Helsinki University Biomedical Dissertations, No. 9

VEGFR-3 in primary lymphedema

Marika J. Kärkkäinen

Molecular/Cancer Biology LaboratoryHaartman Institute and Helsinki University Central Hospital

Biomedicum HelsinkiUniversity of Helsinki

Finland

ACADEMIC DISSERTATION

To be publicly discussed, with the permission of the Medical Faculty of the University of Helsinki,

In the lecture hall 2, Biomedicum Helsinki,Haartmaninkatu 8, Helsinki

On December 14th, 2001, at 9 a.m.

Helsinki 2001

SUPERVISOR

Kari Alitalo, MD, PhDResearch Professor of the Finnish Academy of SciencesMolecular/Cancer Biology Laboratory, Haartman Institute

University of HelsinkiFinland

REVIEWERS

Sirpa Jalkanen, MD, PhDProfessor

MedCity Research Laboratory, TurkuUniversity and National Public Health Institute

Department in TurkuFinland

And

Juha Partanen, PhDDocent

Institute of BiotechnologyUniversity of Helsinki

Finland

OPPONENT

Peter Carmeliet, MD, PhDProfessor of Medicine

The Center for Transgene Technology & Gene TherapyFlanders Interuniversity Institute for Biotechnology

University of LeuvenBelgium

ISBN 952-10-0235-2 (nid.)ISBN 952-10-0236-0 (PDF)

ISSN 1457-8433Multiprint Oy

HELSINKI 2001

You are never given a wish

without also being given

the power to make it come true.

- You may have to work for it, however.

-Richard Bach , Illusions

1

CONTENTSABBREVIATIONS......................................................................................................2

LIST OF ORIGINAL PUBLICATIONS......................................................................3

ABSTRACT................................................................................................................4

REVIEW OF THE LITERATURE ..............................................................................5

1 Development of the circulatory system.............................................................51.1 Vasculogenesis and angiogenesis .............................................................................. 51.2 Growth factors and receptors involved in angiogenesis ............................................. 6

1.2.1 Mechanisms of receptor tyrosine kinase signaling ........................................................61.2.2 VEGFs and their receptors .............................................................................................71.2.3 Angiopoietins and Tie receptors...................................................................................101.2.4 Ephrins..........................................................................................................................11

2 Lymphangiogenesis ...........................................................................................122.1 Formation and function of the lymphatic system ...................................................... 122.2 VEGFR-3 and its ligands in lymphangiogenesis....................................................... 132.3 Other lymphatic endothelial specific factors.............................................................. 15

2.3.1 Prospero-related homeobox protein 1 (Prox1).............................................................152.3.2 Podoplanin ....................................................................................................................162.3.3 Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)...................................16

3 Diseases associated with RTK dysfunction ...................................................173.1 Lymphedema.............................................................................................................. 17

3.1.1 Pathophysiology of lymphedema .................................................................................173.1.2 Classification of lymphedema.......................................................................................173.1.3 Genetic alterations in lymphedema..............................................................................18

3.2 Inactivating RTK mutations in human syndromes .................................................... 20

4 Gene therapy .......................................................................................................214.1 Vectors and approaches............................................................................................ 214.2 Gene therapy in ECs.................................................................................................. 22

AIMS OF THIS STUDY............................................................................................24

MATERIALS AND METHODS................................................................................25

RESULTS AND DISCUSSION................................................................................27

1 The VEGFR3 genomic structure and regulatory region (I) ...........................27

2 Analysis of the lymphedema-linked mutant VEGFR-3s (II, III) .....................282.1 Dominant negative effect of the mutant VEGFR-3s.................................................. 282.2 Interaction of VEGFR-3 with other pathways............................................................ 30

3 The Chy mouse model for human primary lymphedema (IV).......................31

4 Lymphedema therapy in the Chy model (IV)...................................................32

CONCLUDING REMARKS .....................................................................................35

ACKNOWLEDGEMENTS .......................................................................................36

REFERENCES.........................................................................................................37

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ABBREVIATIONSAAV adeno-associated virusABL transforming gene of the Abelson murine leukaemia virusAd adenovirusAng angiopoietinATP adenosine triphosphateCAR coxackie/adenovirus receptorcDNA complementary deoxyribonucleic acidCTFR cystic fibrosis transmembrane regulatorCUB complement-binding domainDC dendritic cellE embryonic dayEC endothelial cellECM extracellular matrixEDA-ID ectodermal dysplasia with immunodeficiencyEgr-1 early growth response factor 1ELC Ebstein-Barr virus-induced ligand for CCR7ENU ethylnitrosoureaFGFR fibroblast growth factor receptorFIGF c-fos-induced growth factorFLK1 fetal liver kinase 1 (mVEGFR-2)FLT1 fms-like tyrosine kinase 1 (VEGFR-1)FLT4 fms-like tyrosine kinase 4 (VEGFR-3)FOXC2 forkhead box C2GDNF glial-derived neurothrophic factorGIST gastrointestinal stromal tumorHA hyaluronanHR homology regionICAM intercellular adhesion molecule 1Ig immunoglobulinIHC immunohistochemistryJM juxtamembranekD kilodaltonKDR kinase insert domain containing receptor (hVEGFR-2)KO knockoutKIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologKL KIT ligandLEC lymphatic endothelial cellLYVE-1 lymphatic vessel endothelial hyaluronan receptor 1MAM meprin, A5, µMAPK mitogen-activated protein kinasesMEN multiple endocrine neoplasiaMET hepatocyte growth factor receptorMFH-1 mesenchyme fork head 1MIM Mendelian inheritance in manMRI magnetic resonance imagingmRNA messenger RNANRP neuropilinPECAM-1 platelet endothelial cell adhesion molecule 1PDGF platelet-derived growth factorPlGF placenta growth factorProx1 prospero-related homeobox protein 1PTB phosphotyrosine bindingRET rearranged during transformationRTK receptor tyrosine kinaseSema semaphorinSH Src homologySLC secondary lymphoid organ chemokineTek tunica interna endothelial cell kinase (Tie2)Tie tyrosine kinase with Ig and epidermal growth factor homology domainsVEGF vascular endothelial growth factorVEGFR VEGF receptorVPF vascular permeability factor (VEGF)VRF VEGF-related factor (VEGF-B)VRP VEGF-related protein (VEGF-C)WT wild-type

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original articles, which are referred to by theirroman numerals.

I

Iljin K, Karkkainen MJ, Lawrence EC, Kimak MA, Uutela M, Taipale J, Pajusola K,Alhonen L, Halmekytö M, Finegold DN, Ferrell RE, and Alitalo K. (2001) VEGFR3gene structure, regulatory region and sequence polymorphisms, FASEB J. 15,1028-1036.

II

Karkkainen MJ, Ferrell RE, Lawrence EC, Kimak MA, Levinson KL, McTigue MA,Alitalo K, and Finegold DN. (2000) Missense mutations interfere with vascularendothelial growth factor receptor-3 signalling in primary lymphoedema. Nat.Genet. 25, 153-159.

IIIIrrthum A*, Karkkainen MJ*, Devriendt K, Alitalo K, and Vikkula M. (2000)Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3tyrosine kinase. Am. J. Hum. Genet. 67, 295-301.

IV

Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, BuelerH, Eichmann A, Kauppinen R, Kettunen MI, Ylä-Herttuala S, Finegold DN, FerrellRE, and Alitalo K. (2001) A model for gene therapy of human hereditarylymphedema. Proc. Natl Acad. Sci. USA 98, 12677-12682.

*) equal contribution

The original articles are reprinted with the permission of the publishers.

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ABSTRACT

The discovery of the first lymphaticendothelial specific receptor, vascularendothelial growth factor receptor-3(VEGFR-3) has facilitated studies onlymphatic vessel formation andregulation. According to recent studies,VEGFR-3 is required for thedevelopment of the blood vasculatureduring embryogenesis. However,VEGFR-3 becomes restricted to thelymphatic endothelium, where itssignaling mediates proliferation,survival and migration of lymphaticendothelial cells (LECs). VEGFR-3 is areceptor for two lymphangiogenicgrowth factors, VEGF-C and VEGF-D.

Abnormal lymphat ic vesseldevelopment and function areassociated with human lymphedema.Lymphedema is characterized bydisfiguring and disabling swelling of thelimbs due to defective lymphaticdrainage. The molecular pathogenesisof various lymphedema phenotypeshas been unclear, but recently,missense mutations within theVEGFR3 region were linked to primarylymphedema. This study presentsfunctional characteristics of thelymphedema-associated mutantVEGFR-3s. In addition, a possibletreatment for lymphedema, withVEGFR-3 as a target, is described.

We show here that missensemutations of the VEGFR3 gene resultin lymphedema in several families.During in vitro analysis of the mutantreceptors, all disease-associatedVEGFR3 alleles were found to encodetyrosine kinase inactive proteins. Theability of the mutant receptors to inducedownstream gene activation was

defective, and the mutant moleculeshad longer cellular half-lives. Theseresults suggest that the mutantVEGFR-3s interfere with the wild-type(WT) receptor signaling and function ina dominant negative manner,consistent with the autosomal dominantinheritance of primary lymphedema.Thus, heterozygous loss of VEGFR-3tyrosine kinase activity results inhuman primary lymphedema.

Because the mutations withinVEGFR3 are likely to impair severalcellular processes, we have analysedthe pathogenesis of lymphedema usingthe Chy mouse model. As in humanlymphedema patients, Chy mice havean inactivating Vegfr3 mutation in theirgermline, and swelling of the limbs dueto hypoplastic cutaneous lymphatics.Lymphedema is the result of aheterozygous genotype both in humansand in the Chy mice, and thus thereremains some VEGFR-3 activity due tothe functional WT allele. We thereforeexplored the possibility of usingVEGF-C ligand overexpression asa therapeutic tool for primarylymphedema. In these studies,VEGF-C gene therapy induced growthof functional cutaneous lymphaticvessels in the Chy mice.

As a conclusion, this studyestablishes the importance ofVEGFR-3 for normal lymphaticvascular development, and thatmutations interfering with VEGFR-3signal transduction are a cause ofprimary lymphedema. Our results alsosuggest that VEGF-C gene therapymay be applicable to treatment ofhuman lymphedema.

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REVIEW OF THE LITERATURE

1 Development of the circulatory system

1.1 Vasculogenesis and angiogenesis

The need for oxygen and nutrients in abody is supplied by the blood vessels.The lymphatic vessels collect theextravasated protein-rich tissue fluid,filter it through the lymph nodes andreturn it back to the circulation (Fig. 1).

The blood vasculature forms in acoordinated manner through acombination of vasculogenesis andangiogenesis, and supports the growthof the developing embryo. In the yolksac, the mesoderm-derived precursors,hemangioblasts, form blood islands,and give rise to both endothelial andhematopoietic lineages (Fig. 2). Invasculogenesis, the endothelial cells(ECs) proliferate, differentiate andassemble to form the early vascularplexus (Carmeliet and Collen, 1999;Flamme et al., 1997). In addition to theextraembryonic formation of the

vasculature, hematopoiesis andvasculogenesis also take placeintraembryonically, and the bloodvessels differentiating in the embryoare connected to the yolk sac byvitelline vessels.

After the development of theprimary vascular plexus, the maturevasculature forms by sprouting andremodeling from pre-existing vessels ina process called angiogenesis(Carmeliet, 2000; Risau, 1997). Themature vasculature consists of acomplex organized network assembledto arteries, capillaries, and veins.Ephrins and their receptors areinvolved in the patterning of bloodvessels from the earliest stages ofangiogenesis (Gale and Yancopoulos,1999; Wang et al., 1998).

Figure 1. Circulatory system. A schematic drawing of the circulatory system indicating theextravasation of fluid and macromolecules from the blood vessels, and their absorbtion into thelymphatic vessels. (Modified from Jones et al., 2001; Karkkainen et al., 2001)

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Angiogenic sprout ing requiresprogrammed proteolytic activity toselectively dissolve the extracellularmatr ix (ECM). The basementmembrane and ECM are degraded byco-operation between adhesive andproteolytic mechanisms (Reviewed inWerb, 1997). For example theplasminogen-plasmin system andmatrix metalloproteinases are involvedin the break-down of many ECM andbasement membrane proteins.Migration of ECs is also facilitated byintegrins, which are ECM receptorswith distinct cellular and adhesivespecificities. Sprouting EC tubes fuseand coalesce into loops, allowing bloodcirculation in the newly vascularizedregion.

Non-sprouting angiogenesis is aprocess whereby pre-existing vesselsare split by transcapillary pillars of ECM(Risau, 1997). In vivo this can occurwhen ECs inside a vessel proliferate,producing a wide lumen that can besplit by transcapillary pillars.

In the course of maturation, thenewly formed vessels are supported bya basement membrane and a pericytelayer and thus, become less vulnerableto vessel regression (Benjamin et al.,1998). The ECs themselves alsosecrete growth factors, such asplatelet-derived growth factor (PDGF)-B, which are important in therecruitment of the supporting pericytes.

It has also been suggested thatcirculating endothelial precursor cellsparticipate in angiogenesis in adults(Shi et al., 1998). These cells areimmobilized after vascular trauma,possibly due to the elevated circulatinglevels of VEGF, further supporting theidea that the endothelial precursors arealso involved in neo-angiogenesis (Gillet al., 2001; Takahashi et al., 1999).Recent findings also suggest that apopulation of VEGFR-2 positive cellsmay give rise to cells of bothendothelial and smooth muscle cell(SMC) lineages (Yamashita et al.,2000).

Figure 2. Differentiation of endothelial and hematopoietic cells. The endothelial and hematopoieticcells are derived from a common precursor, the hemangioblast. The hemangioblasts aggregate and thecells in the interior become hematopoietic cells, whereas the cells in the periphery differentiate intoprimitive ECs, angioblasts. (Modified from Eichmann et al., 2000)

1.2 Growth factors and receptors involved in angiogenesis

1.2.1 Mechanisms of receptor

tyrosine kinase signaling

Targeted EC signaling is achieved byspecific receptor tyrosine kinases(RTKs), which mediate growth factorsignals from the extracellular space tothe cytosol and finally to the nucleus ofthe target cell. RTKs are classified into

subclasses based on sequencesimilarities and distinct structuralcharacteristics (van der Geer et al.,1994). However, the overall basicstructure of most RTKs is very similar.They contain a l igand-bindingextracellular part, a single membranespanning region, and a cytosolicdomain for signal transduction. A

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juxtamembrane (JM) region precedingthe catalytic domain is located on thecytoplasmic side, followed by theconserved kinase domain thatcatalyzes the transfer of the γ-phosphate of ATP to tyrosine residuesof protein substrates.

C r y s t a l i z a t i o n o f t h eunphosphorylated RTKs have revealedmolecular mechanisms by which RTKsare kept in an unphosphorylated stage(Reviewed in Hubbard et al., 1998).The activation loop of the kinase seemsto be autoinhibitory in a monomericreceptor , p revent ing l igand-independent phosphorylation. Recentreports have also elucidated animportant role of the JM domain ininhibition of RTK autophosphorylation(Huse et al., 2001; Wybenga-Groot etal., 2001).

Most known ligands for RTKs aresecreted soluble proteins. In the caseof VEGFs, the dimeric ligand binds tothe extracellular part of the receptor,resulting in a symmetric receptor dimer(Wiesmann et al., 1997). Dimerizationis followed by transphosphorylation ofthe tyrosine recidues in the activationloop, which stabilizes an active state ofthe kinase via a conformation favorablefor catalysis (Hubbard et al., 1998;Schlessinger, 2000).

Receptor phosphorylation generatesdocking sites for downstream signalingproteins. The phosphorylated tyrosineresidues and their adjacent sequencesact as specific activation sites fordownstream signal ing proteinscontaining the Src homology 2 (SH2) orphosphotyros ine-b inding (PTB)domains. Further activation of multiples ignal t ransduct ion cascadeseventually leads to biochemicalchanges in the cell, such asreadjustment of gene expression.

1.2.2 VEGFs and their receptors

VEGF family members and theirreceptors are critical regulators of bothangiogenesis and lymphangiogenesis(Table 1) (Reviewed in Veikkola et al.,2000). The importance of the parentmolecule VEGF in normal angiogenesishas been implicated as inactivation ofeven a single Vegf allele resulted inlethality between E11-12 (Carmeliet etal., 1996; Ferrara et al., 1996). TheVegf+/- mice were growth retarded andexhibited a number of developmentalanomalies. This phenotype appears tobe due to gene dosage and it is the firstcase where the loss of a single allele islethal.

VEGF family members are secretedglycoproteins that form either disulfide-linked or non-covalently bound dimers,whose subunits are arranged in anantiparallel manner (Muller et al.,1997). All the VEGF family memberscontain a VEGF homology domain witheight conserved cysteine residues.

Although VEGFs form structurally aunified family, they differ in theirexpression pat terns, receptorspecificities and biological activities.More diversity within the family isobtained by alternative splicing. Spliceisoforms of VEGF, placenta growthfactor (PlGF), and VEGF-B differ intheir ability to bind heparin and in theirtissue distribution. For example,postnatal myocardial angiogenesis isimpaired in mice expressing only theVEGF1 2 0 isoform, suggesting thatspecific isoforms may be important foran optimal angiogenic response in vivo(Carmeliet et al., 1999). Biologicalfunction of VEGFs is also affected byheterodimerization and by proteolyticprocessing (Cao et al., 1996; DiSalvoet al., 1995; Joukov et al., 1997;Olofsson et al., 1996; Stacker et al.,1999).

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Table 1. VEGF family membersGrowth factor Also known as ReferenceVEGF vascular permeability factor, VPF (Senger et al., 1983)

(Ferrara and Henzel, 1989)(Plouet et al., 1989)

PlGF (Maglione et al., 1991)

VEGF-B VEGF-related factor, VRF (Olofsson et al., 1996)(Grimmond et al., 1996)

VEGF-C VEGF-related protein, VRP (Joukov et al., 1996)(Lee et al., 1996)

VEGF-D c-fos-induced growth factor, FIGF (Orlandini et al., 1996)(Yamada et al., 1997)

NZ2-VEGFNZ7-VEGF

VEGF-Es, ORF-VEGFs(viral VEGF homologues)

(Ogawa et al., 1998)(Wise et al., 1999)(Meyer et al., 1999)

To date, two receptor families,VEGFRs and neuropilins (NRPs), havebeen found to bind VEGFs. VEGFRsare tyrosine kinase receptors, whichare expressed predominantly on thesurface of ECs. The three knownmembers of this family (VEGFR-1-3) allcontain seven extracellular Ig-homology domains, of which the 2n d

and 3rd are necessary for ligand bindingand specificity (Fig. 3) (Davis-Smyth etal., 1996; Fuh et al., 1998; Galland etal., 1993; Matthews et al., 1991;Mäkinen et al., 2001; Pajusola et al.,1992; Shibuya et al., 1990; Terman etal., 1991; Wiesmann et al., 1997). Theintracellular catalytic kinase domain ofVEGFRs is split by a kinase insert.

VEGFR-2 (KDR/Flk1) is a keymarker of hemangioblasts andangioblasts, but its expression isdownregulated in hematopoietic cells.The crucial role of VEGFR-2 for normalvasculogenesis has been shown, asVegfr2-/- embryos fail to develop bloodislands and embryonic vasculature, anddie in utero between embryonic days(E) 8.5-9.5 (Shalaby et al., 1995).Furthermore, analysis of chimeric miceshowed that Vegfr2 is required cellautonomously for both extraembryonicand intraembryonic EC developmentand hematopoiesis (Shalaby et al.,1997). The interaction of VEGF withVEGFR-2 seems to be a criticalrequirement for the full spectrum ofVEGF induced biological responses.Receptor selective VEGF mutantswhich bind only to VEGFR-2 are fully

active EC mitogens, whereas themutants binding only to the VEGFR-1have a substantially reduced ability topromote EC growth (Keyt et al., 1996).In addition, the virus-encoded ORF-VEGFs bind to VEGFR-2 but not toVEGFR-1, and they are capable ininducing EC proliferation, migration andpermeability (Meyer et al., 1999;Ogawa et al., 1998; Wise et al., 1999).EC survival is also mediated viaVEGFR-2 (Gerber et al., 1998).

VEGFR-1 (Flt1) as a weak signalingreceptor, seems to act in concert withVEGFR-2 to negatively regulatephysiological angiogenesis. In mouseembryos, VEGFR-1 deficiency resultsin increased proliferation of endothelialprecursors, whereas EC differentiationis normal (Fong et al., 1995; Fong etal., 1999). Surprisingly, mice lackingthe VEGFR-1 tyrosine kinase domainappeared normal (Hiratsuka et al.,1998). O n l y V E G F - i n d u c e dmacrophage migration was suppressedin these mice, suggesting that VEGFR-1 signaling is not required forvasculogenesis, and that VEGFR-1may play a role in inhibition of ECproliferation. However, VEGFR-1 mayhave an important role in pathologicalangiogenesis. VEGFR-1 is upregulatedby hypoxia at the transcriptional level(Gerber et al., 1997). In addition, It hasbeen shown that PlGF, which bindsonly to VEGFR-1, is required forpathological angiogenesis (Carmeliet etal., 2001).

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Figure 3. Binding specificity of VEGF family members to the VEGFRs. (Achen et al., 1998; DeVries et al., 1992; Joukov et al., 1996; Meyer et al., 1999; Ogawa et al., 1998; Olofsson et al., 1998;Park et al., 1994; Terman et al., 1992; Wise et al., 1999)

VEGFR-1 ligands also act aschemoattractants for cells of themonocyte/macrophage lineage, whichexpress VEGFR-1 (Barleon et al.,1996). VEGFR-1 signaling is requiredfor migration of these cells, andinflammatory cells play an importantrole in pathological angiogenesis andcollateral vessel growth (Hiratsuka etal., 1998). Recently, it was also shownthat VEGF administration enhances theformation of atherosclerotic plaques inan experimental setting (Celletti et al.,2001). This effect may occur viamonocyte/macrophage recruitmentfrom the bone marrow.

NRPs were characterized asreceptors for semaphorins, which havea key role in mediating axonal guidanceduring neuronal development (Chen etal., 1997; He and Tessier-Lavigne,1997; Kolodkin et al., 1997). However,it was demonstrated that in addition tonormal neural development, NRP1 iscrusial for the formation of theembryonic vasculature (Kawasaki etal., 1999; Kitsukawa et al., 1995). Incontrast, mice with a targeted Nrp2

deletion are viable until adulthood(Chen et al., 2000; Giger et al., 2000),despite several problems in theperipheral and central nervoussystems, indicative of a defective axonguidance response.

NRPs have the ability to bindvarious VEGFs in an isoform-specificmanner (Fig. 4) (Gluzman-Poltorak etal., 2000; Migdal et al., 1998; Mäkinenet al., 1999; Soker et al., 1998). It hasalso been demonstrated that NRP-1has a functional role in VEGF/VEGFRsignaling, as it enhances VEGF1 6 5

binding to VEGFR-2, and the VEGFR-2mediated chemotactic response of ECs(Soker et al., 1998). The extracellulardomain of NRP-1 also binds with highaffinity to immunoglobulin homologydomains 3 and 4 of VEGFR-1 (Fuh etal., 2000). In addition, a naturallyoccurring soluble form of NRP-1 bindsVEGF165 and shows antitumor activityin an experimental rat prostatecarcinoma model, possibly byantagonizing VEGF165 binding to its cellsurface receptors (Gagnon et al.,2000).

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Figure 4. NRPs selectively bind members of the semaphorin and VEGF families. NRPs aretransmembrane proteins containing two CUB (complement-binding) domains (ovals), two coagulationfactor (V/VIII) domains (boxes), and a MAM (meprin, A5, µ) domain (arrow head) in the extracellularpart, a single transmembrane domain, and a short cytoplasmic tail.

It seems that the cytoplasmic tails ofNRPs do not possess signalingcapabilities but, instead, NRPs utilizeother receptors in signal transduction.For example plexins, which themselvesdo not bind secreted semaphorins,form complexes with NRPs (Takahashiet al., 1999; Tamagnone et al., 1999).The plexin-1/NRP-1 complex has ahigher affinity to Sema3A than NRP-1alone, and the intracellular signaling istransmitted via this complex andactivation of a plexin-associatedtyrosine kinase. Thus, also in ECsNRPs may signal by formingcomplexes with VEGFRs.

In addition to the cell surfacereceptors for VEGFs, naturallyoccurring, soluble forms of VEGFR-1,NRP-1, and NRP-2 have been found(Gagnon et al., 2000; Kendall andThomas, 1993; Rossignol et al., 2000).By inhibiting ligand binding to themembrane receptors, these solublemolecules may also act as naturallyoccurring regulators of the angiogenicprocesses. Whether addit ionalregulation is achieved by VEGFRheterodimer formation, remains to beseen.

1.2.3 Angiopoietins and Tie

receptors

Tie receptors (tyrosine kinase with Igand EGF homology domains) areanother family of EC specific RTKs. Ingene targeting studies, Tie1 andTie2/Tek (tunica interna endothelial cellkinase) have been shown to havedistinct roles in vascular development(Jones et al., 2001). The orphanreceptor Tie1 is needed for thestructural integrity of blood vascularECs, and its deficiency results inoedema and localized hemorrhaging inembryos (Puri et al., 1995; Sato et al.,1995). Signaling via the Tek receptor isparticularly important in angiogenicsprouting, vessel remodeling andmaturation (Dumont et al., 1994; Satoet al., 1995).

Interestingly, Tek has two ligandswith dual actions, angiopoietin (Ang) 1acts as an agonist for Tek, whereasAng2 usually functions as a naturallyoccurring antagonist (Maisonpierre etal., 1997; Teichert-Kuliszewska et al.,2001). This is supported by the findingthat Tek promoter driven Ang2overexpression in transgenic miceresults in a phenotype similar to that of

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mice deficient in Ang1 or Tek(Maisonpierre et al., 1997; Suri et al.,1996).

VEGF and angiopoietins act in a co-operative and coordinated manner inthe formation and maintenance ofvessels. Whereas VEGF is thought topromote EC proliferation, differentiationand migration, angiopoietin signalingvia Tek mediates vessel maturationand EC interaction with the supportingcells. Recent data suggest that Ang1can inhibit vascular permeability andstabilize existing vessels. Transgenicoverexpression of Ang1 in mouse skinresults in enlarged vessels at normaldensity due to circumferential growth,whereas the dermal microvesselsinduced by VEGF in the same modelwere numerous, tortuous, and leaky(Detmar et al., 1998; Suri et al., 1998).Surprisingly, mice coexpressing bothAng1 and VEGF in the skin showedenlarged and more numerous, butleakage-resistant vessels, suggestingthat Ang1 is capable of inhibiting theVEGF induced permeability (Thurstonet al., 1999). Similar results wereobtained when adenoviral VEGF and/orAng1 were intravenously administeredto mice (Thurston et al., 2000).

In vessels undergoing activeremodeling, the stabilizing function ofAng1 is overcome by an excess ofAng2 expression. It has beenspeculated that Ang2 is able to triggerdetachment of pericytes from the ECs,which would then be more vulnerableto an angiogenic stimulus, for exampleVEGF (Gerber et al., 1999). Indeed,Ang2 expression precedes that ofVEGF at sites of active vessel growthin adults, for example during thevascular remodeling in the ovary and intumors (Holash et al., 1999;Maisonpierre et al., 1997; Stratmann etal., 1998). Surprisingly, recent datasuggest that Ang2 has a role in

lymphatic development. The Ang2knockout (KO) mice, exhibit chylousascites, with defects in the patterningand function of the lymphaticvasculature (G Thurston, C Suri, GYancopoulos, personal comm.).

1.2.4 Ephrins

During vascular network formation,blood vessels assemble to form ahierarchic pattern of arteries and veins,connected by a capillary network.Ephrins and their receptors are aninteresting ligand/receptor system,which has a role in defining thearterious/venous patterning of thevessels, and in the EC/matrixinteractions. A special feature of thissystem is that tyrosine kinase receptorsbind multimeric, membrane-boundligands, resulting in a bidirectionalsignaling in the interacting cells(Reviewed in Schmucker and Zipursky,2001). It has been speculated that theephrin-B2/EphB4 signaling may providea repulsive effect in two adjacent celltypes, as ephrin-B2 and its receptorEphB4 differentially mark the ECs ofarteries and veins, respectively (Wanget al., 1998). It also seems that otherephrins and Eph receptors are involvedin the development of the vasculature(Reviewed in Gale and Yancopoulos,1999).

Surprisingly, mice lacking ephrin-B2or EphB4 resemble mice lacking eitherAng1 or Tek, with aberrant vesselremodeling, sprouting and hearttrabeculation (Adams et al., 1999;Gerety et al., 1999; Wang et al., 1998).These results suggest that theAng1/Tek and ephrin-B2/EphB4signaling pathways may interact.

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2 Lymphangiogenesis

2.1 Formation and function of the lymphatic system

The circulatory system, formed by theheart and blood vessels, requires theadjacent lymphatic system to collectextravasated protein-rich fluid andlymphocytes from the tissues, andtransport them back to the bloodcirculation. These two systemscommunicate with each other and arein t imate ly assoc ia ted dur ingdevelopment.

The deve lopment o f thecardiovascular system precedes thedevelopment of the lymphatic system.One theory postulates that the fetallymphatic sacs are formed from thelarge central veins in the jugular andperimesonephric regions (Sabin, 1912;Sabin, 1902). The lymphatic vesselsthen arise from these sacs by sproutingtowards the peripheral regions. Indeed,the two lymphatic specific markers,VEGFR-3 and Prox1, are expressed atthe sites for the suggested lymphaticsacs during mouse development(Dumont et al., 1998; Kaipainen et al.,1995; Wigle and Oliver, 1999). Alllymphatic vessel links to the venoussystem are disconnected, except forthe region of the left thoracic duct thatdrains the lymph into the subclavianvein.

In contrast to sprouting from pre-existing veins, another theory suggeststhat the lymphatic vessels develop byin si tu d i f f e ren t ia t i on f rommesenchymal cells in different tissues(Huntington and McClure, 1908;Kampmeier, 1912), or by a combinationof sprouting and in situ formation (vander Jagt, 1932). It has recently beens h o w n t h a t m e s o d e r m a llymphangioblasts participate in thedevelopment of the avian lymphaticsystem, supporting the theory that theperipheral lymphatic vessels developby multiple mechanisms (Schneider et

al., 1999; Wilting et al., 2000; Wilting eta l . , 2000). Whe the r theselymphangioblasts also participate inlymphangiogenesis in adults, remainsto be seen.

The lymphatic vessels collect theinterstitial fluid and transport it back tothe blood. Fluid and macromoleculesfrom the stromal compartment enter theinitial lymphatic sinuses and lymphaticcapillaries, which consist of a single,flat EC layer surrounded by anincomplete basement membrane. TheECs of the lymphatic capillaries alsohave large inter-endothelial gaps,which facilitate the trafficking ofmacromolecules. From the capillaries,the fluid is transferred to the collectinglymphatic vessels, and ultimately intothe venous circulation via the thoracicduct. Larger lymphatic vessels aresurrounded by a muscular layer thatcontracts automatically when thelymphatic vessel becomes stretchedwith fluid. In addition, external factorssuch as skeletal muscle movementsand arterial pulsation compress thelymphatic vessels and increase theefficiency of fluid transfer (Guyton,1991). The LECs are attached to thesurrounding connective tissue byanchoring filaments. Furthermore, theluminal side the lymphatic vessels issegmented by valves, which preventlymphatic back-flow.

In addition to fluid transport, thelymphatic system also has an importantrole in immunological responses.Lymphatic vessels take up any foreignmolecules and leukocytes which havegained access to the tissue spaces.The lymph is filtered through the lymphnodes, which trap various antigens andattract antigen presenting cells, such asdendritic cells (DCs) in various epitheliaor Langerhans cells in the epidermis.

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These cells monitor the extracellularenvironment, detect antigens, andtrigger T cell activation (Rescigno andBorrow, 2001). In f lammatorychemokines recruit DC precursors andmediate DC activation. The DCs thenmigrate from the periphery to the lymphnodes via the lymphatic vessels.Activated DCs upregulate the cytokinereceptor CCR7 and become sensitiveto its ligands SLC (secondary lymphoidtissue chemokine) and ELC (Ebstein-Barr virus-induced ligand for CCR7),which are expressed for example in thehigh endothelial venules, and in the Tcell rich areas of lymph nodes,respectively (Cyster, 1999). Also otherspecific chemokines and chemokinereceptors are involved in cell trafficking

in the lymphatic system. For examplethe β-chemokine receptor D6 isexpressed in the skin and intestinallymphatic vessels and in the lymphnodes, suggesting that this receptormay have a role in cell trafficking inthese organs (Nibbs et al., 2001).

As metastasis to the regional lymphnodes occurs via the lymphatic vessels,recent areas of interest regarding theLECs are their interactions with tumorcells, the transfer of cells through theendothelium and their entry into thelymph nodes. Knowledge of themechanisms regulating cell traffickinginto the lymphatic vessels and into thelymph nodes could be helpful in trials toprevent the metastatic process.

2.2 VEGFR-3 and its ligands in lymphangiogenesis

The first characterized marker for thelymphatic endothelium was VEGFR-3(fms -like tyrosine kinase 4, FLT4)(Galland et al., 1993; Pajusola et al.,1992). During embryogenesis, VEGFR-3 is first expressed in a subset of bloodvascular ECs (Kaipainen et al., 1995).Accordingly, mice deficient in theVegfr3 gene show abnormalremodeling of the primary vascularplexus and die at E9.5 (Dumont et al.,1998). However, during furtherdevelopment Vegf r3 expressionbecomes mainly restricted to thelymphatic vessels (Dumont et al., 1998;Kaipainen et al., 1995), and severalstudies have indicated the importanceof VEGFR-3 as a mediator oflymphangiogenesis.

In humans, but not in mice, theV E G F R 3 gene encodes twopolypeptides that differ at their C-terminus due to alternative splicing(Galland et al., 1993; Pajusola et al.,1993). The protein corresponding thelonger transcript is more abundant, andits C-terminus contains three tyrosylresidues, of which Y1337 is importantin signaling (Fournier et al., 1995).

VEGF-C and VEGF-D bind to andactivate VEGFR-3 (Achen et al., 1998;Joukov et al., 1996; Lee et al., 1996;Orlandini et al., 1996; Yamada et al.,1997). During development, thedistribution of the VEGFR-3 mRNAfollows a somewhat similar spatio-temporal pattern to VEGF-Cexpression, suggesting paracrineligand-receptor signaling (Kukk et al.,1996). Knocking out these genesshould reveal their importance duringdevelopment. Although VEGF-C andVEGF–D show different expressionpatterns during embryogenesis, it ispossible that they compensate for eachother at sites of overlappingexpression, such as in the lung and inthe kidney mesenchyme (Avantaggiatoet al., 1998; Kukk et al., 1996).

Both VEGF-C and VEGF-D arelymphangiogenic. VEGF-C is mitogenictowards LECs and shows a selectivelymphangiogenic response indifferentiated avian chorioallantoicmembrane (Oh et al., 1997).Accordingly, overexpression of VEGF-C in transgenic mice induces thedevelopment of a hyperplastic

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lymphatic vessel network (Jeltsch et al.,1997; Mandriota et al., 2001). Recentdata also suggest that VEGF-D, and aVEGFR-3 specific mutant of VEGF-C(VEGF-C156S) are lymphangiogenicwhen overexpressed in the skin oftransgenic mice (Joukov et al., 1998;Veikkola et al., 2001). Conversely,inhibition of lymphatic growth isobtained when a soluble form ofVEGFR-3 is expressed in a similartransgenic mouse model (Mäkinen etal., 2001). These results and theexpression patterns of VEGF-C andVEGFR-3 in the lymphatic vasculaturesuggest that lymphatic growth isinduced by VEGF-C and mediated viaVEGFR-3.

As VEGF-C and VEGF-D bind bothVEGFR-2 and VEGFR-3, they arecapable of activating these receptorson the lymphatic endothelium andVEGFR-2 on blood ECs. Proteolyticcleavage is an important regulator ofthe receptor binding and thus, thebiological activity of VEGF-C andVEGF-D. Unlike the other VEGFs,VEGF-C and VEGF-D have N- and C-terminal extensions, and areproteolytically cleaved upon secretion(Fig. 5) (Joukov et al., 1997; Stacker etal., 1999). Receptor binding affinity isenhanced during the stepwiseprocessing of VEGF-C and VEGF-D,and only the fully processed forms ofthese growth factors bind to VEGFR-2.Binding to VEGFR-2 on blood vesselsmay explain the angiogenic potential ofVEGF-C and VEGF-D under certainconditions (Cao et al., 1998; Marconciniet al., 1999). The ability of VEGF-C toinduce blood vessel permeability is alsomediated via VEGFR-2 (Joukov et al.,1998). Interestingly, mouse VEGF-Ddoes not bind mouse VEGFR-2,suggesting that the biological functionsof VEGF-D differ in mouse and man,and that VEGF-D interaction withVEGFR-2 is not crucial for normaldevelopment (Baldwin et al., 2001).

It has been suggested that VEGF-C

and VEGF-D are involved in tumorangiogenesis and lymphangiogenesis,because their expression isupregulated in certain metastatichuman tumors (Achen et al., 2001;Yonemura et al., 1999). In addition,VEGF-C and VEGF-D may also inducetumor angiogenesis via VEGFR-3,which is upregulated in angiogenicblood vessels in tumors (Valtola et al.,1999). Several experimental modelssuggest that overexpression of thesegrowth factors can increase themetastatic rate of tumors. Doubletransgenic mice having both VEGF-Cand SV40 virus large T-antigenoverexpression in pancreatic β-cells,showed increased metastasis via thelymphatic vessels (Mandriota et al.,2001). VEGF-C overexpression intumor xenografts implanted ontoimmunocompromised mice alsor e s u l t e d i n i n c r e a s e dlymphangiogenesis and metastasisformation (Karpanen et al., 2001;Skobe et al., 2001a), whereas twoother VEGF-C overexpressing tumormodels suggested that VEGF-C alsoinduces angiogenesis (Kadambi et al.,2001; Skobe et al., 2001b). VEGF-Doverexpression in a tumor xenograftmodel resulted in both angiogenic andlymphangiogenic responses (Stacker etal., 2001). Thus, the lymphangiogenic/angiogenic properties of VEGF-C andVEGF-D may depend on the tissuesetting, or on protease expression andprocessing of the growth factors inthese models. For example, onlylymphangiogenesis was enhancedwhen very little of VEGF-C was presentin the fully processed form (Skobe etal., 2001a). Inhibitors of thelymphangiogenic factors could serve aspotential therapeutic tools to inhibittumor metastasis. Indeed, in theexperimental models, both solublereceptors and blocking antibodies havebeen shown to inhibit metastasis viathe lymphatic vessels (Karpanen et al.,2001; Stacker et al., 2001).

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Figure 5. The proteolytic processing of VEGF-C and VEGF-D. The first cleavage site is marked withan open arrowhead, and the second by a black arrowhead. Numbers indicate the approximatemolecular masses (kD) of the corresponding polypeptides in reducing conditions. The regions of theVEGF-C polypeptide are marked as follows: VHD; VEGF homology domain, N-term; N-terminalpropeptide, C-term; C-terminal propeptide, ss; disulphide bonds. (Modified from Joukov et al., 1997;Stacker et al., 1999)

In addition to the importance of VEGF-C in tumor lymphangiogenesis, it hasbeen shown that VEGFR-3 is alsopresent in fenestrated capillaries orveins of several organs including thebone marrow, splenic and hepaticsinusoids, kidney glomeruli and certainendocrine glands (Partanen et al.,2000). VEGF-C was detected inneuroendocrine cells such as the αcells of the islets of Langerhans,

prolactin secreting cells of the anteriorpituitary, adrenal medullary cells, anddispersed neuroendocrine cells of thegastrointestinal tract. These resultssuggest that VEGF-C functions in aparacrine manner, and that the VEGF-C/VEGFR-3 signaling may have a rolein peptide release from secretorygranules of certain neuroendocrinecells to surrounding capillaries.

2.3 Other lymphatic endothelial specific factors

2.3.1 Prospero-related homeobox

protein 1 (Prox1)

In addition to VEGFR-3, there aresome other genes which are largelyexpressed in LECs. The expressionpattern of the homeobox gene Prox1

suggested that it has a functional rolein the developing central nervoussystem (Oliver et al., 1993). However,results with Prox1-/- mice showed thatin addition to the liver and lens-fibredevelopment, Prox1 is essential in theformation of the lymphatic vasculature

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(Sosa-Pineda et al., 2000; Wigle et al.,1999; Wigle and Oliver, 1999).

The marker gene expression in theProx1 targeted mice showed thatstarting as early as E9.5, Prox1 isexpressed in the subpopulation of EC,in the regions that give rise to thelymphatic sacs. In the Prox1 deficientmice, the budding and sprouting of thelymphatic endothelial cells from theseregions were arrested, suggesting thatsome unidentified guidance signalsmay regulate the migration of the Prox1expressing LECs (Wigle and Oliver,1999).

Dur ing embryogenesis, theexpression pattern of Prox1 in thedevelop ing lymphat ic systemresembles that of VEGFR-3 (Dumont etal., 1998; Wigle and Oliver, 1999).Interestingly, Prox1 haplo-insufficiencyin Prox1+/- newborn mice leads tochylous fluid accumulation into theintestine, and death a few days afterbirth (Wigle and Oliver, 1999). Theseresults suggest that Prox1 is requiredfor lymphatic, but not blood vesseldevelopment and that even haplo-insufficiency of Prox1 results inimproper development of the entericlymphatic vessels.

2.3.2 Podoplanin

Podoplanin is an integral plasmamembrane protein first found inglomerular epithelial cells, where itcontrols the shape of podocytes(Breiteneder-Geleff et al., 1997; Matsuiet al., 1999). In addition to podocytes,podoplanin is also expressed in theendothelium of lymphatic capillaries,but not in the blood vasculature(Breiteneder-Geleff et al., 1999;Kriehuber et al., 2001; Mäkinen et al.,2001). In the skin and kidney,podoplanin co-localizes with VEGFR-3,and the ECs of Kaposi's sarcomasexpress both VEGFR-3 andpodoplanin, which supports theirsuggested lymphatic endothelial origin

(Breiteneder-Geleff et al., 1999;Weninger et al., 1999). However, thefunction of podoplanin in LECs is notknown.

2.3.3 Lymphatic vessel endothelial

hyaluronan receptor 1 (LYVE-1)

The ECM glycosaminoglycanhyaluronan (HA) plays an importantrole in the maintenance of tissueintegrity and facilitation of cell migrationduring embryogenesis, wound healingand inflammation. HA is rapidlydegraded within lymph nodes. Theintegral membrane protein LYVE-1 hasbeen identified as a new LEC specificreceptor for HA (Banerji et al., 1999). Itis distributed equally on the luminal andabluminal surfaces of lymphaticvessels, and it transports HA acrossthe lymphatic endothelium into thelymph (Prevo et al., 2001). LYVE-1 ishomologous to CD44, which is requiredfor lymphocyte entry into the adultthymus (Banerji et al., 1999; Protin etal., 1999). Extravasation of CD44expressing lymphocytes is mediated bytheir interaction with HA-coatedvascular endothelium in inflammationand increased amounts of HA in thetumor interstitium predict poor survivalof the patients (Auvinen et al., 2000;DeGrendele et al., 1997). It remains tobe seen whether LYVE-1 plays a role inmetastasis via the lymphatic vessels,for example by assisting theintravasation of HA expressing tumorcells.

The specific roles of Prox-1,podoplanin and LYVE-1 functions in theLECs remain to be studied. However,these markers can now be utilized tostudy lymphatic development and toexplore the etiology of diseases, inwhich the lymphatic vessels areaffected.

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3 Diseases associated with RTK dysfunction

3.1 Lymphedema

3.1.1 Pathophysiology of

lymphedema

Lymphatic vessels play a central role inmaintaining the fluid balance of theinterstitial tissues. If this balance isdisturbed, for exapmple if the amountof lymphatic fluid formed exceeds thelymphatic absorption or transportcapacity, a protein-rich fluid collects inthe interstitium. Lymphedema ischaracterized by a chronic, disfiguringand disabling swelling of one or severallimbs due to insufficient lymphaticdrainage, but not due to increasedvascular permeability (Reviewed inRockson, 2001).

Isotopic lymphoscintigraphy andmagnetic resonance imaging (MRI)have been used to visualize the edemaand functional defects of the lymphaticsystem in lymphedema patients. Inlymphoscintigraphy, a radiolabeledmacromolecular tracer is administeredinto the subcutaneous tissue of theaffected limb, and the major lymphatictrunks and lymph nodes are visualized.In the lymphedematous area, thetransport of the tracer is absent ordelayed, and dermal backflow(progressive dispersion of the tracerinto the soft tissues) can be detected.MRI analysis reveals structuralchanges such as thickening of the skinwith diffuse dermal and subcutaneousedema, an intact subfascia lcompartment and variability in regionallymph node size (Case et al., 1992;Idy-Peretti et al., 1998). These changesare not necessarily distinguishablebetween primary and secondarylymphedema (Idy-Peretti et al., 1998).

3.1.2 Classification of lymphedema

Lymphedema is divided into two maincategories. Primary lymphedema is acondition with no identifiable cause,whereas secondary lymphedemaresults from a disruption of thelymphatic circulation due to an earlierd isease or t rauma. Pr imarylymphedema can be present at birth, ordevelop at puberty or, more rarely, inadulthood (Witte et al., 1997). Incongenital hereditary lymphedema(Milroy’s disease) the superficiallymphatic vessels are usuallyhypoplastic or aplastic. In contrast, themicrolymphatic network in late onsetlymphedema (Meige’s disease) usuallyappears larger than in healthy controls(Bollinger, 1993; Bollinger et al., 1983;Pfister et al., 1990). Approximately 35%of primary lymphedema patients have apositive family history of the disease(Dale, 1985). It has been estimated thatapproximately 1:6000 newborns willeven tua l l y deve lop p r imarylymphedema, with sex ratio of onemale to three females (Dale, 1985).

In contrast to primary lymphedema,secondary or acquired lymphedemadevelops when the lymphatic vesselsare damaged by infection, radiationtherapy or when lymph nodes aresurgically removed. It has beenestimated that 3-5 million people inUSA have secondary lymphedema.

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Figure 6. Lymphatic filariasis. Filariasis is caused by Wuchereria bancrofti microfilarial worm infection(left). The filarial infection in some cases results in massive swelling of the leg (right). Images:WHO/TDR Image Library, photographers: Stammers and Dreyer

The most common form oflymphedema results from filariasis (Fig.6). According to the World HealthOrganization (WHO), over 120 millionpeople suffer from filarial lymphedemaworldwide, mainly in tropical andsubtropical areas. In addition toenlargement of the entire leg, arm, orgenitals, filariasis may also causeinternal damage to the kidneys and thelymphatic system. Lymphatic filariasisis caused by the parasitic filarial wormsWuchereria bancrofti and B r u g i amalayi . These worms lodge in thelymphatic system, where they live for 4-6 years, producing millions of immaturemicrofilariae (minute larvae) thatcirculate in the blood. The disease isfurther transmitted by mosquitoes thatbite infected humans and pick up themicrofilariae, which then develop insidethe mosquito and are transmitted toother humans. The infection isgenerally acquired during childhood,but the development of lymphedematakes several years.

In all forms of lymphedema, thepersistent accumulation of stagnant,protein-rich fluid into the interstitiumreduces oxygen availability in tissuesand the normal immune defences arepartially lost. As a consequence, theaffected area often shows increasedt issue f ibros is and adiposedegeneration, interference with woundhealing, and susceptibility to infections.

3.1.3 Genetic alterations in

lymphedema

Despite having been described over acentury ago, little progress has beenmade in understanding the molecularmechanisms causing hereditarylymphedema. Recently, several groupsreported linkage of the early onsetprimary lymphedema to the VEGFR3region on distal chromosome 5q(Aprelikova et al., 1992; Evans et al.,1999; Ferrell et al., 1998; Galland et al.,1992; Witte et al., 1998), and fivespecific lymphedema-linked missensemutations were found in the VEGFR-3tyrosine kinase domain (Irrthum et al.,2000; Karkkainen et al., 2000).

While mutations which inhibit thebiological activity of VEGFR-3 are onecause of primary lymphedema, thereare several lymphedema families andother lymphedema syndromes whichinvo lve o ther genet ic loc i .Lymphedema-d is t i ch ias is (LD)syndrome has been linked tochromosomal region 16q24 (Bell et al.,2000; Mangion et al., 1999). LD ischaracterized by pubertal lymphedemawith enlarged cutaneous lymphaticvessels, and a double row ofeyelashes. In addition, 16% of the LDpatients also show congenital heartdisease (Corbett et al., 1982).Mutations in the FOXC2 (MFH1) genehas been identified in the LD patients(Fang et al., 2000).

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Figure 7. FOXC2 mutations in the lymphedema distichiasis families. A schematic structure of theFOXC2 protein is presented. All mutations marked with a circle result in a truncated protein with orwithout a novel C-terminus. The one missense mutation discovered (C374T; S125L) is indicated by anarrow.

FOXC2 encodes a member of theforkhead/winged-helix family oftranscription factors, which are involvedin diverse developmental pathways(Miura et al., 1993). Foxc2-/- mice dieduring embryogenesis or perinatally,and exhibit cardiovascular defects,such as interrupted aortic arch, andskeletal defects, such as cleft palate(Iida et al., 1997; Winnier et al., 1997).However, Foxc2+/- mice do not have anobvious phenotype, and they show onlymild defects in ocular development(Smith et al., 2000). So far, nolymphedema has been detected inmice deficient in Foxc2. Results fromKO mouse studies are thus indisagreement somewhat with thesevere phenotype caused by theheterozygous FOXC2 mutations in thehuman LD patients.

Small insertions or deletions havebeen found throughout the FOXC2gene in LD patients, often resulting in apremature termination of the FOXC2protein and production of a novel C-terminus (Fig. 7) (Bell et al., 2001;Finegold et al., 2001). Thus, FOXC2haplo-insufficiency appears to be onecause of LD. The broad phenotypicheterogeneity within the familiessuggests that FOXC2 mutations maybe the cause of lymphedema in families

displaying phenotypes attributed toother lymphedema syndromes(Finegold et al., 2001). Interestingly,FOXC2 expression also counteractsobesity, hypertriglyceridemia, and diet-induced insulin resistance (Cederberget al., 2001), This result may provideconnection to the adipose tissueaccumulation which is often associatedwith lymphedema.

Mutations within V E G F R 3 andFOXC2 are the only ones which havebeen found to cause hereditarylymphedema. In addit ion, twochromosomal regions have been linkedto other lymphedema syndromes(Table 2). The disease locus incholestasis-lymphedema syndrome,characterized by severe neonatalcholestasis and chronic severelymphedema, was mapped tochromosome 15q (Bull et al., 2000).One of the variable features of Turnersyndrome (complete or part absence ofone of the X chromosomes) islymphedema. The locus responsible forthe symptoms of this disease has beenmapped to a region Xp11.2-p22.1 (Zinnet al., 1998). Interestingly, the VEGFD(FIGF) gene is also located in Xp22.1(Rocchigiani et al., 1998), but there areno reports of involvement of VEGF-D inTurner syndrome.

Table 2. Genetic alterations in lymphedema syndromesLymphedema syndrome MIM* Age at onset Gene loci GeneMilroy’s disease 153100 congenital 5q34-q35 VEGFR3Lymphedema-distichiasis (LD)syndrome

153400 puberty 16q24.3 FOXC2

cholestasis-lymphedemasyndrome

214900 puberty 15q Not known

Turner syndrome - congenital Xp11.2-p22.1 Not known* Mendelian inheritance in man (http://www3.ncbi.nlm.nih.gov/Omim/)

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All these reports suggest that otherlymphedema genes exist. Furtherstudies may reveal new factorsaffecting LEC regulation and help us toexplain the genotypic and phenotypicheterogeneity of lymphedema. It shouldalso be noted that not all members of

primary lymphedema families withinactivating VEGFR3 mutations areaffected by the disease (Karkkainen etal., 2000). This suggests that additionalgenetic or environmental factors play arole in the development oflymphedema.

3.2 Inactivating RTK mutations in human syndromes

Among the 20 different RTK families,several inherited mutations have beenfound to be involved in thedevelopment of human diseases. Manyof these occur near the transmembranedomain and lead to constitutiveactivation of the tyrosine kinase(Reviewed in Robertson et al., 2000).This uncontrolled activity often leads tofamilial predisposition to developneoplasia. Inheri ted neoplasiasyndromes can result from activatingMET (hepatocyte growth factorreceptor) mutations in multiple papillaryrenal cell carcinomas and RET(rearranged during transformation)mutations in multiple endocrineneoplasia (MEN). Alternatively,activating mutations may also result indevelopmental abnormalities, such asfibroblast growth factor receptor(FGFR)-3 mutations in dwarfism andmutations in other FGFRs incraniosynostosis syndromes. In theRTKs expressed on ECs, activatingTEK mutations have been found invenous malformations (Vikkula et al.,1996). A single point mutation (R859W)within the Tek kinase domain leads toan increase of RTK activity. Lesionsresult due to large venous channelswith a low number of surroundingsmooth muscle cells.

In contrast to many activatingmutations in RTKs, their inactivatingmutations in inherited conditions arerare. This may be due to the fact thatsignaling via RTKs is essential duringnormal development, and inactivatingmutations are likely to act in a dominantnegative manner, thus quenching

downstream signaling. The KITreceptor provided the first example of aloss-of-function mutation in an inheriteddisease both in humans and in mice(Giebel and Spritz, 1991; Tan et al.,1990). KIT is a member of PDGFRfamily and binds stem cell factor (KITligand, KL). Loss-of-function mutationswithin the KIT receptor have beenfound in piebaldism, characterized by alack of pigmentation leading to whitepatches of skin and hair due todefective melanocyte migration. Milderforms of piebaldism are caused bymutations which lead to disruption ofthe extracellular domain and thus aninability to respond to the ligand. Moresevere disease is caused byinactivating missense mutations in thetyrosine kinase domain due to adominant negative effect (Seereferences in Robertson et al., 2000).Activating mutations within KIT havebeen ident i f ied in inher i tedgastrointestinal stromal tumors (GIST).Interestingly, a (KIT, ABL, andPDGFRβ) specific tyrosine kinaseinhibitor, STI571, has been found to bean excellent therapeutic agent for GIST(Joensuu et al., 2001; Tuveson et al.,2001).

Inactivating mutations have alsobeen found in RET, which is requiredfor the normal development of thekidney, enteric system, and neuralcrest. RET binds the ligands of theglial-derived neurothrophic factor(GDNF) family in a heterodimericcomplex with glycosylphosphatidyl-inositol-linked receptors (GFR-α-1-4).

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Deletions or point mutations within REThave been assoc ia ted wi thHirschsprung disease, which ischaracterized by absence of entericganglia along the gastro-intestinal tractand leads to bowel obstruction inneonates (Reviewed in Eng andMulligan, 1997). The mutations inHirschsprung disease are predicted toencode truncated, inactive or non-expressed RET receptors, and act in adominant negative manner. In addition

to these inactivating mutations, gain-of-function mutations of RET have beenidenti f ied in inheri ted cancersyndromes, such as in MEN and infamilial medullary thyroid carcinoma(Jhiang, 2000). Surprisingly, in somefamilies a single point mutation resultsin both Hirschsprung disease andMEN, suggesting that different cellsmay require different balancing of RTKsignaling (Takahashi et al., 1999).

4 Gene therapy

4.1 Vectors and approaches

Gene therapy aims to transfer genesinto cells to correct genetic defects, orto express other therapeutic geneproducts (Lemoine, 1999). Fortreatment of monogenic diseases, theexpression of the defective gene maybe introduced into the cells involved inthe pathology, or into their precursorseither by ex vivo, in situ, or in vivo genetransfer. So far, only somatic genetherapy has been applied to humanpatients. Germline gene therapy cannotbe attempted until more studies andsafety trials have been performed, anduntil the mechanisms affecting thesuccessful gene therapy are fullyunderstood. The ethical issues alsoneed to be carefully considered beforestudies of germl ine genet icengineering.

All gene therapeutic strategiesdepend on the delivery of geneticmaterial into the cell and thence thenucleus. Methods currently involvegene transfer using viruses, nakedDNA, or DNA-cationic liposomecomplexes. Although naked DNA orcationic liposomes are non-toxic andnon-immunogenic, they are relativelyinefficient. Thus, viral gene therapiesare of most interest if high expressionlevels and long-term therapies are

required. Several viruses, such asadenoviruses, adeno-associatedviruses (AAVs), retroviruses, andHerpes simplex viruses have beenused as vectors to introduce genes intothe cells. The viral genomes aremodified to delete the regions whichare important in viral replication, thuslimiting the process to only a singleinfectious cycle and increasing thesafety of the therapy.

Adenoviruses are commonly usedfor gene transfer. These viruses infectseveral cell types, and have been usedto give high-level, but short-term geneexpression in several tissues, such asthe lung, liver, and muscle (Kresina,2001). Adenoviruses have a naturaltropism for epithelial cells, and caninfect non-dividing cells. Most of thea d e n o v i r u s e s u t i l i z e t h ecoxackie/adenovirus receptor (CAR) forcell attachment, and the exposed RGDmotif in the viral capsid is important inintegrin-mediated virus entry into thecell (Roelvink et al., 1999; Stewart etal., 1997; Tomko et al., 1997; Wickhamet al., 1993). When administratedintravenously, the adenoviruses mostlyinfect the liver. WT adenoviralinfections are associated with veryminor symptoms, such as acute

22

respiratory infections. However, theconsequences may be severe inimmunosuppressed patients. Althoughadenoviruses give a high-level of geneexpression, their major limitation is thattransgene expression is lost within amonth, which is partly due to animmune response to the remaining viralproteins.

Whereas the adenoviral genetransfer only gives short termexpression, AAVs may give transgeneexpression for a year (Daly et al.,2001). AAVs are non-pathogenichuman viruses, which do not elicit aninflammatory reaction or a cytotoxicimmune response, and infect both thedividing and non-dividing cells ofseveral organs (Reviewed in Monahanand Samulski, 2000). AAVs arereplication deficient and require ahelper virus (usually adenovirus orherpesvirus). Viral entry into the cells ismediated by cell surface receptorssuch as heparan sulfates, α vβ5

integrins, and FGFR-1 (Qing et al.,1999; Summerford et al., 1999;Summerford and Samulski, 1998). Onedisadvantage in the use of AAVs istheir limited packaging capacity, asthey cannot accommodate more than4.5 kb of foreign DNA. Whereas WTAAVs integrate site-specifically tochromosome 19 (Kotin et al., 1990),recombinant viruses may integraterandomly into the host genome, andput the patients at risk of insertionalmutagenesis. Despite this, AAVencoded Factor IX has been used totreat haemophilia B and CTFR (Cystic

fibrosis transmembrane regulator) totreat cystic fibrosis (Kay et al., 2000;Wagner et al., 1999). These studiesdemonstrate that there is no vector-related toxicity, germline transmission,or formation of inhibitory antibodies. Itwas also shown that AAV gives long-term expression in humans.

For diseases caused by dominantnegative mutations, very few genetherapeutic trials have been attempted.Overexpression of the WT protein, orinhibition of mutant protein expressionare the two main strategies which havebeen attempted in trials to decrease adominant negative effect. Antisensetechniques are a way to decrease theamount of a mutant protein product.This has been tried in vitro withosteogenesis imperfecta, which resultsfrom a mutation in the proα chains oftype I collagen (Niyibizi et al., 2000). Inthis technique the specific antisenseDNA oliconucleotides bind the targetmRNA, and the complex is digested byintracellular enzymes. A more specificcleavage of the target mRNA has beeninvestigated using ribozymes, whichalso provide a way to target pointmutations. (Lewin and Hauswirth,2001). In Marfan syndrome, whichresults from dominant negativemutations in fibrillin-1, ribozymes havebeen shown to be efficient at cleavageof f ibri l l in RNA (Kilpatrick andPhylactou, 1998). So far, diseasescaused by inactivating dominantnegative RTK mutations have not beentreated with gene therapy.

4.2 Gene therapy in ECs

Angiogenesis is a novel target for genetherapy (Reviewed in Ferrara andAlitalo, 1999). In attempts tomanipulate angiogenesis, VEGFR-2 i sof the highest interest therapeuticallybecause it dominates the angiogenicresponse to VEGF. Depending on thedisease concept , therapeut ic

angiogenesis may aim at induction ofnew vessel growth or inhibition ofangiogenesis. Pro-angiogenic therapyhas been shown to be efficient inischemia models, such as in hindlimbischemia and in myocardial infarction(Harada et al., 1994; Laitinen et al.,1998; Pearlman et al., 1995; Pu et al.,

23

1993; Takeshita et al., 1994). Inaddition, angiogenic factors have beenfound to be beneficial in the preventionof arterial restenosis after balloonangioplasty (Asahara et al., 1995). Onebenefit of the treatments applied so faris that they require only a short-termand local expression of the therapeuticgene.

Adenov i ra l (Ad ) VEGF-Coverexpression has been shown toinduce lymphangiogenesis in the earsof nude mice (Enholm et al., 2001). TheVEGF-C receptors VEGFR-2 andVEGFR-3 were upregulated in ECsafter AdVEGF-C infection, but therewas no effect on angiogenesis. Inaddition, VEGF-C gene transfer hasbeen studied both in a restenosismodel and in ischemic hind limb model.It was shown that AdVEGF-Cexpression is able to reduce intimalthickening after balloon injury in arabbit model (Hiltunen et al., 2000). Inthe rabbit ischemic hindlimb model,VEGF-C was shown to promotecollateral formation (Witzenbichler etal., 1998), suggesting that VEGF-Cmay be angiogenic in vivo undercertain conditions. One explanation forthese results may be that the matureform by binding to VEGFR-2, maydirectly induce angiogenesis. Theproteases involved in VEGF-Cprocessing are not known. If some ofthe proteases are upregulated byhypoxia and cytokines, more of the

mature VEGF-C, capable of bindingVEGFR-2 would then be produced. Onthe other hand, VEGF levels arestrongly upregulated in hypoxicconditions, and it has been shown thatVEGF and VEGF-C synergize in theirin vitro angiogenic response (Pepper etal., 1998). Thus, it is possible thatVEGF-C acts in concert with VEGFunder these conditions, or that theangiogenic properties of VEGF-C maybe indirect via a synergistic effect withVEGF and/or v ia VEGFR-2upregulation.

As a conclusion, detailed studies ofthe factors involved in EC growth andregulation could allow us to manipulatethe various steps in angiogenesis andlymphangiogenesis. In the future,therapeutic approaches may includetargeting of therapeutic geneexpression to the desired ECs, forexample into the tumor endothelium.This goal may be achieved by targetingviral entry to the desired cells only. Thisapproach is facilitated by the findingthat the ECs are heterogenic and thus,distinct targeting molecules can beused (Ruoslahti and Rajotte, 2000).Alternatively, the promoters of theTIE1, TEK, KDR, ICAM1, or endoglingenes could be used to achieve ECspecific expression of the therapeuticgene. The lymphatic endothelial cellspecific promoters could also be usedto target lymphatic endothelium.

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AIMS OF THIS STUDY

This study was undertaken to analyse the role of VEGFR-3 in inheritedlymphedema. A further aim was to characterize a mouse model and treatmentpossibilities for primary lymphedema.

In order to explore these questions, I have studied:

1. The structure and polymorphisms of the VEGFR3 gene

2. Functional abnormalities of lymphedema-linked mutant VEGFR-3s

3. Phenotypic features of the Chy mouse model for human primary lymphedema

4. Effects of growth factor therapy on Chy mice

25

MATERIALS AND METHODS

A brief summary of the methods used will be given here. Methods are describedand discussed in more detail in the original publications.

1 The VEGFR3 genomic structure and regulatory region (I)

We characterized the VEGFR3 geneand its regulatory 5' flanking regionusing a genomic cosmid clonecontaining these sequences. The exon-intron boundaries were sequenced intheir entirety. Polymorphic variations inthe VEGFR3 gene were identified byre-sequencing the gene from aminimum of 50 chromosomes, andallele frequencies were estimated fromthe sequencing results. In order tostudy EC specific transcription ofVegfr3, sequences upstream of Vegfr3gene were isolated. For in vitro

analysis, we used a downstreamreporter activation assay. Endothelialand non-ECs were transfected withconstructs containing the luciferasereporter gene driven by fragments ofthe Vegfr3 upstream sequences. For invivo analysis, we used transgenic micewith V e g f r 3 promoter regulatedexpression of the LacZ marker gene.These mice were analysed for β-galactosidase expression to studylymphat ic endothel ia l speci f icexpression of VEGFR-3.

2 Analysis of the lymphedema-linked mutant VEGFR-3s (II, III)

We used genetic linkage analysis anddirect sequencing to ident i fylymphedema-linked VEGFR3 missensemutations in primary lymphedemafamilies. To test whether the singleamino acid substitutions identified alterVEGFR-3 function we generated thecorresponding mutant receptor cDNAsin a VEGFR3 expression vector, andanalysed their function in vitro intransiently transfected cells and instable cell lines expressing the mutantand/or WT receptors. The effect of themutations on tyrosyl phosphorylation of

VEGFR-3 was s tud ied byimmunoprecipitation and Westernblotting. In analyses of the turnovertime of mutant and WT receptors,pulse-chase labeling experiments werecarried out. The effects of mutantVEGFR-3 on downstream signalingwere studied using a transcriptionalreporter assay. An array of differentreporter vectors containing specific cis-acting DNA enhancer elementsupstream of a luciferase reporter genewere assayed.

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3 The Chy mouse model for human primary lymphedema (IV)

In order to study the role of inactivatingVEGFR3 mutations in vivo, we usedthe Chy mouse model of lymphedema.The Chy m ice have anethylnitrosourea-induced mutationwhich has been linked to the Vegfr3locus, on mouse chromosome 11 (Lyonand Glenister, 1984; Lyon andGlenister, 1986). By direct sequencingwe found that the Chy mice wereheterozygous for a Vegfr3 pointmutation (A3157T). This correspondsto an I1053F substitution in theVEGFR-3 tyrosine kinase domain. Themouse and human receptors are highlyconserved and, as the expression

vector for mouse VEGFR-3 was notavailable, the A3157T mutation wasgenerated in the human VEGFR-3exp ress i on vec to r . Ty rosy lphosphorylation of the mutant receptorwas analysed by transient transfectionand Western blotting. The phenotypicfeatures of lymphatic vessels in theChy mice were analysed usingimmunoh i s tochem is t r y ( IHC) ,fluorescent microlymphography, andMRI. The blood vessels were analysedusing platelet endothelial cell adhesionmolecule-1 (PECAM-1) IHC andLycopersicon esculentum l ec t instaining.

4 Lymphedema therapy in the Chy model (IV)

Chy mice are heterozygous for theVEGFR-3(I1053F) mutation andtherefore there remains some signalingvia the WT allele. Hence, we wanted toanalyse whether VEGFR-3 stimulationwith an excess of ligand is sufficient toovercome lymphatic hypoplasia causedby the mutant receptor. We exploredthis possibility using both adenovirusand AAV mediated VEGF-C gene

therapy in the skin of the Chy mice.The effects of VEGF-C overexpressionwere analysed by f luorescentmicrolymphography and IHC. Theresu l ts f rom v i ra l VEGF-Coverexpression studies were furtherconfirmed by crossing the Chy micewith mice expressing VEGF-C156S inskin keratinocytes (Veikkola et al.,2001).

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RESULTS AND DISCUSSION

The main results are briefly described here. The results are described anddiscussed in more detail in the original publications.

1 The VEGFR3 genomic structure and regulatory region (I)

Previous studies have shown linkage ofcongenital primary lymphedema to theVEGFR3 region in chromosome 5q(Evans et al., 1999; Ferrell et al., 1998;Witte et al., 1998). In order to clarifywhich mutations are associated withlymphedema, we first resolved thegenomic structure of the V E G F R 3gene. This gene was found to consistof 31 exons, and its structure closelyresembles those of Vegfr1 andVEGFR2 (Kondo et al., 1998; Yin et al.,1998) (Fig. 8). Exons 30a and 30b ofthe VEGFR3 gene are alternativelyspliced. In mice, only the longer isoformexists (Pajusola et al., 1993), andinterestingly it was reported that theshorter transcript in humans is theresult of a retroviral insertion (Hughes,2001). This insertion may contribute to

diversity in VEGFR-3 signaling, as theshorter form lacks three tyrosylresidues, that are important indownstream signaling (Fournier et al.,1995; Fournier et al., 1996).

VEGFR3 exon-intron boundarieswere sequenced from cosmidsubclones, and the polymorphic singlebase pair transversions within VEGFR3were examined. We found 22intragenic polymorphic variations, ofwhich four resulted in amino acidsubstitutions (N149D, T494A, P641Sand R1146H). Phosphorylation of theP641S mutant of VEGFR-3 was testedin vitro, and it appeared to bephosphorylated in a similar manner tothe WT receptor (Karkkainen et al.,2000).

Figure 8. Location of the VEGFR3 polymorphisms and missense mutations. Genomic structure ofVEGFR3 consists of 31 exons, of which 30a and 30b are alternatively spliced. Above the genomicstructure is shown the VEGFR-3 domain structure, consisting of the signal sequence (ss), seven Ighomology domains (Ig), a transmembrane domain TM and two tyrosine kinase domains (TK). Below theexon structure are indicated the locations of the VEGFR3 polymorphisms and lymphedema-linkedamino acid substitutions (highlighted in grey). The introns are not drawn in scale.

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As VEGFR-3 is specifically expressedon a lymphatic endothelium, theregulatory region of VEGFR3 mayprovide an important tool for molecularbiology in any attempt to target thelymphatic system. On the other hand,regulation of VEGFR-3 expression maybe affected in lymphatic disorders.Thus, we wanted to analyse VEGFR3promoter regions which mediateLEC/EC specific expression, both incell cultures and in vivo. Two homologyregions (HRs) were found, whichshowed 70% identity between mouseand human VEGFR3 promotersequences.

The luciferase reporter assayshowed that the Vegfr3 promoter hadmuch stronger activity in ECscompared with non-ECs. Progressive5’-deletions were made to the promoterto further characterize the sequencescritical for activity. Results using 3.0,1.6, 0.8, and 0.5 kb promoter fragmentsshowed that the region upstream of theHR1 contains inhibitory elements,

whereas the HR1 itself containstranscriptional enhancer elements. Thecell culture experiments were carriedout using blood vascular ECs, becauseno LEC lines were available at the time.

The LEC specificity of variousVegfr3 promoter fragments was furtherconfirmed in transgenic mice. LacZexpression was controlled by 3.6, 1.6,or 0.8 kb promoter fragments, andreporter gene expression wascompared to that of Vegfr3+/- mice atE15.5, when the lymphatic vessels arevisible. The longer promoter fragmentsinduced LEC specific expression,whereas LacZ expression driven by the0.8 kb fragment was also seen in bloodvessels. Overall, staining wasvisualized in only a subset of thetransgenic embryos, suggesting thatfurther regulatory/enhancer elementsmay be located within the first intron ofthe Vegfr3 gene. Such enhancers havepreviously been reported within theVegfr2 and Tek genes (Kappel et al.,1999; Schlaeger et al., 1997).

2 Analysis of the lymphedema-linked mutant VEGFR-3s (II, III)

2.1 Dominant negative effect of the mutant VEGFR-3s

As we had resolved the genomicstructure of VEGFR3, it now becamepossible to identify the V E G F R 3mutations in the Milroy’s diseasefamilies. We found four new missensemutations to add to the one publishedpreviously (Ferrell et al., 1998). Thegoal of this study was also to reveal themechanisms by which the mutantVEGFR-3s result in a lymphedemaphenotype.

To test the properties of the mutantVEGFR-3s, the correspondingexpression vectors were generated andtheir phosphorylation was analysed intransiently transfected cells. No tyrosylphosphorylation was detected in theG857R, H1035R, R1041P, L1044P or

P1114L mutant proteins, indicating thatthe lymphedema-linked mutations blockVEGFR-3 tyrosine kinase function. Themutant receptors were kinase inactivealso in an autokinase assay (MKunpublished data). Upon co-expressionof epitope-tagged WT and mutantreceptors in a transient transfectionsystem, the mutant protein did notsignificantly quench phosphorylation ofthe WT receptor. The WT and mutantreceptors formed heterodimers, and theWT receptor was ab le totransphosphorylate the mutantreceptor. The results were alsoconfirmed in a ligand-dependentsystem.

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Figure 9. A model for mutant VEGFR-3 function in the lymphedema patients. The patients areheterozygous for the mutant allele and, thus have three different types of VEGFR-3 dimers on the LECsurface. Whereas the WT-WT dimers are internalized after activation, the dimers containing a mutantreceptor are not phosphorylated normally. These dimers seem to accumulate on the cell surface,causing a dominant-negative effect and leading to hypoplasia of the lymphatic vessels in the patients.

As tyrosine kinase signaling is alsoregulated via internalization of theactivated ligand-receptor complex(Reviewed in Wiley and Burke, 2001),we studied the turnover time of themutant and WT receptors by pulse-chase analysis. The decay of WTVEGFR-3 polypeptide was faster thanfor the mutant receptors, indicating thatthe mutant receptors were more stableon the cell surface. These resultssuggest that the activated WT receptoris internalized, and degrades at a fasterrate than the mutant receptor, and thatthe mutant receptor may accumulateon the cell surface (Fig. 9).

Downstream signaling mediated byWT and mutant receptors wasanalysed using a transcriptionalreporter assay. An array of differentreporter constructs were tested, andenhanced reporter expression afterVEGFR-3 stimulation was induced viathe AP-1 and NFκB transcription factorcomplexes, but not via other elementstested. In this assay, the ability of

VEGFR-3 to activate the AP-1 or NFκBcomplexes decreased with increasingmutant VEGFR-3 concentration,consistent with the defective signalingfunction of the mutant receptor.

Interestingly, it has recently beensuggested that lymphedema in theEDA-ID (ectodermal dysplasia withimmunodeficiency) patients may becaused by defective VEGFR-3signaling via the NF-κB transcriptionfactor (Döffinger et al., 2001).

In contrast to the inactivatingmissense mutations, VEGFR-3 haplo-insufficiency probably doesn’t lead tol y m p h e d e m a . C h r o m o s o m a labnormalities involving deletion of theVEGFR3 interval do not seem to resultin lymphedema (Barber et al., 1996;Groen et al., 1998). This is compatiblewith the result that Vegfr3-/- mice dieduring embryogenesis due to a failureof cardiovascular development, whilethe heterozygotes have no obviousphenotypic abnormality (Dumont et al.,1998). However, this protection may

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depend on the genetic background ofthe mice (T. Mäkinen et al.,unpublished observations), indicatingthat genetic factors may have an effecton the appearance of lymphedema.

In conclusion, these results suggest

that the signaling via the lymphedema-linked VEGFR-3s is defective. Thereceptor encoded by the mutant alleleseems to act in a dominant negativemanner due to the accumulation of themutant receptor onto the LEC surface.

2.2 Interaction of VEGFR-3 with other pathways

Recent reports have revealed severalproteins which are important in theformation of the lymphatic vasculature.Integrin α9β1 seems to be required fornormal lymphatic development,possibly in concert with VEGFR-3. Thefirst evidence of this was the report thatthe α9-/- mice die 6-12 days after birthdue to severe chylothorax and resultingrespiratory failure (Huang et al., 2000).Whereas β1 integrin is ubiquitouslyexpressed and forms dimers withseveral α subunits, α9 integrin onlyforms dimers with β1 integrin chains,and is expressed only in a subset oftissues, for example transiently in thethoracic duct during embryogenesis.Thus, α9β1 integrin appears to berequired for normal development of thelymphatic system (Huang et al., 2000).In addition, recent results indicate thatβ1 integrin associates with VEGFR-3and is able to induce its tyrosinephosphorylation, albeit weakly (Wanget al., 2001). The ECM proteins(collagen and fibronectin) were able toinduce VEGFR-3 phosphorylation,which was mediated by β1 integrin.Both VEGFR-3 and β1 integrinactivation were required for cellmigration, while a lymphedema-linkedmutant VEGFR (G857R) did not inducecell migration on collagen. Theseresults suggest co-operation betweenβ1 and VEGFR-3 signaling in cell

migration. Thus, it is possible that someof the defects in lymphedema may beas a result of impaired LEC migration.

Net, a member of the Ets-domaintranscription factor and ternary complexfactor families also has a role in thedevelopment of the lymphatic system.One function of Net is to represstranscription of the immediate earlygenes (c-fos, egr-1, and Jun-B) whenthe mitogen-activated protein kinases(MAPK) are not activated. Net isexpressed in the embryonicvasculature, and its EC expressionpersists during development (Ayadi etal., 2001a). Net is co-localized withVEGFR-3 in the thoracic duct, and thelymphatic vessels of the intestine andskin at E16.5 (Ayadi et al., 2001b).Netδ/δ mice, which lack the DNA bindingdomain die soon after birth because ofa chylothorax suggesting a possibleinteraction in the signaling pathways ofNet and VEGFR-3 in lymphaticendothelium (Ayadi et al., 2001b).

These results suggest that severalregulatory and structural proteins havea role in the correct development of thelymphatic vasculature. Furtherknowledge of the interactions betweenthe different signaling pathways mayhelp in revealing the mechanisms oflymphatic vessel development andregulation.

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3 The Chy mouse model for human primary lymphedema (IV)

Since mutations within VEGFR3 arelikely to impair several cellularprocesses, it is necessary to study therole of VEGFR-3 in lymphedema by invivo methods. We have analysed thepathogenesis of lymphedema using theChy mouse model.

The Chy mice , showingaccumulation of chylous fluid into theabdomen, were generated by treating amale mouse with ethylnitrosourea, andscreening the F1 generation forlymphatic phenotypes (Reviewed inAnderson, 2000). The mutation in theChy mouse line has been mapped tochromosome 11 (Lyon and Glenister,1984; Lyon and Glenister, 1986). Wesequenced the Vegfr3 candidate geneof this chromosome in Chy mice(Watkins-Chow et al., 1997) and foundan A3157T mutation resulting in anI1053F substitution in the tyrosinekinase domain of VEGFR-3. Thismutation is located in a highlyconserved catalytic domain of thereceptor, in close proximity to theVEGFR-3 mutations in human primarylymphedema (Irrthum et al., 2000;Karkkainen et al., 2000).

It has been estimated that using theENU mutagenesis technique, each F1animal is heterozygous for 100 newinactivating mutations (Anderson,2000). Thus, the presence of otherpossible mutations affecting the Chyphenotype cannot be ruled out.However, the other mutations shouldbecome segregated out during thenumerous generations of micescreened in the course of maintainingthe Chy mouse strain. In addition, theinactivating VEGFR3 mutations arecausative for human lymphedema, andalso the phenotypic features in the Chymice are likely to result from the Vegfr3mutations.

Heterozygous Chy pups havechylous fluid in the abdomen after

suckling, indicating possible defects inthe lymphatic system. In common withhuman lymphedema patients, the limbsof Chy mice are also swollen. In IHCanalysis, we found that the superficiallymphatic vessels are hypoplastic inChy mice, whereas the intestinalsubserosal lymphatics are enlargedwhen compared with WT mice.Similarly, the pathology of primarylymphedema is characterized by non-functional (usually hypoplastic oraplastic) superficial lymphatic vessels(Kinmonth et al., 1957), and in somerare human cases, lymphedema isassociated with accumulation ofchylous ascites and enlargement of thesubserosal lymphatic vessels (Lee andYoung, 1953; McKendry et al., 1957).

The molecular mechanisms of thelocal defects in the lymphatic vessels inlymphedema are not well known, butone possible explanation is the regionaldiversity of the lymphatic endothelium.This hypothesis is supported by thefinding that inhibition of VEGFR-3signaling in transgenic mice resulted intransient loss of the lymphatic vessels,but these vessels started to re-growafter three postnatal weeks (Mäkinen etal., 2001). However, there was no re-growth of the skin lymphatics. Such aresult cannot be explained by K14-promoter driven transgene expression,because there are also ligand-saturating levels of the transgene-encoded protein in the bloodstream ofadult transgenic mice. Thus, it ispossible that VEGFR-3 signaling isrequired for embryonic and early post-natal development of the lymphaticvessels, which then become mature orresistant to loss of VEGFR-3 ligandsand use other signals to regenerate.

Some di f ferences in geneexpression within the lymphatic vesselshave been detected. For example,podoplanin is expressed only in

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lymphatic capillaries devoid of asmooth muscle layer (Breiteneder-Geleff et al., 1999). A recent studyshows that the β-chemokine receptorD6 is expressed only in the lymphaticvessels of the skin and intestine and inthe lymph nodes (Nibbs et al., 2001). Inaddition, the β-chemokine receptor D6ligands bind to dermal afferentlymphatic vessels (Hub and Rot, 1998).

Lymphatic vessels in both the skinand in the intestine are located near thesurface of the body, in places that areusually richly supplied with lymphaticvessels. These “superficial” lymphaticvessels function as an importanttransport route for Langerhans cells orDCs, which process pathogenicantigens and carry them to the draininglymph nodes. Chemokine binding to theLECs may be involved in the process ofcell entry into the afferent lymphaticvessels. Thus, the proteins needed forthis are expressed in sites where thereare a lot of lymphocytes and DCtrafficking into the lymphatic vessels.

One possible explanation for thelack of lymphatic hypoplasia in thevisceral organs of the Chy mice is thatVEGF-C interacts with a secondreceptor in these organs. BecauseNRP-2 has been found to bind some ofthe VEGF family members, we testedwhether it could be a receptor forVEGF-C. In our studies, VEGF-C wasable to bind NRP-2. In addition,VEGFR-3 was able to interact with theNRP-2 receptor itself (MK, unpublished

data). Although no direct signaling viaNRP-2 has been detected, it may havea role as a co-receptor, which may besimilar to what has been publishedabout NRP-1 and plexins (Takahashi etal., 1999). One could speculate thatNRP-2 has a role in modulatingVEGFR-3 activity in cells where theyare co-expressed. To support thishypothesis, we detected a strong NRP-2 signal from the intestinal lymphaticendothelium, but we did not detectedNRP-2 staining in the lymphatic vesselsof the skin. These results suggest thatthere is a difference in the VEGF-Creceptor expression between theaffected and unaffected lymphaticvessels, which may contribute to thedevelopment of the lymphedemaphenotype.

Interestingly, Ang2 deficient micealso have defects in the intestinallymphatic vessels (G Thurston, C Suri,G Yancopoulos, personal comm.). Incommon with Chy mice, the peritonealcavity of Ang2 KO mice is filled withchylous ascites, and they exhibitdefects in the intestinal vasculature.The lymphatic vessels of the skin arealso abnormal. Thus, Ang2 is alsoneeded for proper lymphat icdevelopment. These results againsuggest co-operation between theVEGFR and Tie families in regulatingthe ECs, and suggest that signaling viaboth the VEGFR and Tie receptors isrequired for proper development andfunction of the lymphatic vessels.

4 Lymphedema therapy in the Chy model (IV)

As lymphedema patients, the Chy micehave some VEGFR-3 activity due to theWT Vegfr3 allele. We thus wanted toanalyse the possibility of using VEGF-Coverexpression as a therapeutic tool forprimary lymphedema. To explore thisopportunity, we infected the ears of theChy mice by intradermal injections ofadenoviruses or AAVs encoding VEGF-

C. Using either of these viruses, wewere able to induce the growth oflymphatic vessels in the skin. Thenewly formed vessels were alsofunctional as shown by the uptake of afluorescent dye into the collectinglymphatic vessels.

For possible future applications, wealso confirmed that a VEGFR-3 specific

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ligand (VEGF-C156S) (Joukov et al.,1998; Veikkola et al., 2001) is sufficientfor the induction of the growth oflymphatic vessels in the Chy mice.These results indicate that VEGFR-3stimulation with an excess of itsspecific ligand is sufficient to overcomelymphatic hypoplasia caused by themutant Vegfr3 allele.

Where do the newly formedlymphatic vessels originate from in theChy mice? The Chy mice show a lackof lymphatic vessels in the skin,although a few lymphatic vessels havebeen detected in some of the mice.Whole-mount staining also show thatthere are some scattered LYVE-1positive LECs in the skin, but noorganized lymphatic vasculature (G.Thurston and MK, unpublished data).Thus, it is possible that viral VEGF-Cstimulation induces proliferation ofthese pre-existing LECs and theformation of a functional lymphaticvascular network. Alternatively, it istempting to speculate that bone-marrow derived precursors might alsohave a role in this process. It has beensugges ted tha t mesoderma llymphangioblasts originating from thesomites can participate in thedevelopment of the lymphaticvasculature (Wilting et al., 2000). Onthe other hand, it has been shown thatthe angioblasts from the bone marrowalso contribute to the angiogenesis inadults (Reviewed in Carmeliet andLuttun, 2001), and a subset of thesecells express VEGFR-3 (S. Rafii and K.Alitalo, unpublished data). Thus, it ispossible that lymphatic endothelialprecursors exist in adults and thatthese cells may participate in thelymphangiogenic processes uponVEGF-C stimulation.

As VEGF-C gene therapy inducedthe growth of new lymphatic vessels inthe Chy model, we speculated thatsuch a therapy could be used to treatlymphedema. The treatment oflymphedema should be local, but in

many cases lymphedema is detected inlarge areas, e.g. in the lower limb.Potential gene therapy for lymphedemacould involve the administration ofVEGFR-3 ligand in the affected sites ortransfer of remaining lymphatic tissuefrom internal sites to affected skin,combined with growth factoradministration. Alternatively, ex vivoVEGF-C gene transfer into thekeratinocytes could be utilized inattempts to induce the growth of thecutaneous lymphat ic vessels(Christensen et al., 2001).

Yet, an important question iswhether the lymphatic vesselsstimulated by VEGF-C exhibit sufficientdrainage capacity to treat the swellingassociated with lymphedema. Thestability of the new lymphatic vesselsshould also be carefully assessed, andit is possible that long-term growthfactor expression is needed to maintainthe newly formed vessels. In patientswith Milroy’s disease, lymphedema isalready present after birth. Therefore,the treatment should be started asearly as possible to avoid thesecondary changes associated withlymphedema, such as fibrosis,thickening of the skin, and cellulitis.

VEGF-C gene therapy should alsobe evaluated very carefully, becauseVEGF-C also binds VEGFR-2 on theblood vascular endothelium and iscapable of stimulating vascularpermeability and, in some conditions,angiogenesis (Cao et al., 1998; Joukovet al., 1997). Because of the possiblecomplications due to tissue edema oraccelerated tumor angiogenesis, theVEGFR-3 specific growth factor VEGF-C156S (Joukov et al., 1998) wouldtherefore be a more attractive choicefor therapeutic applications. Anadditional concern would be the factthat tumor lymphangiogenesis hasbeen associated with enhanced lymphnode metastasis (Karpanen et al.,2001; Mandriota et al., 2001; Skobe etal., 2001a; Stacker et al., 2001). Thus,

34

treatment of secondary lymphedemaarising after lymphadenectomy maypose a problem, because it couldenhance the growth and spread ofdormant metastases. However, thehalf-life of VEGF-C in the bloodcirculation is short (Veikkola et al.,

2001) and local VEGF-C therapy isthus likely to function without systemiceffects. In the studies of lymphedematherapy, genetic mouse models suchas the Chy mice, will be beneficial andessential.

35

CONCLUDING REMARKS

During the past few years, researchershave been able to answer some of thequestions about lymphatic vesselformation and function, issues whichhave been unknown for decades. Twolymphangiogenic molecules, VEGF-Cand VEGF-D have been identified, andtheir signaling via VEGFR-3 has beenshown to induce lymphangiogenesis.Some other proteins, such astranscription factor Prox1, and cellsurface proteins LYVE-1 andPodoplanin have also been found to beinvolved in regulation and function ofthe lymphatic vessels, although theirexact functions are not yet known.

During embryogenesis, VEGF-Cbecomes restricted to cells adjacent toVEGFR-3 posi t ive endothel ia,suggesting paracrine VEGF-C/VEGFR-3 signaling as a mediator oflymphangiogenesis. In vivo studieshave also indicated that VEGFR-3s igna l i ng i s su f f i c i en t f o rlymphangiogenic response. Inlymphedema, the normal developmentor function of the lymphatic vessels isdisturbed, resulting in hypoplasticsubcutaneous lymphatic network, andaccumulation of fluid into theextremities. Lymphedema is a chronic,disfiguring and disabling condition thathas mainly been treated by manuallymphatic drainage and by compressivecarments.

We have shown that disturbedVEGFR-3 signaling results in hereditarylymphedema in some families. Wehave also described the Chy mousemodel for Milroy’s disease with swellingof the limbs due to a inactivating Vegfr3mutation. Using the Chy mice, we haveshown that a potential gene therapy forlymphedema may involve theadministration of VEGF-C or VEGF-D

to patients who have insufficientVEGFR-3 signaling. Another possibilityis to transfer remaining lymphatic tissuefrom internal sites to affected skin,combined with growth factoradministration. Interesting recentevidence indicates that there may beEC progenitors circulating in the blood,and lymphatic endothelial precursorcells have been detected in avianembryos (Schneider et al., 1999). Suchcells would provide an important toolfor the further development of cell andgene based therapies for lymphedema.Our results suggest that VEGFR-3ligand administration alone or inc o m b i n a t i o n w i t h o t h e rlymphangiogenic factors could be apowerful tool in the therapy of variousforms of human lymphedema. It alsogives a paradigm for treatments ofother diseases associated with mutantRTKs.

The identification of other crucialmodifying factors in the process oflymphangiogenesis will be the nextchallenge in lymphatic research. Thisresearch may be facilitated by thefinding that LECs can be separatedfrom the blood vascular ECs (Kriehuberet al., 2001; Mäkinen et al., 2001).Dif ferential gene and proteinexpression between blood vascularECs and LECs can now be analysed indetail. In addition, it may be possible toisolate LECs from lymphedemapatients in order to gain a moredetailed knowledge of the molecularmechanisms of lymphedema, and todevelop more targeted therapies forthis disease. These methods probablyhelp us in understanding thepathogenesis of different diseases inwhich lymphatic vessels are involved.

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ACKNOWLEDGEMENTS

This work was carried out at theMolecular/Cancer Biology Laboratory,Haartman Institute, University ofHelsinki during the years 1997-2001. Iam grateful to professor Eero Sakselafor creating a pleasant atmosphere atthe Department of Pathology.

I wish to thank my supervisor KariAlitalo for the challenges he set me andfor the opportunity to learn so manynew things during these years. I amalso greatful for the first-class facilitiesprovided by the M/CBL, for the chanceto meet all the people at the field’sfrontiers, and to visit some of the mostinteresting places in the world.

This study would not have beenpossible without an exel lentcollaboration with several people. Iexpress my special thanks to BobFerrell and David Finegold whooriginally initiated the studies on thegenetic alterations in the lymphedemafamilies. I also want to acknowledgeKara Levinson, Elizabeth Lawrence,Mark Kimak, Miikka Vikkula, AlexandreIrrthum, Kalevi Pulkkanen, Seppo Ylä-Herttuala, Mikko Kettunen, KatriPajusola, and Michele McTigue for afruitful collaboration. It has been apleasure to work with you!

I am thankful to Jorma Keskioja,Katri Koli, and Terhi Kulmala for theopportunities provided by the HelsinkiBiomedical Graduate School, and TomiMäkelä and Marikki Laiho for the thesisfollow up.

I warmly thank Juha Partanen andSirpa Jalkanen for reviewing my thesisand for giving beneficial comments.

Mari Elemo, Tarja Taina, EijaKoivunen, Sirke Haaka-Lindgren, andPaula Turkkelin are acknowledged fortheir important assistance with mice,and Ilkka Vanhatalo and Antti Huittinenfor their help with computers.

I want to thank all members of theM/CBL for the time we have spenttogether, and for making the lab a“special” place during these years.Especially I want to thank Anne, foralways cheering me up, for helpfuldiscussions, and for collaboration;Marko for his interesting stories duringour lunch breaks; “pikku”-Kaisa for herenthusiastic attitude, Tapio for all thehelp and for refreshing breaks; Kristiinafor collaborative work; and Lotta forfree time activities and for sharing withme the last months of writing andwriting…

I want to express my warm thanksto Sanna Karttunen, Kaisa Makkonen,Pipsa Yli-Kantola, Paula Hyvärinen,Mari Helanterä, Seija Kajander, MiiaPutkinen, Tarja Pitkänen and Marja-Leena Saastamoinen for yourindispensable help! Special thanks toAlun Parsons for technical help and forchecking the language for so manytimes!

I am obliged to my friends for theircompany and back-up.

My hearty thanks belong to myfamily; my father Eero, my mother Arja,my sisters Pauliina and Mira, mybrother Markus, and my brother-in-lawLauri. Thank you for your love andsupport!

I have been financially supported bythe Finnish Cancer Organization,Finnish Cultural Foundation, IdaMontini Foundation, Emil AaltonenFoundation, Paulo Foundation,Research and Science Foundation ofFarmos, Ella and Georg Ehrnrooth’sFoundation, and City of Kotka.

Helsinki, December 2001

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