacultyofMedicine,ThammasatUniversity,95PaholyotinRd,
Klongluang,Pathumthani12120,Thailand;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:DerekJ.McPhee
Received:30July2016;Accepted:17September2016;Published:21September2016
Abstract: Vascularendothelialgrowthfactorreceptor2(VEGFR2)isavitaltargetfortherapeutic
intervention in cancer. We have recently described a computer-based drug design for a small
moleculeVEGFR2inhibitornamedVH02(1-((1-(1H-indazol-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-3-
(3-chloromethylphenyl)urea).Thisstudyaimedtofurtherexploretheanti-angiogenicactivityofVH02
bothinvitroandinvivo.Theinvitroassaysincludecellviability,capillary-liketubeformation,MMP
activity,andwesternblotanalysesofsignalingthroughVEGFR2whiletheinvivoanti-angiogenic
response were performed to evaluate the effect on vascularization in Matrigel plug applied in
C57BL/6Lmice. VH02reducedangiogenesisbehaviorofEA.hy926includingcellviability,migration,
adhesion,capillary-liketubeformation,andMMP-2activityinducedbyVEGF.Furthermore,VH02
regulated angiogenesis by directly inhibiting VEGFR2 on Tyr1175 signaling pathway leading to
the inhibition of Akt-mediated cell survival and migration. Disruption of phosphorylation at
VEGFR2-Tyr1175byVH02abolishedFAK-Tyr397signalingbutnotphosphorylationofp38MAPK.
ThissuggeststhatblockadeofFAKbyVH02apparentlyassociatedwithreductionofendothelial
cellmotility. ActincytoskeletonrearrangementwasdiminishedbyVH02inhumanendothelialcells.
The anti-angiogenic effect of VH02 was confirmed in the invivo model, revealing the reduction
of vascular density in Matrigel plug after VH02 treatment. Additionally, the pericyte-like cells
surroundingbloodvesselsintheplugsweresignificantlyreducedaswellasvasculardensityand
p-Aktintensity. OurfindingsindicatethatVH02successfullyinhibitsVEGF-inducedangiogenesis
bothinvitroandinvivomodels. Thecompoundcouldbefurtherdevelopedasanantiangiogenesis
agentforcancertherapy.
Keywords: vascularendothelialgrowthfactorreceptor2(VEGFR2);structure-baseddrugdesign;
ureaderivatives;angiogenesis;endothelialcells
1. Introduction
Angiogenesisisanimportantfacilitatorforthedevelopmentofsolidcancerbysproutingnew
bloodvesselsfrompre-existingendotheliumtotransportgrowthfactors,oxygen,andvariousnutrients
totumors. Theprocessofangiogenesisiscomplexrequiringmanyinducersandreceptors;among
Molecules2016,21,1258;doi:10.3390/molecules21091258 www.mdpi.com/journal/molecules
Molecules2016,21,1258 2of17
those,thevascularendothelialgrowthfactor(VEGF)familymembersarekeysignalmediatorsthat
controlendothelialcellsinmanyaspects,particularly,insupportingtumorinvasionandmetastasis[1].
TheVEGFsubtypeA(VEGFA)supersedesotherfamilymembers(VEGFB,VEGFC,VEGFD)inthe
fieldofantiangiogenesistherapybecausethefirstgenerationofdrugsavailableinthemarketwas
targetingVEGF(commonlyreferredtoVEGFA)andVEGFreceptors(VEGFR).
VEGF specifically binds to VEGFR1 (Flt-1) or VEGFR2 (Flk-1/KDR), presented on the cell
surface of endothelial cells. However, VEGFR2 is the main signal transducer responsible for
angiogenesis because it has a strong intrinsic kinase activity and plays a decisive role in the
regulationoftumorangiogenesis[2]. TheinteractionofVEGFwithVEGFR2causescellproliferation,
survival, migration, capillary-tube formation and permeability increase that occurs in rapidly
growingtumors[3]. VEGF/VEGFR2signalingpathwayisinitiatedbythedimerizationofVEGFR2
and subsequent phosphorylations of several tyrosine residues, e.g., Y951, Y1175, and Y1214.
Among these residues, VEGFR2-Tyr1175 is the major autophosphorylation site following VEGF
binding that controls angiogenic responses in vascular endothelial cells [4,5]. Phosphorylation
of VEGFR2-Tyr1175 regulates downstream signaling through phospho-Akt at Ser473 promoting
endothelial cell survival and migration [6,7]. The Akt activation is an almost indispensible signal
transductionforregulatingendothelialcellsurvivalandmigrationthroughthePI3K/AKTandeNOS
pathways[4,8]. TheendothelialcellmotilityactivatedbyVEGFR2alsoassociateswithfocaladhesion
molecule(FAK)viatheinteractionofVEGFR2andShbadaptorprotein. Particularly,VEGF-induced
FAKphosphorylationatTyr397inendothelialcellsisahighlyconservedautophosphorylationsiteby
transmittingdifferentsignalstotheirtargets. Itcansustainanincreasedformationofactinstressfibers
andnewfocaladhesionresultinginseveralbiologicalprocesses,includingendothelialcelladhesion,
migration,invasion,tubeformationandsurvival[6,9–11].
Thefirstantiangiogenesisdrugbevacizumab(amonoclonalantibodyforVEGFA)waslaunched
in2004andmanysmallmoleculesthatinhibittheactivityandsignalingcascadeofVEGFreceptors
(VEGFRs) have been developed and approved by the US FDA for the treatment of various
types of cancer [12]. Currently, the antiangiogenesis drugs against VEGF are mostly effective in
clinical practice when combined in chemotherapy [13–16]. The small molecule VEGFR2 tyrosine
kinase inhibitors (TKIs, e.g., sorafenib, sunitinib, pazofinib) usually have urea derivatives in
their chemical structures which are related to their biological effects [17]. Sorafenib, an orally
available angiogenesis drug for multi-tyrosine kinase inhibition (VEGFR2, VEGFR3, PDGFR-beta
and c-kit), is a representative compound that comprises a biaryl urea in its structure [18]. It was
approvedbyUSFDAforadvancedrenalcancerin2005. Thus,thedevelopmentofnovelVEGFR2
inhibitors using structure-based drug design targeting high affinity for VEGFR2 is of interest. We
have recently reported synthesis of a small molecule VEGFR2 tyrosine kinase inhibitor VH02 or
1-((1-(1H-indazol-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-3-(3-chloromethylphenyl)ureabyusingaback
tofrontapproachfrominsilicodrugdesign[19]. VH02isatypeIIkinaseinhibitorwhichismore
selectivetoVEGFR2thantypeIkinaseinhibitors. ThecoremotifofVH02contains1-((3-chloromethyl)
phenyl)-3-(2-propynyl)ureathatisburiedinthebackpocketofVEGFR2. ThedesignofVH02allows
forstronghydrogenbondingtothekinasedomainofVEGFR2whiletwobondsinteractwiththekey
residuesattheATPbindingsite(frontpocket)namelyGlu917andCys919. Thus,weaimtoconfirm
andevaluatetheantiangiogenicactivityofVH02bothinvitroandinvivobytargetingthekeysignals
ofVEGF-inducedangiogenesis. WeexploredVEGFR2-Tyr1175signalingpathwayontheactivationof
Akt,FAK,andp38inhumanendothelialcellsEA.hy926. TheinvivomousemodelofMatrigelplug
assaywasusedtoevaluatetheeffectofVH02onvasculardensityandα-SMAcoverageofthevessels.
Inthisstudy,wefoundthatVH02inhibitedangiogenesisuponVEGFstimulationbothinvitroand
invivowhichhashighpotentialforfurtherleadoptimizationasacancertherapyagent.
Molecules2016,21,1258 3of17
Molecules 2016, 21, 1258 3 of 17
2. Results
2. Results
2.1. VH02InhibitedVEGF-InducedEA.hy926CellViability
2.1. VH02 Inhibited VEGF-Induced EA.hy926 Cell Viability
VH02, a small molecule VEGFR2 TKI (Figure 1A) had a negative influence on endothelial
cell vViaHb0il2it, ya usmndaellr mseorluemculset aVrvEaGtFioRn2 aTnKdI V(FEigGuFres t1imA)u hlaatdio an .neUgnadtievre lionwfluseenrcuem onco ennddiotitohne,liValH c0e2ll
sviiganbiilfiitcya nutnldyerc asuersuemd setnadrvoatthioenli aalndce VllEdGeFa sthtimautlathtieonc. oUnncdenert rlaotwio snesru1m0 cµoMndiotironh,i VghHe0r2 (sFigignuifriecan1tBly).
WcahuisleedV EenGdFo(t2h5elniagl/ cmeLll) daeloanthe saitg nthifie ccaonntlcyenintcraretiaosneds 1ce0l lμvMia boirli thyigtohe1r1 7(.F1i%gu±re8 1.7B%). tWhehciole- inVcEuGbaFt i(o2n5
wngit/hmVLH) a0l2osnieg nsiifigcnainfitclaynitnlyh ibinitcerdeaVseEdG Fce-ilnl dvuiacbeidliteyn dtoo th11e7li.a1l%c e±ll 8v.i7a%bi ltithye actoa-lilnccounbcaetniotrna twioinths tVesHte0d2
(sFigignuifricea1nCtl)y. Tinhhuibsi,tefudr tVhEeGrFe-xipnedruicmeden etnsdwoethreelpiael rcfeolrlm veiadbiulistiyn gat tahlel cmoanxceimntaraltcioonnsc etnesttreadti o(FnigoufrVe H1C02).
aTth5uµs,M fu.rther experiments were performed using the maximal concentration of VH02 at 5 μM.

Figure 1. Effects of VH02 on EA.hy 926 cell viability. EA.hy 926 cells were treated with various
Figure 1. Effects of VH02 on EA.hy 926 cell viability. EA.hy 926 cells were treated with various
concentrations of VH02 and co-incubated with or without VEGF (25 ng/mL) for 24 h. Cell viability
concentrationsofVH02andco-incubatedwithorwithoutVEGF(25ng/mL)for24h.Cellviability
wwaass mmeeaassuurreedd bbyy MMTTTT aassssaayy aass ddeessccrriibbeedd inin ththee MMaateterriaialsls aanndd MMeeththooddss sesecctitoionn.. ((AA)) CChheemmiiccaall
ssttrruuccttuurree ooff VVHH0022;; ((BB)) CCyyttoottooxxiicc eeffffeecctt ooff VVHH0022 oonn tthhee eennddootthheelliiaall cceellllss iinn tthhee aabbsseennccee ooff VVEEGGFF
((2255 nngg//mmLL));; ((CC)) CCyyttoottooxxiicc eeffffeecctt ooff VVHH0022 iinn tthhee eennddootthheelliiaall cceellllss iinn tthhee pprreesseennccee ooff VVEEGGFF ((2255 nngg//mmLL))..
** pp << 00..0055 ccoommppaarreedd wwiitthh ccoonnttrrooll ((vveehhiiccllee ttrreeaatteedd cceellllss));; ∆Δ pp << 00..0055 ccoommppaarreedd wwiitthh VVEEGGFF ttrreeaatteedd cceellllss
((VVEEGGFF ccoonnttrrooll))..
22..22.. VVHH0022 IInnhhiibbiittss VVEEGGFF--SSttiimmuullaatteedd CCeellll MMiiggrraattiioonn,, AAddhheessiioonn aanndd CCaappiillllaarryy--LLiikkee TTuubbee FFoorrmmaattiioonn
The effect of VH02 on cell migration was quantified by tracking the intracellular esterase enzyme
TheeffectofVH02oncellmigrationwasquantifiedbytrackingtheintracellularesteraseenzyme
activity of cells that passed through the porous membrane of a transwell chamber after 16 h. VH02
activityofcellsthatpassedthroughtheporousmembraneofatranswellchamberafter16h. VH02
ssiiggnniifificcaannttllyy iinnhhiibbiitteedd VVEEGGFF--ssttiimmuullaatteedd eennddootthheelliiaall cceellll mmiiggrraattiioonn aatt tthhee ccoonncceennttrraattiioonn 33 aanndd 55 µμMM
compared with VEGF group (Figure 2A). Adhesion of endothelial cells also associates angiogenesis
comparedwithVEGFgroup(Figure2A).Adhesionofendothelialcellsalsoassociatesangiogenesis
and metastasis at an early attachment step. Under VEGF stimulation, VH02 decreased EA.hy 926
and metastasis at an early attachment step. Under VEGF stimulation, VH02 decreased EA.hy 926
adhesion by approximately 35% when compared with VEGF alone (Figure 2B).
adhesionbyapproximately35%whencomparedwithVEGFalone(Figure2B).
Capillary-like tubules are formed in later stage of angiogenesis which generated from complex
Capillary-liketubulesareformedinlaterstageofangiogenesiswhichgeneratedfromcomplex
multiple step process including proliferation, migration, adhesion, and differentiation of endothelial
multiplestepprocessincludingproliferation,migration,adhesion,anddifferentiationofendothelial
cells. The tubular network was closely aligned to form a branched polygonal cell network similar to
cells. Thetubularnetworkwascloselyalignedtoformabranchedpolygonalcellnetworksimilarto
a mesh-like structure in the presence of VEGF (Figure 2C). In contrast, when VH02 was added with
amesh-likestructureinthepresenceofVEGF(Figure2C).Incontrast,whenVH02wasaddedwith
VEGF, a network of capillary-like tube formation was progressively lost and the tubes were generated
VEGF,anetworkofcapillary-liketubeformationwasprogressivelylostandthetubesweregenerated
irregularly without lumen. Endothelial capillary tube formation was remarkably abrogated by VH02
irregularlywithoutlumen. EndothelialcapillarytubeformationwasremarkablyabrogatedbyVH02
iinn aa ccoonncceennttrraattiioonn--ddeeppeennddeenntt mmaannnneerr aatt aass llooww ddoossee aass 11 µμMM.. WWiitthh VVEEGGFF ssttiimmuullaattiioonn,, VVHH0022 aatt tthhee
concentrations of 1, 3 and 5 μM significantly decreased tube lengths by 38.71%, 77.28%, and 78.02%,
respectively (Figure 2C).
Molecules2016,21,1258 4of17
concentrationsof1,3and5µMsignificantlydecreasedtubelengthsby38.71%,77.28%,and78.02%,
respectively(Figure2C).
Molecules 2016, 21, 1258 4 of 17

FigFuirgeur2e. 2In. flInufleunecneceo foVf VHH0202o nonV VEEGGFF-i-nindduucceedd eennddootthheelliiaall cceellll mmiiggrraatitoionn, ,ceclell ladadhehseisoino nanadn dtutbueb e
forfmoramtiaotnio.n(.A (A) V) HVH0202i nihnihbibitieteddV VEEGGFF--iinndduucceedd EEAA..hhyy 992266 mmiiggrraattiioonn. .EEAA.h.yh y92962 6wwereer ealalollwoewde dto to
migmriagtreatfero fmromth ethue puppeprecr hcahmambebrero offt rtarannsswweellllss ppllaattee ttoo tthhee lloowweerr cchhaammbbeer rtrtereaatetded wwithit horo wrwithitohuot ut
VEVGEFG(F2 5(2n5 gn/gm/mLL)) aanndd vvaarryyiningg ddosoesse osfo VfHV0H2.0 A2f.teAr cftuelrtucruinltgu froirn 1g6f ho,r th1e6 mhi,gtrhaetedm cigelrlas twederec emllosvwede re
motvherodutghhr othueg hpotrhoeups omroemusbrmaneem abnrda nqeuaanntdifiqeuda bnyt imfieedasbuyrinmge iansturarcinelgluilnatrr eascteelrlauslea renezstyemraes eacetinvzityym; e
acti(vBi)t yA;d(hBe)sAiodnh oefs EioAn.hoyf 9E2A6 .ohny M92a6troigneMl-caotartiegde lp-claotaetse. dEfpfelacttes so.fE VfHfe0c2ts oonf cVelHl a0d2hoenrecneclel awdehreer menecaesuwree re
by MTT assay at 2 h in the presence or absence of VEGF (25 ng/mL); (C) Effects of VH02 on VEGF-
measurebyMTTassayat2hinthepresenceorabsenceofVEGF(25ng/mL);(C)EffectsofVH02
induced capillary-like tube formation. EA.hy 926 suspended in different concentrations of VH02-
onVEGF-inducedcapillary-liketubeformation. EA.hy926suspendedindifferentconcentrations
containing media were seeded onto a growth factor-reduced Matrigel. VEGF (25 ng/mL) was added
ofVH02-containingmediawereseededontoagrowthfactor-reducedMatrigel. VEGF(25ng/mL)
to stimulate capillary-like tube formation. Representative images were captured at a magnification of
was added to stimulate capillary-like tube formation. Representative images were captured at a
100X on an Olympus inverted microscope. Quantification of percentage of tube lengths was assessed
magnification of 100X on an Olympus inverted microscope. Quantification of percentage of tube
at least six randomly selected fields by Cell B imaging software. Values were presented as mean ±
lengthswasassessedatleastsixrandomlyselectedfieldsbyCellBimagingsoftware. Valueswere
SEM of at least three separate experiments. * p < 0.05 when compared to vehicle-treated cells (control);
presented as mean ± SEM of at least three separate experiments. * p < 0.05 when compared to
Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001 compared with VEGF-treated cells alone (VEGF control).
vehicle-treatedcells(control);∆p<0.05;∆∆p<0.01;∆∆∆p<0.001comparedwithVEGF-treatedcells
alone(VEGFcontrol).
2.3. VH02 Suppressed VEGF-Induced Gelatinase Activity
In the presence of VEGF, VH02 at various concentrations was able to suppress the gelatinase
2.3. VH02SuppressedVEGF-InducedGelatinaseActivity
activity of MMP-2 in a dose-dependent manner, with strong significant inhibition at 1–5 μM (Figure 3).
VHIn02t htreeaptrmeseennt cgeavoef aV cEleGaFrl,yV vHis0ib2lea btavnadr iaotu 7s2 ckoDnac ebnuttr tahteio bnasndw oafs MabMleP-t9o (9su2 pkpDrae)s dsidth neogt ealpaptienaar se
actiovni ttyheo fgMelaMtiPn -s2uibnstaradtoe.s e-dependentmanner,withstrongsignificantinhibitionat1–5µM(Figure3).
VH02treatmentgaveaclearlyvisiblebandat72kDabutthebandofMMP-9(92kDa)didnotappear
onthegelatinsubstrate.
Molecules2016,21,1258 5of17
Molecules 2016, 21, 1258 5 of 17
Molecules 2016, 21, 1258 5 of 17

Figure 3. MMP-2 inhibitory effect of VH02 on EA.hy 926 cells. Represen tative gelatin zymography
Figure3. MMP-2inhibitoryeffectofVH02onEA.hy926cells. Representativegelatinzymography
shows a clear visible band of MMP-2 activity at 72 kDa while MMP-9 activity at 92 kDa was not
showFisguarcel e3a. rMvMisPi-b2l einbhaibnidtoroyf eMffeMctP o-2f VaHct0iv2 iotyn aEtA7.h2yk 9D26a cwelhlsi.l eReMprMesPe-n9taaticvteiv giteylataitn 9z2ymkDogarawphays not
observed. The activity of MMP-2 was quantified by the band density and analyzed by Image J
obsesrhvoewd.s Tah celeaacrt ivviistiybloef bManMdP o-2f MwaMsPq-u2 aancttiifiveitdy bayt 7th2 ekbDaan dwhdielen sMityMaPn-9d aacntaivlyitzye adt b9y2 IkmDaag ewJass onfotwt are.
software. The data are expressed as percentage of reference control. * p < 0.05 versus vehicle control;
Theodbastearvareed.e xTphree sascetidviatsy poefr cMenMtPag-2e wofarse fqeuraenntciefiecdo nbtyro tlh.e* pba<n0d. 0d5evnesritsyu sanvde hainclaelyczoendt rboyl; ∆Im∆apge< J0 .01;
ΔΔ p < 0.01; ΔΔΔ p < 0.01 versus VEGF (25 ng/mL) alone.
∆∆∆sopft<w0a.r0e1. Tvheer sduastaV aErGe eFx(p2r5esnsged/ masL p)earlcoenneta.ge of reference control. * p < 0.05 versus vehicle control;
ΔΔ p < 0.01; ΔΔΔ p < 0.01 versus VEGF (25 ng/mL) alone.
2.4. VH02 Inhibited VEGFR2-Dependent Cell Signaling
2.4. VH02InhibitedVEGFR2-DependentCellSignaling
2.4. VVHE0G2 FIn htirbeiatetmd VenEtG FdRra2m-Daetpicenaldleyn ts Ctiemll uSliagtneadl inVg EGFR2 autophosphorylation on Tyr1175 and
dVoEwGnsFtretarema tsmigennaltindgr baym Aaktitc aanlldy FAstKim (Fuilgautreed 4)V. EGFR2 autophosphorylation on Tyr1175 and
VEGF treatment dramatically stimulated VEGFR2 autophosphorylation on Tyr1175 and
downstreamsignalingbyAktandFAK(Figure4).
downstream signaling by Akt and FAK (Figure 4).

Figure 4. Effects of VH02 on VEGF-mediated signaling pathway through VEGFR2, Akt , FAK and P38
in EA.hy926 cells. The protein phosphorylation of VEGF receptor-2 (p-VEGFR2), Akt (p-Akt), FAK
Figure 4. Effects of VH02 on VEGF-mediated signaling pathway through VEGFR2, Akt, FAK and P38
Figu(rpe-F4A.K)E, fafnedct sP3o8f (pV-HP3082) woans VanEaGlyFz-emd ebdyi awteesdtesrnig bnlaolti nagnalpyastish wasa dyestchrriboeudg hin VthEeG MFaRte2r,iaAlsk atn,dF AK
in EA.hy926 cells. The protein phosphorylation of VEGF receptor-2 (p-VEGFR2), Akt (p-Akt), FAK
andMPe3t8hoinds EseAc.thioyn9. 2(6A)c Relelps.resTenhteatipvreo bteloints pshhoowsp phrootreyinla ptihoonspohfoVryElaGtiFonr seicgenpatlos rr-e2la(tipv-eV tEo GthFeR n2o)n,-Akt
(p-FAK), and P38 (p-P38) was analyzed by western blot analysis as described in the Materials and
(p-Apkhto),spFhAoKryl(apte-FdA pKro)t,eianns;d (BP) 3C8ha(pn-gPes3 8in) VwEaGsFaRn2a plhyozsepdhobryylwatieosnt;e (rCn) Cblhoatngaensa ilny Asikst apshodsepshcorribyleadtioinn; the
Methods section. (A) Representative blots show protein phosphorylation signals relative to the non-
Mate(Dri)a ClshaanngdesM ine tFhAoKd sphsoescptihoonry.la(tAio)n;R (eEp) Crehsaenngteast iivne Pb38lo pthsosshpohworyplartoioteni.n Vaplhuoess pwheorer yrelaptrieosnenstiegdn als
phosphorylated proteins; (B) Changes in VEGFR2 phosphorylation; (C) Changes in Akt phosphorylation;
relataivs emteoanth ±e SnEoMn.- p* ph o< s0p.0h5o; r*y* lpa <te 0d.0p1 rvoetresiunss v;e(Bhi)cCleh caonngtreosl; iΔn pV <E 0G.0F5R; Δ2Δp hp o<s 0p.0h1o; rny.sla. ntioonn-;si(gCn)ifCichaannt ges
(D) Changes in FAK phosphorylation; (E) Changes in P38 phosphorylation. Values were represented
inAvketrpsuhso VspEhGoFr yalloantieo. n ;(D)ChangesinFAKphosphorylation;(E)ChangesinP38phosphorylation.
as mean ± SEM. * p < 0.05; ** p < 0.01 versus vehicle control; Δ p < 0.05; ΔΔ p < 0.01; n.s. non-significant
Valuveesrswuse rVeErGeFp raelosenne.t e dasmean ± SEM.*p<0.05; **p<0.01versusvehiclecontrol; ∆ p<0.05;
∆∆p<0.01;n.s.non-significantversusVEGFalone.
Molecules2016,21,1258 6of17
Molecules 2016, 21, 1258 6 of 17
TThhee pphhoosspphhoorryyllaattiioonn ooff VVEEGGFFRR22 wwaass ddeeccrreeaasseedd bbyy VVHH0022 aatt ccoonncceennttrraattiioonn 33,, aanndd 55 μµMM iinn aa ddoossee
ddeeppeennddeenntt mmaannnneerr,, ccoommppaarreedd ttoo VVEEGGFF aalloonnee ((FFiigguurrees 44AA,,BB)).. TThhee pphhoosspphhoorryyllaattiioonn ooff AAKKTT aatt SSeerr447733
ssiittee,, aa kkeeyy mmoolleeccuullaarr ttaarrggeett ddoowwnnssttrreeaamm ooff VVEEGGFFRR22,, wwaass ccoommpplleetteellyy bblloocckkeedd bbyy VVHH0022 aatt bbootthh 33 aanndd
55 μµMM uunnddeerr VVEEGGFF ssttiimmuullaattiioonn ((FFiigguurrees 44AA,,CC)).. TThheessee rreessuullttss iinnddiiccaatteedd tthhaatt VVHH0022 hhaadd aann aabbiilliittyy ttoo
aabboolliisshh VVEEGGFF//VVEEGGFFRR2/2A/kAt-kdt-edpeepnednednet netndenodthoetlhiaell icaelllc esullrsvuivrvali.v Nale.xNt, ewxet, fuwrethfeurr itdheenrtiidfieendt itfiheed FAthKe
aFcAtiKvaatcitoinv aotino nToynr3T9y7,r 3w97h,icwhh iisc hmisajmora jtoarrgtaertg oeft oefnednodthoethliealli acleclle lmlmotoiltiitlyit yanandd aaddhheessioionn. .VVHH0022 aatt aa
ccoonncceennttrraattiioonn ooff 55 μµMM ssiiggnniiffiiccaannttllyy iinnhhiibbiitteedd FFAAKK pphhoosspphhoorryyllaattiioonn iinn pprreesseennccee ooff VVEEGGFF iinndduuccttiioonn;;
hhoowweevveerr,, tthheerree wwaass nnoo cchhaannggee iinn tthhee pphhoosspphhoorryyllaattiioonn ooff pp3388 MMAAPPKK ((FFiigguurrees4 A4A,D,D,E,)E.).
22..55.. VVHH0022 IInnfflluueenncceedd AAccttiinn CCyyttoosskkeelleettoonn ooff EEAA..hhyy 992266
TThhee ssttrruuccttuurreess ooff vveehhiiccllee--ttrreeaatteedd ccoonnttrrooll cceellllss wweerree rreeaarrrraannggeedd iinnttoo mmeemmbbrraannee rruufffflleess ((FFiigguurree 55AA))..
IInn rreessppoonnssee ttoo VVEEGGFF ssttiimmuullaattiioonn,, cceellllss sshhoowweedd rreeoorrggaanniizzaattiioonn ooff aaccttiinn cchhaarraacctteerriizzeedd bbyy pprroommiinneenntt
eexxtteennssiioonn ooff ssttrreessss ffiibbeerrss iinnttoo ppaarraalllleell ddiissppeerrssiioonn tthhaatt eevvookkeedd aa mmiiggrraattoorryy rreessppoonnssee aatt 1155 mmiinn ((FFiigguurree 55BB))..
IInn ccoonnttrraasstt,, pprreettrreeaattmmeenntt ooff cceellllss wwiitthh vvaarryyiinngg ccoonncceennttrraattiioonnss ooff VVHH0022 ffoorr 3300 mmiinn bbeeffoorree aaddddiinngg VVEEGGFF
iinnhhiibbiitteedd ssttrreessss ffiibbeerr ffoorrmmaattiioonn iinn aa ddoossee--ddeeppeennddeenntt mmaannnneerr ((FFiigguurree 55CC––EE)).. TThhee aaccttiinn ccyyttoosskkeelleettoonn
wwaass cchhaannggeedd iinn ssiizzee aanndd sshhaappee aanndd aa lloossss ooff ppoollyymmeerriizzeedd aaccttiinn wwaass oobbsseerrvveedd aafftteerr ttrreeaattmmeenntt wwiitthh
VVHH0022.. TThheessee rreessuullttss ssuuggggeesstteedd tthhaatt VVHH0022 ccoouulldd rreessttrraaiinn rraappiidd aaccttiinn ccyyttoosskkeelleettoonn rreeaarrrraannggeemmeenntt iinn
VVEEGGFF--iinndduucceedd ssttrreessss fifibbeerr ffoorrmmaattiioonn aanndd rreessuulltt iinn tthhee iinnhhiibbiittiioonn ooff cceellll mmiiggrraattiioonn ooff EEAA..hhyy 992266..

Figure 5. Immunofluorescence images of actin cytoskeleton in EA.hy926. Cells were treated with
Figure5. ImmunofluorescenceimagesofactincytoskeletoninEA.hy926. Cellsweretreatedwith
VH02 for 30 min, and then stimulated with or without VEGF (50 ng/mL) for 15 min. Actin fibers were
VH02for30min,andthenstimulatedwithorwithoutVEGF(50ng/mL)for15min. Actinfibers
vwiesureavliizseuda liinze cdelilns cfeixllesdfi xweidthw 4it%h 4f%ormfoarmldaelhdyedhey daendan tdagtaggegde dbyb yanatni-tri-arbabbibt itcoconnjujuggaatetedd AAlleexxaa FFlluuoorr
((ggrreeeenn)) aass ddeessccrriibbeedd iinn tthhee MMaatteerriiaallss aanndd MMeetthhooddss sseeccttiioonn.. TThhee nnuucclleeii wweerree ccoouunntteerrssttaaiinneedd wwiitthh DDAAPPII
((bblluuee)).. AAllll iimmaaggeessw weereret atkaeknenin inth tehsea msaemeex peoxspuorseubrey Obyly Omlpyumspimusm imunmofluunoorfeluscoernescceemncicer omscicorpoyscwopityh
w60i0th× 6m00a×g nmifiagcantiifoicna.tSiocna.l eScbaalre= ba1r0 =µ m10. μ(Am). C(Aon) tCroolncterlolls cuenllds eurnbdaesra lbcaosnald citoionnd;it(iBo)nc; e(Blls) tcreelalst etdrewatietdh
wVEitGh FV(E5G0Fn g(5/0m nLg)/;m(CL));c e(Cll)s ctreellast etdrewateitdh wVHith0 2V(H10µ2M (1) aμnMd)V aEnGdF V(E5G0Fn g(/50m nLg);/m(DL));c e(Dlls) tcreelalst etdrewatietdh
wVHith0 2V(H30µ2M (3) aμnMd)V aEnGdF V(E5G0Fn g(5/0m nLg)/;m(EL))c; e(Ells) ctreellast etrdeawteitdh wViHth0 2V(H50µ2M (5) aμnMd) VaEnGd FV(E5G0Fn g(5/0m nLg)/.mL).
2.6. Confirmation of VH02 Action on HUVECs
2.6. ConfirmationofVH02ActiononHUVECs
The in vitro antiangiogenesis activity of VH02 was confirmed in HUVECs under serum
TheinvitroantiangiogenesisactivityofVH02wasconfirmedinHUVECsunderserumstarvation.
sVtHar0v2at(i1ona. nVdH502µ (M1 )anedff e5c tμivMe)ly efifnehctiibvietelyd itnhheibpitheods pthheo pryhloastipohnoroyflaVtiEoGn FoRf 2VEanGdFRA2k atnudn Adekrt uVnEdGeFr
VstEimGuFl asttiiomnuilnataiodno sien- dae dpoensed-ednetpmenadnennert cmoamnpnaerre dcowmiptharVeEdG wFiathlo VneE.GSFor aalfoenneib. S(5oµraMfe)nwiba s(5p μerMfo)r mweads
performed as a reference drug for suppressing VEGF-induced phosphorylation of VEGFR2 and Akt.
asareferencedrugforsuppressingVEGF-inducedphosphorylationofVEGFR2andAkt. Thelevels
TofhVe EleGveFl-ss toimf VuElaGteFd-sVtimEGuFlaRte2dp VhoEsGpFhRo2r yplhatoisopnhworayslasitgionnifi wcaans tsliygndiefcicraenastleyd dferocrmea4s3e7d. 0f9ro%m± 43572..0091%%
±d o5w2.0n1t%o 7d1o.w88n% to± 711.68.84%8% ±, 1568..4981%%, ±58.1951.%61 %± 1a5n.6d1%31 .a1n1d% 3±1.191.%98 %± 9b.9y8%VH b0y2 V(H1 0µ2M ()1, μVMH)0,2 V(H50µ2M (5),
μM), and sorafenib (5 μM), respectively (Figure 6A). The intensity of p-VEGFR2 was influenced by
both factor 1, VEGF stimulation, (PBS or VEGF) and factor 2, drug treatment, (vehicle, VH02, or
Molecules2016,21,1258 7of17
andsorafenib(5µM),respectively(Figure6A).Theintensityofp-VEGFR2wasinfluencedbyboth
Molecules 2016, 21, 1258 7 of 17
factor1,VEGFstimulation,(PBSorVEGF)andfactor2,drugtreatment,(vehicle,VH02,orsorafenib)
wsohriacfhenhiabd) wahsiigchn ihfiacdan at isnigtenriaficctaionnt iantteFr(a3c,8ti)o=n 5a.t1 F0(,3p,8<) 0=. 055.1.0T, hpe <t r0e.0a5tm. Tehnet wtreitahtmVeHn0t 2woitrhs oVrHaf0e2n iobr
aslosroafiennhiibb iatelsdo VinEhGibFi-tsetdim VuElaGtFe-dstAimktulpahteodsp Ahkotr yplhaotisopnhforroymlattihoen lefrvoemlo tfh3e1 l9e.v1e9l% of± 31492..1799%% ±d o4w2.n79t%o
1d3o8w.5n1% to± 13283.5.118%% ±, 1273..5198%%,± 176..5297%% ,±a 6n.d271%2.,8 a4n%d ±12.68.146%% ±, 6re.1s6p%ec,t riveseplyec(Ftiivgeulrye (6FBig)u.Treh e6Bc)h. aTnhgee cshinanAgkets
pinh oAskpth pohroyslapthioonrywlaetrioent hweerrees tuhlets roesfuthltes oinf ttehrea cintitoenrabcetitowne beentwfaecetonr fa1c(tVorE 1G (FV-pErGimF-epdricmoendd citoionnd)itaionnd)
faancdto fra2ct(odrr u2 g(dtrreuagt mtreeanttm)wenhti)c hwwhiecrhe wobersee rovbesderavteFd(3 a,8t )F=(31,80). 4=1 1,0p.4<10, .p0 1<. 0.01.
FF--aaccttiinn oorrggaannizizaatitoionnw waassv visiusualailziezdedb ybys tsatianiinnigngw withithA lAexleaxFal uFlouro-cro-cnojungjuagteadtepdh palhloalidloiind(iFni g(Fuirgeu6r)e.
I6n).u nIntr euantetdreHatUedV EHCUs,VFE-aCctsi,n Fst-aaicntiinn gswtaaisnisnegen wasasa csoeretnic aalsn eatw coorrkt.icVaEl GnFetpwroomrko. teVdErGeoFr gparnoimzaotitoend
orefothrgeaancitzinatcioynto oskf ethleet oanctiinnt ocysttorseksselfiebtoenrs inaltoon sgtrtehses lfoibnegrist uadloinnagl tahxei slownigthitiund3i0naml ianx.isC wo-ittrheiant m30e nmtionf.
HCUo-VtrEeaCtsmwenitth 5ofµ MHUsoVrEafCesn ibwwitiht h5o rμwMit hosuortaVfeEnGibF iwnhitihb iteodr Fw-aicthtionurte aVrrEaGngFe minehnitb.itTehde FF--aaccttiinn
cryetaorsrkaenlgeetomnewnta. sTchhea nFg-eadctiinn sciyzetoasnkdelsehtoanp ewaansd cthheanlogsesdo finF -saicztein apnodl ysmhaerpiez aatinodn wthaes loobssse rovf eFd-aacftteinr
cpeolllsymweerreiztarteiaotne dwwasi tohb1searnvded5 aµfMterV cHel0ls2 w. ere treated with 1 and 5 μM VH02.

Figure6.Cont.
Molecules2016,21,1258 8of17
Molecules 2016, 21, 1258 8 of 17

Figure 6. Effects of VH02 on VEGFR2 and Akt phosphorylation, and actin polymerization in HUVECs
Figure 6. Effects of VH02 on VEGFR2 and Akt phosphorylation, and actin polymerization in
in normal control group (PBS) and VEGF-induced condition. (A) Representative confocal
HUVECsinnormalcontrolgroup(PBS)andVEGF-inducedcondition. (A)Representativeconfocal
immunofluorescence images of HUVECs stained by fluorescent-tagged anti-p-VEGFR2 Tyr1175 antibody
immunofluorescence images of HUVECs stained by fluorescent-tagged anti-p-VEGFR2 Tyr1175
(green) in the presence and absence of VEGF treatment (control); (B) Representative confocal
antibody(green)inthepresenceandabsenceofVEGFtreatment(control);(B)Representativeconfocal
immunofluorescence images of HUVECs stained with fluorescent-tagged anti-p-Akt Ser473 antibody
immunofluorescenceimagesofHUVECsstainedwithfluorescent-taggedanti-p-AktSer473antibody
(green) in the presence and absence of VEGF treatment (control). HUVECs were pretreated for 30 min
(green)inthepresenceandabsenceofVEGFtreatment(control).HUVECswerepretreatedfor30min
with VH02 or sorafenib as indicated in the figure. The slides were also counter-stained with antibodies
withVH02orsorafenibasindicatedinthefigure.Theslideswerealsocounter-stainedwithantibodies
against F-actin (pink). Cell nuclei were identified by Topro-3 (blue). All images were taken at the same
againstF-actin(pink).CellnucleiwereidentifiedbyTopro-3(blue).Allimagesweretakenatthesame
eexxppoossuurree ttiimmee bbyyc coonnfofoccaallm micircorsocsocpoypyw withitZh -Zst-asctkacpkr opjreoctjeiocntison(6s0 0(6×00m× amgnaigfinciaftiicoanti)o.nS)c.a Slecablaer sb,a3r0s,µ 3m0 .
μFmlu.o Freluscoernecseceinncteen isnitteynvsaitlyu evsawlueerse wreeprere rseepnrteesdeanstemde aasn m±eaSnE M± SoEfMd uopf ldicuaptelicseaptea sraetpeaerxaptee erixmpeenritmswenitths
with five randomly images from each group. ** p < 0.01, *** p<0.001 compared with PBS of each pair
fiverandomlyimagesfromeachgroup. **p<0.01,***p<0.001comparedwithPBSofeachpairof
of treatment (analyzed by two-way ANOVA).
treatment(analyzedbytwo-wayANOVA).
2.7. VH02 Blocked New Blood Vessel Formation in Matrigel Plug in Vivo
2.7. VH02BlockedNewBloodVesselFormationinMatrigelPluginVivo
The in vivo antiangiogenic activity of VH02 was shown in a Matrigel plug mouse model. After
The invivo antiangiogenic activity of VH02 was shown in a Matrigel plug mouse model.
day 10, Matrigel plugs in the control group showed very sparse blood vessel formation. In contrast,
After day 10, Matrigel plugs in the control group showed very sparse blood vessel formation.
plugs supplemented with 1 μg VEGF demonstrated dramatically increased new blood vessel
Incontrast,plugssupplementedwith1µgVEGFdemonstrateddramaticallyincreasednewblood
formation marked by the dark red color in the plug (Figure 7A). Under VEGF stimulation, VH02 (5
μM) strongly reduced Matrigel plugs neovascularization by 12-fold, similar to the activity of
sorafenib (5 μM). The Matrigel plugs showed enhanced immunofluorescent stained for CD31 after
Molecules2016,21,1258 9of17
vessel formation marked by the dark red color in the plug (Figure 7A). Under VEGF stimulation,
MVHole0cu2le(s5 20µ1M6, 2)1s, t1r2o5n8 glyreducedMatrigelplugsneovascularizationby12-fold,similartotheac9ti ovfi 1ty7
of sorafenib (5 µM). The Matrigel plugs showed enhanced immunofluorescent stained for CD31
VEGF stimulation and were significantly blocked by VH02 or sorafenib (Figure 7B). There was a
afterVEGFstimulationandweresignificantlyblockedbyVH02orsorafenib(Figure7B).Therewas
significant interaction effect between VEGF stimulation (factor 1) and treatments with VH02 or
a significant interaction effect between VEGF stimulation (factor 1) and treatments with VH02 or
sorafenib (factor 2) on angiogenesis as detected by CD31 (F(2,12) = 28.12, p < 0.0001). Similarly, Akt
sorafenib (factor 2) on angiogenesis as detected by CD31 (F(2,12) = 28.12, p < 0.0001). Similarly,
phosphorylation was significantly reduced by VH02 treatment in the presence of VEGF. We also
Akt phosphorylation was significantly reduced by VH02 treatment in the presence of VEGF.
identified the pericyte-like cells within the plugs which were positively stained with anti-αSMA
We also identified the pericyte-like cells within the plugs which were positively stained with
antibodies.
anti-αSMAantibodies.

Figure7.Cont.
Molecules2016,21,1258 10of17
Molecules 2016, 21, 1258 10 of 17

Figure7. AntiangiogenesiseffectsofVH02Matrigelplugsinamousemodel. Matrigelplugswere
Figure 7. Antiangiogenesis effects of VH02 Matrigel plugs in a mouse model. Matrigel plugs were
implantedintothehidelimbsofmicewithvarioustreatments,includingvehicle(PBS),VEGF(1µg),
implanted into the hide limbs of mice with various treatments, including vehicle (PBS), VEGF (1 μg),
VH02(5µM),sorafenib(5µM),orcombinationasindicatedinthefigure.(A)Representativephotosof
VH02 (5 μM), sorafenib (5 μM), or combination as indicated in the figure. (A) Representative photos
Matrigelplugsfrommicewithdifferenttreatments;(B)Representativeimmunostainingoftransverse
of Matrigel plugs from mice with different treatments; (B) Representative immunostaining of
cryosections of Matrigel plugs assessed with antibodies against CD-31 (green), p-Akt (pink), and
transverse cryosections of Matrigel plugs assessed with antibodies against CD-31 (green), p-Akt
DAPI(blue);(C)RepresentativeimmunofluorescenceimagesofMatrigelplugsstainedwithantibodies
(pink), and DAPI (blue); (C) Representative immunofluorescence images of Matrigel plugs stained
against CD-31 and alpha-SMA. The cell nuclei were identified by Topro-3 iodide (blue) staining.
with antibodies against CD-31 and alpha-SMA. The cell nuclei were identified by Topro-3 iodide
Arrowsindicatemicrovessel-likeformation.Scalebars,250µm.Thequantificationofimmunostaining
(blue) staining. Arrows indicate microvessel-like formation. Scale bars, 250 μm. The quantification of
intensitywasperformedincomparisonwithvehicle-treatedgroups(PBS).Valuesaremean±SEMof
immunostaining intensity was performed in comparison with vehicle-treated groups (PBS). Values
atleastsixrandomlyselectedimagesfromeachplug.**p<0.01,***p<0.001comparedwithPBSof
are mean ± SEM of at least six randomly selected images from each plug. ** p < 0.01, ***; p < 0.001
eachpairoftreatment(analyzedbytwo-wayANOVA).
compared with PBS of each pair of treatment (analyzed by two-way ANOVA).
The pericyte-like cells were localized surrounding the endothelial cell layer. The coverage of
blood vessels with pericyte α-SMA was significantly decreased after treatment with VH02 when