VEGF-DIs the Strongest Angiogenic and LymphangiogenicEffector Among VEGFs Delivered Into Skeletal Muscle via Adenoviruses
Angiogenesis (bloodvessel growth), lymphangiogenesis (lymph system growth) are allintrinsicallyconnected with lymphedemaand share many of the same genes. Wehave several pages onboth processes.
April 24, 2003
Fromthe Department of Biotechnology and Molecular Medicine (T.T.R.,J.E.M., M.G., T.H., A.P., I.K., S.Y.-H.), A.I. Virtanen Institute,KuopioUniversity, Finland; National NMR facility (M.I.K., R.A.K.), A.I.VirtanenInstitute, Kuopio University, Finland; Ludwig Institute for CancerResearch (M.G.A.,S.A.S.), Royal Melbourne Hospital, Victoria, Australia; and theMolecular/CancerBiology Laboratory (K.A.), Haartman Institute, University of Helsinki,Finland.
Correspondenceto Seppo Ylä-Herttuala, MD, PhD, Departmentof Biotechnology and Molecular Medicine, A.I. Virtanen Institute,University ofKuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala@uku.fi
Optimalangiogenic and lymphangiogenic gene therapy requires knowledgeof the best growth factors for each purpose. We studied thetherapeutic potential of human vascular endothelial growth factor(VEGF)family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D aswell as aVEGFR-3–specific mutant (VEGF-C156S) usingadenoviralgene transfer in rabbit hindlimb skeletal muscle. Thesignificance ofproteolytic processing of VEGF-D was explored usingadenovirusesencoding either full-length or mature (NC)VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands,VEGF-A andVEGF-DNC,induced the strongest angiogenesis and vascular permeabilityeffectsas assessed by capillary vessel and perfusion measurements,modifiedMiles assay, and MRI. The most significant featureof angiogenesisinduced by both VEGF-A and VEGF-DNCwas a remarkable enlargement of microvessels with efficientrecruitmentof pericytes suggesting formation of arterioles orvenules. VEGF-Aalso moderately increased capillary density andcreated glomeruloidbodies, clusters of tortuous vessels, whereasVEGF-DNC–inducedangiogenesis was more diffuse. Vascular smoothmuscle cellproliferation occurred in regions withincreased plasma proteinextravasation, indicating that arteriogenesismay be promoted by VEGF-Aand VEGF-DNC.Full-length VEGF-C and VEGF-D inducedpredominantly and the selectiveVEGFR-3 ligand VEGF-C156Sexclusively lymphangiogenesis.Unlike angiogenesis, lymphangiogenesis was notdependent on nitricoxide. The VEGFR-1 ligand VEGF-B did notpromote either angiogenesisor lymphangiogenesis. Finally, we found apositive correlationbetween capillary size and vascularpermeability. This studycompares, for the first time, angiogenesis andlymphangiogenesisinduced by gene transfer of different humanVEGFs, and shows thatVEGF-D is the most potent member when deliveredvia an adenoviralvector into skeletal muscle.
Key Words: vascular permeability •magnetic resonance imaging• edema • pericyte • arteriogenesis
Angiogenesis(capillary growth), lymphangiogenesis (lymphatic vesselgrowth), arteriogenesis (enlargement of arteries), and vasculogenesis(in situ formation of blood vessels from vascular stemcells) arecrucial for normal embryonic development, growth, andtissue repair.Further understanding of these processes mayalso contribute to thetreatment of many disorders, such as cancer,tissue ischemia, andlymphedema. Vascular endothelial growth factors(VEGFs) are involvedin all types of vascular growth.1–6Currently, the human VEGF family consists of 5members: VEGF-A, -B,-C, -D, and placenta growth factor (PlGF),7–13which differ in their ability to bind to VEGF receptors thatareprimarily expressed in endothelial cells (ECs): VEGFR-1 (Flt-1),VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt-4), and neuropilin-1. VEGF-Abindsto VEGFR-1 and VEGFR-2 as well as to neuropilin-1, whereasPlGF andVEGF-B bind only to VEGFR-1 and neuropilin-1.1,14,15VEGF-C and VEGF-D are synthesized and secreted as largeprecursor formsthat are proteolytically processed into mature forms comprisingthecentral VEGF homology domain.16,17Unprocessed forms preferentially signal throughVEGFR-3, whereas onlythe mature forms efficiently trigger VEGFR-2signaling.16,17Both VEGF-C and -D have been suggested to bemainly lymphangiogenesisfactors; their angiogenic potential has beenreported to beconsiderably weaker than that of VEGF-A.6,13,16In addition to the naturally occurring forms, Joukovet al18have generated a mutant factor (VEGF-C156S) thatbinds toVEGFR-3 but not to VEGFR-2.
VEGFreceptors have distinct biological roles. VEGFR-2 is consideredtomediate most of the angiogenic, survival, and vascular permeabilityeffectsof VEGFs and effects on endothelial progenitor cells (EPCs),1,3,19whereas the role of VEGFR-1 is controversial. Moststudies indicatethat VEGFR-1 is mainly a decoy receptor existingalso as a solubleform, and that it may downregulate VEGFR-2–mediatedmitogenesis.1,19–21However, others have reported that the VEGFR-1ligand PlGF mobilizesEPCs, hematopoietic, and inflammatory cells,and that it may amplifyVEGFR-2–mediated effects.22There is extensive evidence that VEGFR-3–mediated signalingaloneis sufficient for the growth and maintenance oflymphaticvasculature.2,23,24
Nitricoxide (NO) is crucial in VEGF-A–mediated angiogenesis andvascular permeability,25but it is notknown whether NO is necessary forlymphangiogenesis.
Foroptimal gene therapy, it is essential to determine which VEGFfamily member is most suitable for angiogenesis/lymphangiogenesisina given tissue. To address this issue, we compared adenovirusesencodinghuman VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-C156S inrabbithindlimb skeletal muscle. The objective was to study thedirecteffects of these VEGFs on local blood and lymphatic vesselgrowth,blood perfusion, and vascular permeability in theinjected musclesand thus establish a basis for the selection ofmost suitable growthfactors for each purpose. In addition, thesignificance ofproteolytic processing of VEGF-D in vivo wasstudied by usingadenoviruses expressing both its full-length andmature (NC)forms. We further explored the effects of these growthfactors onvascular smooth muscle cells (SMCs) and pericytes, andevaluated therole of NO in vascular permeability, angiogenesis, andlymphangiogenesis induced by VEGF-D in vivo.
Material and Methods
Thesuperficial femoral artery of New Zealand White rabbits (n=66)was excised and two reentry arteries for collaterals nearthe kneejoint were ligated in order to induce collateral arterygrowth in thehindlimb as described.4The nonischemicthigh region lacking confounding endogenous upregulation ofVEGF-Aand VEGFR-226was used for the analysis ofthe effects induced by VEGFs. Seven days aftersurgery, animalsreceived intramuscular (IM) injections of humanclinical gradeadenoviruses (total dose 1011viral particles [vp])encoding human VEGF-A165,8VEGF-B167,10full-length VEGF-C,11full-length or mature(NC)VEGF-D,13or VEGF-C156S18(n=3 to 8 in each group). N-Nitro-L-argininemethyl ester (L-NAME, NO synthase inhibitor)27was used to study whether angiogenesis,lymphangiogenesis, andvascular permeability promoted either by thefull-length or matureform of VEGF-D are dependent on NO production.Transduced hindlimbswere monitored for vascular permeability andsubsequent edema withgadodiamide (GdDTPA-BMA)-contrastagent-enhanced T2*-weightedMRI (MRI)4,265 days after gene transfer (GT). Muscle perfusion andvascular permeabilitywere assessed quantitatively with fluorescent microspheres andamodified Miles assay, respectively, 6 days after GT.4Thereafter, muscle samples were collected for histologicalanalyses.4,26All animal experiments were approved by the ExperimentalAnimal Committee,University of Kuopio.
Forfurther details, see the expanded Materials and Methods intheonline data supplement available at http://www.circresaha.org.
VEGFR-2Ligands VEGF-A and VEGF-DNCInduce the Strongest Angiogenesis Effects in Skeletal Muscle
Effects of VEGFs were evaluated in nonischemic muscles in areasoutsidethe needle track in order to exclude the confounding effectsofendogenous growth factors induced by injection trauma orischemia.Control virus (1011 vp/mL) encoding the LacZmarker genedid not induce angiogenesis or significant inflammation inrabbitskeletal muscle (Figures 1a and 1c),whereas an angiogenesisresponse characterized by a remarkable microvesselenlargement wasinduced by both AdVEGF-A and AdVEGF-DNC(Figures 1b, 1d, and 1i).In AdVEGF-DNCtransduced muscles, the size of enlarged vesselssometimes exceededthat of surrounding skeletal myocytes. Adenovirusesencodingfull-length VEGF-C and VEGF-D also enlarged themicrovessels,although less than AdVEGF-A and AdVEGF-DNC(Figures 1f and 1h),whereas AdVEGF-B and AdVEGF-C156Shad no effects on blood vessels (Figures 1e and 1g).VEGFR-2 and vß3integrin were not detectable with immunostaining incapillaries ofintact and AdLacZ transduced muscles (data notshown). In contrast,both VEGF-A (data not shown) and VEGF-DNCupregulated VEGFR-2 and vß3integrin expression in the ECs (Figures 1jand 1k).
Interestingly,there were differences in the angiogenesis patterns promotedby AdVEGF-A and AdVEGF-DNC.The strongest effects after AdVEGF-A treatmentoccurred in connectivetissue between muscle bundles and in musclefascias, whereasangiogenesis stimulated by AdVEGF-DNCwas more diffuse (Figures 2b, 2c, 2f, and 2g).Furthermore,AdVEGF-A generated more glomeruloid bodies (clusters oftortuousvessels28)than AdVEGF-DNC(Figure 2b).
VEGFR-3Ligands VEGF-C, VEGF-C156S, and VEGF-D PromoteLymphangiogenesis
Histochemical staining of lymphatic endothelial cells for 5'nucleosidaseactivity29and an intraarterial injectionof Ricinus Communis lectin (data not shown)revealed that theCD31-positive but -SMA–negativevessels in the interstitial connective tissuebetween the musclebundles were lymphatics, not blood vessels (Figures2d through 2j).
Adenovirusencoding the full-length VEGF-D induced the strongest lymphaticvessel growth as almost all the interstitial connective tissueintransduced muscles was filled with lymphatic vasculature 6days afterGT (Figures 2d and 2h). AdVEGF-DNC,AdVEGF-C, and AdVEGF-C156Swere also potentlymphangiogenesis inducers (Figures 2g,2i, and 2j). Remarkably,the adenovirus encoding the VEGFR-3 selectivemutant VEGF-C156Sstimulated exclusively lymphangiogenesis, whereasAdVEGF-A (Figures2f and 2j) and AdVEGF-B (data not shown)had no effects onlymphatic vessels.
VEGF-Aand VEGF-DNCInduce Arteriogenesis
In AdLacZ muscles, only a portion of the capillarieshad -SMA–positivepericytes and there were few proliferating SMCs (Figures2k and 2o). In contrast, both inAdVEGF-A– and AdVEGF-DNC–transducedmuscles, nearly all enlarged capillaries had a complete oralmost complete-SMA–positive pericyte coverage,indicating that -SMAexpression was upregulated (Figures 2b,2c, and 2l).Furthermore, BrdU labeling showed a highproliferation rate of ECsand pericytes after AdVEGF-A (data not shown)and AdVEGF-DNCGT (Figure 2n). Inthese muscles, collateralarteries showed active remodeling with abundantnumbers ofproliferating SMCs (Figure 2p),especially inregions of plasma protein extravasation (see following sections).
AdVEGF-Aand AdVEGF-DNCIncrease Vascular Permeability and Cause Edema
Extensive vascular permeability and subsequent edema were observedinAdVEGF-A and AdVEGF-DNCtransduced muscles 5 days after GT by contrastagent–enhanced MRI (Figures3b and 3g). Extravasated contrastagent was detected under theskin, in the semimembranosus muscle and in itsfascias, and in thefat tissue between the medial and lateralmuscle compartments. Incontrast, AdLacZ or otherAdVEGFs did not induce significantvascular permeability effects (Figures 3a, 3c through3f, 4a, 4c, and 4e).The modified Miles assay demonstrated strongand quite diffusevascular permeability in AdVEGF-DNCtransduced muscles (Figure 4b).Microscopically, extravasatedplasma proteins were retained, especially in connective tissuebetween muscle bundles, around large blood vessels, as wellas withinmuscle fibers surrounded by enlarged, leaky microvessels (Figures4d and 4f).
Angiogenesisand Lymphangiogenesis Profiles of Adenovirally DeliveredVEGFs
A quantitative comparison of capillary density and mean area,totalcapillary area, plasma protein extravasation, regional muscleperfusion, and total lymphatic vessel area in the transducedmuscles6 days after GT gave an angiogenesis or lymphangiogenesis profileforeach VEGF. Surprisingly, only AdVEGF-A GT significantly increasedthecapillary density (Figure 5a).In contrast, AdVEGF-DNCenlarged the mean capillary area as much as 14-fold ascompared withAdLacZ control without inducing any increase in thecapillary number(Figures 5a and 5b). Thecorresponding increase in the meancapillary size was 6.4-fold with AdVEGF-A. Effects on microvesselenlargementreached a statistical significance also with AdVEGF-C andAdVEGF-Dwith 2.5- and 2.2-fold increases, respectively (Figure5b).
AdVEGF-DNCand AdVEGF-A GT resulted in dramatic 24- and 13-fold increases,respectively, in plasma protein extravasation as measuredby themodified Miles assay (Figure 5b).Although AdVEGF-C andAdVEGF-D had weak effects on capillary enlargement, they didnotsignificantly enhance plasma protein extravasation.
About6% and 8% of the AdVEGF-A– and AdVEGF-DNC–transducedmuscles were covered by microvessel lumens, respectively (Figure5c). These figures were much greaterthan in AdVEGF-C– (1.8%),AdVEGF-D– (1.7%), and AdLacZ-treated muscles (0.7%).Regionalperfusion was increased accordingly in the transduced muscles.AdVEGF-A,AdVEGF-DNC,and AdVEGF-C induced statistically significant4.0-, 3.2-, and2.0-fold increases in perfusion, respectively (Figure5c). As shown in Figure 5d,lymphatic vessel areaof muscles (%) was strongly increased with adenoviruses expressingthe VEGFR-3 ligands VEGF-C (1.5%), VEGF-C156S(1.3%), VEGF-D(2.6%), and VEGF-DNC(1.4%) as compared with AdLacZ (0.12%).
Finally,angiogenesis and lymphangiogenesis indices were calculated foreach VEGF. These indices illustrate the balance between bloodandlymphatic vessel growth (Figure 6).VEGF-A was the onlyto induce angiogenesis but not lymphangiogenesis, whereas full-lengthVEGF-C and VEGF-D were found to be mainly, and VEGF-C156S exclusively,lymphangiogenic. The mature form VEGF-DNCinduced significant growth of both vesseltypes. VEGF-B wasinefficient both for angiogenesis andlymphangiogenesis.
Angiogenesisbut not Lymphangiogenesis Is Dependent on NO Production
NO synthase inhibitor L-NAME significantly blocked capillaryenlargementand increases in vascular permeability after AdVEGF-DNCtreatment as shown in Figures3h and 7a.In contrast, NO synthase inhibition did notaffect lymphangiogenesisinduced by full-length VEGF-D (Figure 7b).
VascularPermeability Correlates With Microvessel Size
A positive correlation was found when the mean microvessel areacalculatedfrom each transduced muscle was plotted against the respectivevascular permeability ratio (Figure 7c).A positive correlationalso existed between the total capillary area and theregional muscleperfusion, indicating that the anatomical observationabout capillaryvessel growth is in line with a physiologicalmeasure of blood flow (Figure7d). However, the correlation wasbest with physiological (small)capillary sizes because microspheres (diameter15 µm) do not likelyget stuck in strongly enlarged capillaries(diameter >15 µm),which may underestimate perfusion values obtained with thismethod.
Weperformed GT of different VEGFs in the nonischemic thigh musclesin this rabbit model of collateral artery growth4because necrosis, inflammation, and expressionof endogenous growthfactors caused by ischemia26may maskdifferences between transduced growth factorsand make this kind ofcomparison impossible. Furthermore, therapiesdesigned foraugmentation of arteriogenesis should also beeffective innonischemic muscles upstream to ischemia, whichis usually the siteof collateral growth.
Whereasthis in vivo study supports the role for VEGFR-2 signaling asthe major regulator of angiogenesis and vascular permeability,ourdata suggest that VEGFR-1 signaling alone may not be capableofinitiating angiogenesis because its ligand VEGF-B was ineffective.Nevertheless,distinct histological characteristics were associated withAdVEGF-A–inducedangiogenesis in comparison with that obtainedwith AdVEGF-DNC.AdVEGF-A increased capillary density andinduced the formation ofglomeruloid bodies28more frequentlythan AdVEGF-DNC,which exerted more diffuse effects and stronglyenlarged thepreexisting microvessels. These findings mayrelate to the fact thatVEGF-A binds to VEGFR-1 and VEGFR-2,1whereasVEGF-DNCbinds to VEGFR-2 and VEGFR-3.13,17However, it is more likely that the differentaffinities of VEGF-Aand VEGF-DNCfor the extracellular matrix are responsible for these biologicaldifferences. In contrast to VEGF-A165, there isno heparan-bindingdomain in the sequence of VEGF-DNC.1,13Furthermore, the selective VEGFR-1 and VEGFR-3ligands, VEGF-B andVEGF-C156S, respectively,appeared inert toward bloodvessels. Nevertheless, the possibility ofsubtle interspeciesdifferences on receptor activation, which couldmodify the biologicaloutcomes, cannot be excluded because humanVEGFs were tested in therabbit. However, according to our previousstudies and this work,human and mouse VEGF-A and human VEGF-C haveall shown expectedpotency in rabbits.4,30Further studies are needed to clarify the role of VEGFR-1 inangiogenesisbecause PlGF, another VEGFR-1 ligand, is angiogenic atleast in somecircumstances.22
Asexpected from in vitro receptor binding profiles11,13,17,23and recent in vivo work,2we foundthat VEGFs activating VEGFR-3 inducedlymphangiogenesis in rabbitskeletal muscle. The counting of lymphaticvessels was done from CD31and -SMA double-immunostainedsectionsbecause of the possibility that molecular markers used forthedetection of lymphatic endothelium, such as VEGFR-3 andLYVE-1, mayalso be expressed on activated ECs of blood vesselsor may not beexpressed in all lymphatic ECs.6,23,31,32VEGF-C stimulated predominantly, and the VEGFR-3–specificmutantVEGF-C156S exclusively, lymphatic vessel growth.However, lymphangiogenesisinduced by the full-length VEGF-D was strongest asnearly all thespace between muscle bundles was filled with lymphaticsin AdVEGF-D–treatedmuscles. The ability of the processed formVEGF-DNCto induce substantially more angiogenesis andless lymphangiogenesisthan the full-length form can be explained bythe fact thatproteolytic processing increases its affinitytoward VEGFR-2 more(290-fold) than toward VEGFR-3 (40-fold).17Our data also suggest that, at least in nonischemic skeletalmuscle,VEGF-D is not efficiently cleaved by proteases. Takentogether, ourresults implicate that VEGF-C, VEGF-D, and VEGF-C156Scould be applicable in the treatment of lymphatic disorderssuch asprimary and secondary lymphedema.24
Bloodvessels formed in response to VEGF-A have been suggested tolack pericytes,33and it has been proposedthat because of this defect they are prone toregression when VEGF-Alevels are decreased.34However,our data demonstrate that the expanded vesselsinduced both by AdVEGF-Aand AdVEGF-DNCare accompanied by an -SMA–positivepericyte coverage, which, together with thelarge size (diameter upto 50 µm), suggests a shift from themidcapillary phenotype towardarterioles, venules, or possibly arteriovenousshunts. Nevertheless,in spite of the efficient pericyte recruitment,our recent studyshows that at least in nonischemic skeletalmuscle, where excessblood perfusion is not necessary, the enlargedcapillaries inducedby AdVEGF-A or AdFGF-4 regress after the termination of geneexpression.4To the best of our knowledge, it has not been shown thatVEGFs couldinduce SMC or pericyte proliferation in vivo andthus these actionsare likely indirect. Arterial SMCs and pericyteproliferationoccurred in areas of increased plasma proteinextravasation, whichsuggests that protein efflux from thevasculature to theextravascular space may contribute to theseprocesses.4For example, extravasation of plasma proteins triggersthe clottingcascade, leading to deposition of fibrin gel inthe interstitialspace. In addition to causing edema, thehydrated fibrin gel isproangiogenic because it provides matrix forcell migration andgrowth.35Additional growth factors thatare mitogenic for SMCs, such as PDGFs, may also be upregulated.Furthermore,increased shear stress in the enlarged microvessels andenhancedintegrin signaling36may further contributeto the transformation of capillaries towardbigger vessels.
TheNO synthase inhibitor L-NAME27significantly blocked AdVEGF-DNC–inducedvascular effects including capillary enlargement andextravasation ofplasma proteins, indicating that, like in the case of VEGF-A,19,25NO is crucial for blood vascular effects stimulated byVEGF-DNC.Furthermore, VEGFR-2 and vß3integrin were upregulated on ECs by VEGF-DNC,suggesting that its angiogenic signaling mechanismsare similar tothose of VEGF-A.1Furthermore, efficientangiogenesis and VEGFR-2 upregulation by its ligands innonischemic skeletalmuscle shows that ischemia is not requisite for blood vesselgrowth.In contrast to angiogenesis, inhibition of NOS didnot affectlymphangiogenesis. To the best of our knowledge, thisis the firstdemonstration that NO synthases are not crucial forlymphangiogenesis.Perivascular cells may be important for such aspecific requirementof NO in angiogenesis as blood vascular, butnot small lymphaticvessels, have a pericyte coverage.37
Wefound an interesting correlation between mean capillary sizeandplasma protein extravasation. VEGFR-2 ligands induce plasma proteinextravasation probably by multiple mechanisms. For example, theyincrease EC surface area, capillary blood flow and pressure,and alsohave direct effects on EC ultrastructure and vesicle transportation.38As shown in this study, cell proliferation isinvolved in capillaryenlargement. Thus, in addition to hemodynamic changesin enlargedmicrovessels, an increased number of intercellular cleftsbetweendividing ECs, and possibly decreased integrity ofthe basementmembrane may be of importance in vessel leakiness occurring5 to 6days after AdVEGF GT. In contrast to this kind ofvascularpermeability related to angiogenesis, ultrastructural changesandvesicle transport in ECs may be more important in acuteplasmaprotein leakage induced by VEGFs.4Ang-1 hasbeen reported to improve the EC barrierfunction to plasma proteins.39However, our data suggest that edema after VEGF GT could bereducedby hindering the excess enlargement of developing vessels. Ontheother hand, this may not be possible without the restrictionof ECand SMC proliferation, which would likely compromise the successoftherapeutic angiogenesis. Furthermore, as discussed above,increasedplasma protein extravasation may be essential forefficientangiogenesis35and arteriogenesis.
Ourfindings have several important implications. Firstly, they showthat at least in nonischemic skeletal muscle the angiogenic effectsof VEGF family members comprise microvessel enlargement, pericyterecruitment, and increases in vascular permeability, andnotnecessarily increases in capillary density. This relationshipbetweenangiogenesis and vascular permeability could be used forthe judgmentof successful gene transfer and for the evaluation ofedema afterclinical VEGF gene therapy with noninvasive imaging methodssuch asMRI or ultrasound. Secondly, unlike angiogenesis, lymphangiogenesisis not dependent on VEGFR-2 and NO. Most importantly, thisuniquecomparison of the biological effects generated by differenthumanVEGF family members in vivo provides a classification ofhuman VEGFsas blood or lymphatic vessel growth factors depending ontheirreceptor specificity. The VEGFR-1 ligand VEGF-B is notable totrigger either angiogenesis or lymphangiogenesis inrabbit skeletalmuscle, whereas VEGFR-2 and VEGFR-3 ligands arestrongly angiogenicand lymphangiogenic, respectively. In conclusion,VEGF-A, VEGF-C,VEGF-C156S, VEGF-D, and VEGF-DNChave potential as vascular therapeutics and the mostsuitable VEGFfor each treatment can be chosen based on the need for angiogenesisand/or lymphangiogenesis.
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