In vivo effects and therapeutic potential ofVEGF-C

Anne Saaristo

Molecular/Cancer Biology Laboratory andLudwig Institute for Cancer Research

Haartman Institute and Helsinki University Central HospitalBiomedicum HelsinkiUniversity of Helsinki

Finland

Academic dissertation

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

In the lecture hall 3 of the Biomedicum Helsinki,Haartmaninkatu 8, Helsinki

On August 16th, 2002, at 12 o’clock noon.

Helsinki, 2002

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Supervised by

Kari Alitalo, M.D., Ph.D.Research Professor of the Finnish Academy of SciencesMolecular/Cancer Biology LaboratoryHaartman Institute, Biomedicum HelsinkiUniversity of HelsinkiFinland

Reviewed by

Olli Saksela, M.D., Ph.D.DocentDepartment of DermatologyHelsinki University Central HospitalFinland

Risto Renkonen, M.D., Ph.D.ProfessorDepartment of Bacteriology and ImmunologyHaartman InstituteUniversity of HelsinkiFinland

Opponent

Michael Detmar, M.D.Associate ProfessorDepartment of DermatologyHarvard Medical SchoolBoston, MAUSA

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Contents

ABSTRACT .................................................................................................................................... 7

REVIEW OF THE LITERATURE ............................................................................................ 8

The formation of blood and lymphatic vessel networks during embryonic development....................8Vasculogenesis and angiogenesis................................................................................................................ 8Endothelial cell differentiation.................................................................................................................... 8Lymphangiogenesis................................................................................................................................... 10Characteristics of lymphatic vessels......................................................................................................... 11

Molecular regulation of the blood and lymphatic vessel growth............................................................14VEGF and its receptors.............................................................................................................................. 14VEGF-B and PlGF..................................................................................................................................... 16VEGF-C, VEGF–D and their receptors.................................................................................................... 18Angiopoietins and their Tie-receptors....................................................................................................... 19Ephrins........................................................................................................................................................ 20Lymphatic vessel markers......................................................................................................................... 20

Diseases associated with lymphatic vessel function..................................................................................23Lymphedema.............................................................................................................................................. 23Tumorigenesis and metastasis................................................................................................................... 26

AIMS OF THE STUDY..............................................................................................................30

MATERIALS AND METHODS ............................................................................................... 31

RESULTS AND DISCUSSION................................................................................................. 34

I Expression of VEGF-C and its receptor VEGFR-3 in nasal mucosa and in nasopharyngeal tumors 34

II Expression of VEGF-C, VEGF-D and VEGFR-3 in normal human tissues..................................... 34

III In vivo effect of VEGF-C on blood and lymphatic vessels.............................................................. 35

IV Characterization of the VEGFR-3 specific mutant form of VEGF-C (VEGF-C156S) as alymphangiogenic factor............................................................................................................................ 38

CONCLUSIONS.......................................................................................................................... 42

ACKNOWLEDGEMENTS ........................................................................................................ 44

REFERENCES............................................................................................................................ 45

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Abbreviations

AAV adeno-associated virusAd adenovirusAng angiopoietinBEC blood vascular endothelial cellCAR coxsackie/adenovirus receptorCFTR cystic fibrosis transmembrane regulatorE embyonic dayEC endothelial cellECM extracellular matrixFlk-1 fetal liver kinase 1 (mouse VEGFR-1)Flt-1 fms-like tyrosine kinase-1 (VEGFR-2)Flt-4 fms-like tyrosine kinase-4FOXC2 forkhead box C2HA hyaluronanHEV high endothelial venuleHIF-1 hypoxia-inducible factor 1Ig immunoglobulinkDa kilodaltonKDR kinase insert domain containing receptor (human VEGFR-2)LEC lymphatic endothelial cellLYVE-1 lymphatic vessel endothelial hyaluronan receptor-1mRNA messenger ribonucleid acidNRP neuropilinPDGF platelet-derived growth factorPDGFR platelet-derived growth factor receptorPECAM-1 platelet endothelial cell adhesion molecule-1PlGF placenta growth factorProx-1 prospero-related homeobox protein-1SLC secondary lymphoid organ chemokineTek tunica interna endothelial cell kinase (Tie-2)Tie tyrosine kinase with Ig and EGF homology domains (Tie-1)TK tyrosine kinaseVEGF vascular endothelial growth factorVEGFR vascular endothelial growth factor receptorVPF vascular permeability factor (VEGF)

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List of Original Publications

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

I Saaristo A, Partanen TA, Jussila L, Arola J, Hytonen M, Vento S, Kaipainen A,Malmberg H, Alitalo K. 2000. Vascular endothelial growth factor-C and its receptorVEGFR-3 in nasal mucosa and in nasopharyngeal tumors. Am J. Pathol., 157: 7-14.

II Partanen TA, Arola J * , Saaristo A*, Jussila L, Ora A, Miettinen M, Alitalo K. 2000.VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3in fenestrated endothelia in human tissues. FASEB J., 14: 2087-2096.

III Saaristo A, Veikkola T, Enholm B, Hytonen M, Arola J, Pajusola K, Turunen P, JeltschM, Karkkainen M, Bueler H, Yla-Herttuala S, Alitalo K. Adenoviral VEGF-Coverexpression induces blood vessel enlargement, tortuosity and leakiness, but nosprouting angiogenesis in the skin or mucous membranes. FASEB J., 16: 1041-1049.

IV Saaristo A * , Veikkola T * , Tammela T, Enholm B, Karkkainen M, Pajusola K, BuelerH, Yla-Herttuala S, Alitalo K. Lymphangiogenic gene therapy without blood vascularside-effects. Submitted.

* Equal contribution

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ABSTRACT

The lymphatic vasculature is essential for the maintenance of normal fluid balance and forthe immune response, but it is also involved in a variety of diseases. Hypoplasia ordysfuction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia orabnormal growth of these vessels are associated with lymphangiomas andlymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemicmetastasis of cancer cells. Recent novel findings on the molecular mechanisms involved inlymphatic vessel development and regulation allow the modulation of the lymphangiogenicprocess and specific targeting of the lymphatic endothelium. So far, two peptide growthfactors have been found which are capable of inducing the growth of new lymphatic vesselsin vivo in a process called lymphangiogenesis. These growth factors, VEGF-C and VEGF-Dbelong to the VEGF family of growth factors which also includes VEGF, placenta growthfactor (PlGF) and VEGF-B. VEGF-C and VEGF-D are ligands for the endothelial cellspecific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as inmouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells whichline the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the mainmediator of angiogenesis, VEGFR-3 signaling is crucial for the development andmaintenance of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosinekinase leads to primary lymphedema due to defective lymphatic drainage in the limbs.

In order to develop targeted therapy approaches for diseases involving the lymphaticvasculature it is important to understand the basic biology of the growth factors modulatinglymphatic vessels. The present study was undertaken to characterize the expression patternsof VEGF-C and VEGF-D and their receptor VEGFR-3 in human tissues and to further studytheir in vivo effects on blood and lymphatic vessel growth. VEGFR-3 was confirmed to bespecific for the lymphatic endothelium in most tissues, but its expression was also detected incertain fenestrated and discontinuous blood vessel endothelia. In experimental animal modelsVEGF-C induced lymphatic vessel growth, i.e. was lymphangiogenic, but high levels ofVEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutantform of VEGF-C called VEGF-C156S lacked these side effects but was sufficient fortherapeutic lymphangiogenesis in a mouse model of lymphedema. The results show thatVEGF-C156S is a specific lymphatic endothelial growth factor in the skin and an attractivemolecule for pro-lymphangiogenic therapy.

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REVIEW OF THE LITERATURE

The formation of blood and lymphatic vessel networks during embryonicdevelopment

Vasculogenesis and angiogenesis

All proliferating or developing tissues and tumors, are dependent on oxygen and nutrientssupplied by the vascular system. During embryogenesis, the development of the vascularsystem occurs via two processes, vasculogenesis and angiogenesis (Figure 1). Vasculogenesisinvolves the de novo differentiation of endothelial cells (ECs) from mesoderm-derivedprecursor cells, called hemangioblasts (Risau and Flamme, 1995). The EC precursors(angioblasts) and the hematopoietic cell precursors are thought to be derived from commonprecursor cells. According to this theory, the hemangioblasts aggregate to form primary bloodislands in which the cells in the interior differentiate into hematopoietic stem cells whereas thecells in the periphery differentiate into angioblasts. The angioblasts then cluster andreorganize to form capillary-like tubes. Circulating endothelial progenitor cells (EPCs) havebeen isolated in peripheral blood of adult tissue, and some data suggests that these cells canparticipate in postnatal formation of new blood vessels (postnatal vasculogenesis/angiogenesis) (Asahara et al., 1997; Shi et al., 1998; Springer et al., 1998).

Once the primary vascular plexus is formed, new capillaries form by sprouting or by splitting(intussusception) from pre-existing vessels in the process called angiogenesis (Figure1)(Risau, 1997). The newly formed vasculature is further remodeled into a more mature tree-like hierarchy of vessels containing vessels of different sizes. Excess branches are pruned,some vessels regress and others fuse to form larger ones. In the primary capillary plexus theECs start to differentiate into arterial or venous type (Yancopoulos et al., 1998). ECs becomesurrounded by pericytes and smooth muscle cells and formation of the extracellular matrix(ECM) and particularly the basal lamina gives support to the vessels. In pathologicalangiogenesis maturation and stabilization of the vessels occur improperly and the vesselsremain immature (Hashizume, 2000; Shunichi, 2002). Tumor blood vessels are leaky and asunstable vessels they are dependent on continuous growth factor stimulation for survival.

Endothelial cell differentiation

In adults the blood vessel network consists of a very heterogeneous group of ECs. ECsfunction in a variety of physiological situations, and therefore the capillary endothelium ofeach individual normal tissue is highly specialized (Cotran, 1999; Ruoslahti and Rajotte, 2000).Tumor vasculature has also been shown to express its own specific markers (Ruoslahti andRajotte, 2000). Tissue-specific vascular markers provide new opportunities for the targeting oftherapeutic compounds, such as genes and drugs, to the endothelial cells thus avoidingunwanted systemic toxicity.

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Figure 1 Schematic illustration of vasculogenic and angiogenic processes in developing embryos. Invasculogenesis, mesodermal cells first differentiate into hemangioblasts, whereafter endoderm-mesoderm interactions are required for further blood island differentiation (Risau and Flamme, 1995).After the primary capillary plexus has been formed, new vessels are generated via angiogenesis (Risau,1997). During sprouting angiogenesis, ECs degrade the underlying basement membrane, migrate,proliferate and reassemble into tubes. In non-sprouting angiogenesis, new vessels are formed byintussusceptive growth or the existing vessels increase in size through intercalated growth. The formedvasculature is remodelled into a more mature tree-like hierarchy containing vessels of different sizeswhen excess branches are pruned, some vessels regress and others fuse to form larger vessels. Thevessels further differentiate by recruitment of pericytes and smooth muscle cells. Formation of the extracellular matrix and particularly the basal lamina gives support to the vessels. Arterio-venousdifferentiation is regulated by Ephrin, Notch and Neuropilin family members (Adams, 1999; Wang,1998; Lawson, 2001; Herzog, 2001). Some growth factors or their receptors mediating blood vesselgrowth and maturation are indicated on the right.

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Blood vessels are divided into arteries, arterioles, capillaries, venules and veins depending ontheir size, function and morphology. Capillaries are the smallest vessels and they areresponsible for nutrient and oxygen diffusion to the tissues. The degree of permeability ofcapillaries depends on the nature of the intercellular junctions adjoining the cells, and also onthe transendothelial transport properties and morphology of the ECs (Dejana, 1995). In general,the endothelia of capillaries in adult tissues can be subdivided into three groups: continuous,fenestrated and discontinuous or sinusoidal capillary endothelia (Figure 2). Typical organsthat contain continuous capillary endothelia are skeletal muscle, skin and the central nervoussystem. The exchange of molecules is strictly controlled in these tissues and in the centralnervous system the permeability of blood vessels is further restricted by a special blood brainbarrier.

Fenestrated capillaries are characterized by the presence of fenestrations, special channelsacross the endothelial cells, 80-100 nm in diameter. These channels appear to be closed by athin diaphragm. Fenestrated capillaries also have pinocytotic vesicles. One theory suggeststhat fenestrations are formed when a pinocytotic vesicle spans a narrow cytoplasmic layerand opens, simultaneously, on both surfaces (Ross, 1995). In the gastrointestinal tract and inthe gallbladder the capillaries are thicker and have few fenestrations when absorption is notoccuring. However, during the absorption of nutrients and production of bile in thegallbladder, the numbers of both pinocytotic vesicles and fenestrae increases rapidly (Ross,1995). Fenestrated capillary endothelia can also be found in other sites where there is aspecial need for regulation of blood vessel permeability and molecule transport across the ECwall, including the endocrine glands and nasal respiratory mucosa. Special discontiuous, orsinusoidal, capillary endothelia can be found in the liver, spleen and bone marrow. Basallamina and occasional pericytes are present in continuous and fenestrated capillary endotheliabut in sinusoidal capillaries these structures can be totally absent and unusually wide gapscan be found between ECs.

Lymphangiogenesis

In humans, the first lymph sacs have been found in 6 to 7 week old embryos (van der Putte,1975). This is nearly 1 month after the development of the first blood vessels. There are twotheories about the origin of the lymphatic vessels. A century ago Sabin proposed that theprimitive lymph sacs originate by EC budding from the pre-existing embryonic veins (Figure3) (Sabin, 1902). The peripheral lymphatic system would then spread from these primarylymphatic sacs by sprouting. Later during the development most of the lymph sacsdifferentiate to form primary lymph nodes and the blood is removed from the lymphaticnetwork to the veins as they become functional (Clark, 1912). An alternative model suggeststhat the initial lymph sacs arise in the mesenchyme from precursor cells, independent ofveins, and that the connection to the venous system is formed later in development(Huntington and McClure, 1908). Recently two lymphatic specific markers, VEGFR-3 andProx1, have been show to be expressed in the endothelium lining the budding lymphatic sacsin mouse embryos, supporting Sabin’s theory of lymphatic development (Dumont, 1998;Kaipainen, 1995; Wigle and Oliver, 1999). However, in a quail-chick chimera model mesodermallymphangioblasts, lymphatic precursor cells, were shown to participate in the development of

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the lymphatic system, supporting the theory that the peripheral lymphatic vessels develop bymultiple mechanisms (Schneider, 1999; Wilting, 1999; Wilting, 2000). Whetherlymphangioblasts can participate in lymphangiogenesis in adult mammals is still to bedetermined.

Figure 2. Schematic illustration of capillary endothelia types of blood and lymphatic vessels. In theupper lane light grey/yellow marks pericytes and dark grey/red marks blood vessel ECs. In thecontinuous and fenestrated capillaries the basal lamina is continuous, whereas in the sinusoidalcapillaries the basal lamina is discontinuous. Lymphatic capillaries are irregular and thin-walled andcontain anchoring filaments that attach them to the ECM.

Characteristics of lymphatic vessels

Lymphatic vessels differ from blood vessels in several ways. Lymphatic capillaries areessentially thin-walled and blind-ended endothelial tubes that, unlike typical bloodcapillaries, lack pericytes and continous basal lamina and contain large interendothelial pores(Figure 2) (Barsky, 1983; Casley-Smith, 1980; Ezaki, 1990; Oh, 1997). Lymphatic capillaries alsocontain anchoring filaments that connect the vessels to the ECM (Casley-Smith, 1980). Thesefilaments are thought to maintain the patency of the vessels during increased tissue pressureand inflammation. Due to their greater permeability, lymphatic capillaries are more effectivethan blood capillaries in removing protein-rich fluid from intercellular spaces. In the smallintestine, lymphatic vessels serve as conveyers of large proteins and lipids that can not getacross the fenestrae of the absorptive capillaries (Ross, 1995). Large collecting lymphatic

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Figure 3. Scheme illustrating the formation of lymphatic vessels. A widely accepted theory suggeststhat during the embryonic period lymphatic vessels are generated from the veins. A subpopulation ofthe ECs in the embryonic veins differentiate to lymphatic ECs (lymphatic commitment) and lymphaticsacs are formed by sprouting or budding from the veins in a process that is called“lymphvasculogenesis”. Lymphatic vessels sprout, expand, remodel and establish a blind-ended vesselsystem that is connected to the venous system. In addition, lymphatic precursor cells may differentiateto lymphatic ECs and form new vessels. Transcription factor Prox1 and tyrosine kinase receptorVEGFR-3 are thought to participate in lymphatic differentiation and growth as discussed in laterchapters. Angiopoietins may play role in the remodelling of the primary lymphatic vessel network.

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vessels contain connective tissue and bundles of smooth muscle in their wall as well asvalves, which prevent the backflow of lymph (Ross, 1995). Unlike in the blood vessel systemthere is no central pump in the lymphatic vessel network in mammals. Lymph is driven in thetissues by the compression of the primary lymphatic vessels by adjacent skeletal muscles.Contractility of the collecting lymphatic vessel wall also contributes to lymphatic vesselfunction (Berens von Rautenfield, 1993; Wilting, 1999). When the lymph vessel becomesstreched with fluid the wall of the vessels automatically contracts. Valves in the lymphaticvessels further aid the unidirectional flow in the vessel network. Some tissues, such as brain,retina, bone marrow and cartilage totally lack the lymphatic vessels.

Lymphoid organs such as lymph nodes, tonsils, Peyer’s patches, spleen and thymus, are partof the lymphatic system. Besides fluid transport, the lymphatic system has an important rolein immunological responses. As the lymph circulates in the lymphatic vessels, it passesthrough lymph nodes, where it is exposed to cells of the immune system. In the lymph nodesforeign substances (antigens) are concentrated by the dendritic cells and presented tolymphocytes. This leads to a cascade of steps that results in immune responses. Lymphocytescirculate between the lymphatic and blood vasculature. Lymphocytes that enter lymphaticvessels in peripheral tissues, enter the lymph nodes via the afferent lymphatic vessels.Lymphocytes may also enter the lymph node through the wall of special postcapillaryvenules that are called the high-endothelial venules (HEV). Lymphocytes are thenrecirculated to the blood circulation along with lymph via the efferent lymphatic vessels andthe thoracic duct. Lymphocyte passage across the endothelium is guided by several adhesionmolecules, including the integrins, selectins and their ligands (Butcher, 1999; Kunkel, 2002;Rosen, 1999; Tedder , 1995). The trafficking of the antigen presenting cells, the dendritic cellsand Langerhans cells, from the peripheral tissues to the lymphatic vessels is also regulated bydifferent cell adhesion molecules and chemokine signalling cascades. For example, theactivated dendritic cells upregulate the cytokine receptor CCR7 in order to become sensitiveto secondary lymphoid tissue chemokine (SLC) that is constitutively produced by thelymphatic endothelial cells in the skin (Cyster, 1999; Gunn, 1998). Interestingly, it has recentlybeen shown that certain human breast cancer cell lines also express the CCR7 receptor(Muller, 2001). In an experimental animal model expression of CCR7 enhanced lymphaticmetastasis of the melanoma cells 10-fold as compared to control tumor cells, neutralizinganti-SLC were capable of blocking this effect (Viley, 2001). Tumor cells may therefore usethe same trafficking pathways as the lymphocytes and antigen presenting cells in order togain access to the lymphatic vessels.

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Molecular regulation of the blood and lymphatic vessel growthBoth angiogenesis and lymphangiogenesis are tightly regulated by intercellular signallingmechanisms, growth factors and cytokines. The angiogenic switch, in tumors for instance, isthought to be caused by a shift in the net balance of positively acting angiogenic mediatorsand negatively acting angiogenesis inhibitors. The mechanisms underlying this shift ofbalance are incompletely understood, but several factors including oncogenes, tumorsuppressor genes and hypoxia are known to contribute to the regulation of this balance bycausing up- or down-regulation of endogenous angiogenesis inhibitors and pro-angiogenicgrowth factors (Hanahan, 1996; Kerbel, 1998). Regulation of the lymphangiogenesis is howevermuch less well understood.

VEGF and its receptors

VEGF, or VEGF-A, is a major regulator of vasculogenesis and angiogenesis, but it is alsorequired for generation of blood cells and for homing of leukocytes to sites of inflammation(Ferrara, 1999b; Yancopoulos, 2000). VEGF has been shown to play a role in pathologicalangiogenesis in many diseases, including tumors, psoriasis, rheumatoid arthritis and severalintraocular syndromes (Ferrara, 1999a, Folkman, 1995). VEGF inhibitors are currently beingtested in numerous clinical trials (Ferrara and Alitalo, 1999). On the other hand VEGF genetransfer has been used in proangiogenic therapy trials in ischemic diseases (Blau and Banfi,2001; Isner, 2002; Ylä-Herttuala, 2001).

VEGF is a highly specific mitogen for vascular ECs (Conn, 1990; Connolly, 1989; Ferrara andDavis-Smyth, 1997; Ferrara and Henzel, 1989; Gospodarowicz, 1989; Keck , 1989; Leung, 1989;Plouet, 1989). Inactivation of only a single VEGF allele in mice resulted in embryonic lethaltydue to defective angiogenesis (Carmeliet, 1996; Ferrara, 1996). There is also strong evidencethat VEGF is a survival factor for ECs, both in vitro and in vivo (Alon, 1995; Benjamin andKeshet, 1997; Gerber, 1998; Yuan, 1996). It has been proposed that pericyte coverage of newlyformed vessels is the critical event that determines when ECs no longer require VEGF forsurvival in vivo (Benjamin, 1998).

VEGF is also known as vascular permeability factor, as it is a potent inducer of the vascularleak (Bruce, 1987; Dvorak, 1995; Senger, 1983). It has been shown to induce fenestrations inadrenal cortex capillary ECs in culture (Esser, 1998; Roberts and Palade, 1995). Furthermore,inhibition of VEGF activity by specific monoclonal antibodies reduces vascular permeability(Dvorak, 1995; Mesiano, 1998).

VEGF is expressed as several isoforms of different amino acid chain lengths (VEGF121,VEGF145, VEGF165, VEGF183, VEGF189, VEGF206) that differ in their ability to bind heparinand neuropilin–1 (NRP-1) (Houck, 1991; Jingjing, 1999; Poltorak, 1997; Soker, 1998; Tischer,1991). VEGF121, that fails to bind to NRP-1 and heparin, is a freely diffusible protein (Houck,1992; Soker, 1998). Due to heparin binding, a significant fraction of secreted VEGF165 remainsbound to the extracellular matrix (Houck, 1992). The VEGF isoforms VEGF189 and VEGF206

bind heparin with the highest affinity and are almost completely sequestered in the ECM(Park, 1993). Results from several in vivo models have suggested that VEGF165 is the most

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efficient VEGF isoform for inducing angiogenesis (Grunstein, 2000; Stalmans, 2002). The factthat VEGF165 is present in ECM and as a diffusing molecule may facilite the formation of theconcentration gradient of the ligand that is required for EC migration. In addition, binding tothe NRP-1 receptor may also explain why VEGF165 is a more efficient EC mitogen thanVEGF121.

VEGF receptors

VEGF binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR) receptors in the ECs with highaffinity (Figure 4) (De Vries, 1992; Millauer, 1993; Quinn , 1993; Terman, 1992). Both VEGFR-1and -2 have seven immunoglobulin (Ig) –homology domains in the extracellular domain, asingle transmembrane region and a tyrosine kinase (TK) domain, which is interrupted by akinase-insert domain (Matthews, 1991; Shibuya, 1990; Terman, 1991). Embryos lacking VEGFR-2 fail to develop blood islands, embryonic vasculature and mature hematopoietic cells(Shalaby, 1997; Shalaby, 1995). VEGF mutants and viral VEGF-E that bind selectively toVEGFR-2 are able to induce mitogenesis, chemotaxis and increased vessel permeability invivo (Keyt, 1996; Meyer, 1999; Wise, 1999; Gille, 2001). In vitro VEGFR-2 undergoes strongtyrosine phosphorylation after VEGF stimulation, whereas VEGFR-1 phosphorylation is veryweak (Waltenberger, 1994; Seetharam., 1995). In VEGFR-1 deficient mice there is excess of ECsthat fail to assemble into tubes to form a functional vessel network whereas mice expressingVEGFR-1 lacking the tyrosine kinase domain show no vascular phenotype (Fong,, 1995;Hiratsuka, 1998; Fong, 1999). These studies suggest that during the development VEGFR-1may be non-signalling and function as a regulator of the bioavailability of VEGF. VEGFR-2has been considered to be the key signalling receptor for VEGF in the ECs, but recent resultshave suggested that VEGFR-1 mediated signalling may play an important role inpathological angiogenesis and inflammation (Carmeliet, 2001).

In addition to ECs, VEGFR-1 is expressed in monocytes, macrophages, pericytes, placentaltrophoblasts, renal mesangial cells and in some bone marrow derived hematopoietic stemcells whereas VEGFR-2 can be found in megakaryocytes, platelets, retinal progenitor cells,some hematopoietic stem cells and in circulating endothelial precursor cells (Barleon, 1994;Charnock-Jones, 1994; Sundberg,, 2001a; Clauss, 1990; Katoh, 1995; Ziegler, 1999; Ziegler, 1993;Hattori, 2002). VEGFR-1 signalling induces monocyte migration and recent data suggest thatVEGFR-1 signalling also promotes the survival and recruitment of bone marrow derivedstem cells (Clauss, 1996; Gerber, 2002; Hattori, 2002; Luttun, 2002).

Neuropilins (NRP-1 and -2) are receptors for the collapsin/semaphorin family which regulateneuronal cell guidance (Fujisawa, 1997; Fujisawa, 1998). In past years NRPs have been shownto be expressed in certain blood and lymphatic vessel ECs and they bind to VEGF in anisoform specific manner (Soker, 1996; Soker, 1998; , Karkkainen, 2001; Gluzman-Poltorak, 2000).In the ECs NRPs seem to function as accessory receptors that enhance or regulate thesignalling of VEGFs via VEGF receptors by forming receptor complexes (Soker, 1998;Yamada, 2001; Gluzman-Poltorak, 2001). Mice lacking NRP-1 exhibit deficiences in thedevelopment of the cardiovascular system suggesting that NRP-1 is required for VEGFinduced vasculogenesis and angiogenesis (Kawasaki, 1999; Yamada, 2001). NRP-2 acts as areceptor for splice isoforms VEGF145 and VEGF165 (Gluzman-Poltorak, 2000) NRP-2 isexpressed by human ECs but mice lacking NRP-2 do not have cardiovascular malformations

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(Chen, 2000; Giger, 2000; Gluzman-Poltorak, 2000).

Regulation of the VEGF expression

Expression of VEGF is upregulated by hypoxia and VEGF mRNA is often upregulated nearareas of tumor necrosis (Plate, 1992; Shweiki, 1992). In hypoxic tissues the hypoxia induciblefactor-1 (HIF-1) transcription factor has a central role in inducing the transcription of genesthat are involved in glycolysis and angiogenesis, including VEGF (Gleadle, 1998). VEGFexpression is also stimulated by oncogenes, for example by members of the Ras and erbBfamilies (Okada, 1998; Viloria-Petit, 1997), whereas certain tumor suppressor genes, includingthe LKB1 and the von Hippel-Lindau (vHL) tumor suppressor gene products, appear to limitthe production of VEGF (Gnarra., 1996; Maxwell, 1999; Siemeister, 1996; Ylikorkala, 2001).VEGF expression has also been shown to be regulated by estradiol in human breast cancercells and a functional estrogen response element was identified in the regulatory region of theVEGF gene (Hyder, 2000).

VEGF-B and PlGF

VEGF-B has two splice isoforms, VEGF-B167 and VEGF186, that are differentially expressedwith a predominant expression of VEGF-B167 (Li, 2001; Olofsson, 1996). VEGF-B is a ligandfor VEGFR-1 and its isoforms differ in their binding to heparin and to NRP-1 (Olofsson, 1998;Mäkinen, 1999). Expression of VEGF-B is not upregulated by several studied growth factors,hypoxia or oncogenes (Enholm, 1997). In adult tissues, VEGF-B expression is abundant in theheart and skeletal muscle (Olofsson, 1996). VEGF-B deficient mice are otherwise healthy andfertile, but they display atrial conduction defects or reduced heart size (Aase, 2001; Bellomo,2000). In addition, VEGF-B knock out mice show impaired recovery and vascular functionafter experimentally induced myocardial ischemia (Bellomo, 2000).

PlGF is another member of the VEGF family of growth factors that binds specifically toVEGFR-1 (Maglione, 1991; Park, 1994). PlGF was originally discovered in the human placentaand it has two isoforms, PlGF-1 and -2 (Cao, 1997; Maglione, 1991; Maglione, 1993; Migdal,1998). PlGF-2 is able to bind to NRP-1 and heparin. Both VEGF-B and PlGF formheterodimers with VEGF, and VEGF/PlGF heterodimers have been shown to bind to theVEGFR-2 receptor (Olofsson, 1996b; Cao, 1996; DiSalvo, 1995). In culture PlGF homodimersare chemotactic for monocytes and ECs (Clauss, 1996), but PlGF alone is not capable ofinducing EC proliferation or vascular permeability (Park, 1994). Interestingly, highconcentrations of PlGF that saturate the VEGFR-1 sites for binding, have been shown topotentiate the activity of VEGF both in vivo and in vitro, suggesting that VEGFR-1 may actas a decoy receptor for VEGF in the ECs (Park, 1994). PlGF deficient mice or even doubleknockout mice lacking both PlGF and VEGF-B do not have an obvious phenotype (Carmeliet,2001). However, loss of PlGF impairs angiogenesis, plasma extravasation and collateralgrowth during ischemia, inflammation, wound healing and cancer (Carmeliet, 2001).Transplantation of wild type bone marrow rescued the impaired angiogenesis and collateralgrowth in PlGF deficient mice, indicating that PlGF might contribute to blood vessel growthby mobilizing the bone-marrow derived EC precursor cells (Carmeliet, 2001). Recent reportsalso show that VEGFR-1 is expressed in some bone marrow derived hematopoietic stem cells

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and that PlGF promotes and anti-VEGFR-1 antibody inhibits the recruitment of the myeloidstem cells from the bone marrow (Hattori, 2002; Luttun, 2002). VEGFR-1 is also expressed inmonocyte/macrophages and in pericytes (Sundberg, 2001a; Clauss, 1990). Recent reportssuggest that PlGF and possibly also VEGF-B play role in pathological angiogenesis byincreasing the recruitment of bone marrow derived myeloid and ECs precursor cells,inflammatory cells and pericytes and by enhancing the effects of VEGF (Carmeliet, 2001;Luttun, 2002; Hattori, 2002).

Figure 4. Receptor binding specificity of VEGF family members. VEGFR-2 is the main receptor forVEGF in the ECs. VEGFR-1 signalling mediates monocyte migration and according to recent data,recruitment of VEGFR-1+ stem cells from the bone marrow. Role of VEGFR-1 signalling in the ECs ispoorly defined. VEGFR-3 signalling regulates lymphatic vessel growth. Neuropilins (NRPs) function asisoform specific accessory receptors for some VEGF family members.

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VEGF-C, VEGF–D and their receptors

VEGF-C and VEGF-D were the first characterized growth factors capable of inducinggrowth of new lymphatic vessels in vivo (Achen, 1998; Joukov, 1996; Jeltsch, 1997; Oh, 1997).VEGF-C and VEGF-D activate the endothelial cell -specific tyrosine kinase receptorsVEGFR-2 and VEGFR-3 (Achen, 1998; Joukov, 1996). VEGF-C is mitogenic for lymphaticECs and induces a selective lymphangiogenic response in differentiated avian chorioallantoicmembrane (Oh, 1997). Accordingly, overexpression of VEGF-C or VEGF-D in transgenicmice induces development of a hyperplastic lymphatic vessel network (Jeltsch, 1997; Veikkola,2001). Recent data also suggest that a VEGFR-3 specific mutant of VEGF-C (VEGF-C156S)is lymphangiogenic when overexpressed in the skin of transgenic mice (Joukov, 1998;Veikkola., 2001). Conversely, inhibition of lymphatic growth was obtained when VEGF-C/VEGF-D binding to their receptors was blocked by a soluble form of the extracellulardomain of VEGFR-3 in a similar transgenic mouse model (Mäkinen, 2001a).

Figure 5. Proteolytic processing of VEGF-C (and VEGF-D).. The growth factors are synthesized asprepropolypeptides containing signal sequence, N- and C- terminal propeptides and the VEGF-homology domain (VHD). Proteolytic processing increases the binding affinity to VEGFR-3 and onlythe fully processed 21kDa form of VEGF-C is able to bind to VEGFR-2. The numbers indicate theapproximate molecular masses (kDa) of the corresponding polypeptides under reducing conditions.Modified from Joukov, 1997

Proteolytic cleavage, by as yet uncharacterized proteases, is an important regulator ofreceptor binding and thus, the biological activity of VEGF-C and VEGF-D (Figure 5)(Joukov, 1997; Stacker, 1999). Partially processed forms of VEGF-C and VEGF-D are able tobind and to activate VEGFR-3, while the fully processed short forms are also potentstimulators of VEGFR-2. Presumably, via VEGFR-2 VEGF-C can induce capillary EC

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migration and proliferation in culture (Joukov, 1996; Joukov, 1997) and stimulate angiogenesisin the cornea and ischemic muscle (Cao, 1998; Witzenbichler, 1998). The proinflammatorycytokines IL-1ß and TNF-α upregulate VEGF-C mRNA, whereas both dexamethasone andan IL-1 receptor antagonist inhibited this effect (Ristimaki, 1998). The short form of VEGF-Chas been shown to increase blood vessel permeability in vivo as a recombinant protein(Joukov, 1998). However, very little is known about the proteolytic processing of VEGF-C/Din different tissues.

The VEGFR-3 tyrosine kinase receptor is expressed predominantly in the ECs lining theinner surface of lymphatic vessels in adult murine tissues (Aprelikova, 1992; Galland, 1993;Pajusola, 1992; Kaipainen, 1995). During embryogenesis, VEGFR-3 is first expressed in bloodvascular ECs (Kaipainen, 1995). Accordingly, mice deficient in the VEGFR-3 gene showabnormal remodelling of the primary vascular plexus and die at E9.5 (Dumont, 1998).However, during further development VEGFR-3 is abundant in the lymphatic endotheliumand downregulated elsewhere. The lymphangiogenic effect of VEGF-C/VEGF-D is thoughtto be mediated via VEGFR-3 (Veikkola, 2001). Interestingly, NRP-2, a receptor for variousVEGFs on venous endothelia and semaphorins on neural cells, may act as a co-receptor forVEGF-C in some lymphatic vessels (Herzog,, 2001; Karkkainen, 2001).

Angiopoietins and their Tie-receptors

In addition to VEGFs, angiopoietins have also been shown to play a role in the formation ofthe vascular system. To date there are four known angiopoietins, which all bind to the Tie-2receptor, mediating vessel stabilization signals, whereas the Tie-1 receptor has no knownligand (Davis, 1997; Kim, 1999; Maisonpierre, 1997; Valenzuela, 1999). The phenotypes of Tie-2and Ang1 deficient mice suggest a role for this ligand-receptor system in maintaining thecommunication between ECs and the surrounding mesenchyme, in order to establish stablecellular and biochemical interactions between ECs and pericytes/smooth muscle cells(Dumont, 1994; Puri, 1995; Suri, 1996). An activating mutation of Tie-2 was shown to causehereditary venous malformations characterized by dilatation of blood vessels and deficientsmooth muscle coverage of vessels (Vikkula, 1996). On the other hand, overexpression ofAng1 in the skin of transgenic mice demonstrated that Ang1 can induce a hypervascularphenotype with increase in the size but not number of vessels (Thurston, 1999). Ang1 reducesvascular leakage even in the presence of excess VEGF (Thurston, 1999). Adenovirally mediatedAng1 administration also protected adult vasculature against plasma leakage, but it essentiallylacked the effects on blood vessel morphology seen in the trangenic model (Thurston, 2000).The expression of Ang2, an antagonist of the Tie-2 receptor, has been detected at sites ofactive angiogenesis, including tumors (Holash, 1999; Maisonpierre, 1997). Ang2 is thought toplay a role in destabilizing quiescent adult vessels, and thus to be involved in the initiation ofvascular remodelling. The study of the Ang-2 null mouse has revealed that the angiopoietinsare also likely to play roles in the lymphatic development (Gale, 2002). Mice lacking Ang-2have lymphatic defects, but the expression of Ang-1 in the Ang-2 locus is sufficient to rescuethe lymphatic phenotype, suggesting that both Ang1 and Ang2 may play role inlymphangiogenesis as agonists.

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Ephrins

Recently, Eph receptor tyrosine kinases and their cell-surface-bound ephrin ligands werefound to have a role in defining boundaries between arterial and venous vascular domains(Adams, 1999; Holder, 1999; Wang, 1998). In addition to vascular development, members of thisgrowth factor family also participate in the regulation of axon guidance and bundling in thedeveloping brain, control of cell migration and adhesion, and in the tissue patterning of theembryo (Wilkinson, 2001). A characteristic feature of the Eph/ephrin family is that membranebound tyrosine kinase receptors bind to their multimeric, membrane bound ligands, whichresults in bidirectional signalling between the interacting cells (Schmucker and Zipursky, 2001).Ligands of the Ephrin B family were also found to induce capillary sprouting in vitro (Adams,1999). Interestingly, the phenotypes of the mice lacking ephrin-B2 or EprinB4 resembles thephenotype of the mice lacking either Ang1 or Tie-2 (Adams, 1999; Gerety, 1999; Wang, 1998).Deficient signalling of both cascades leads to aberrant vessel remodelling and sprouting andto abnormal heart trabeculation, suggesting that the Ang1/Tie2 and ephrin-B2/EphB4signalling cascades may interact.

During development NRP-1 expression is restricted to the arteries whereas NRP-2 isexpressed in veins, suggesting that neuropilins may also play role in arterio-venousdifferentiation (Herzog, 2001). In addition, signalling via Notch induces expression of arterialgenes and suppresses venous specific genes (Lawson, 2001)

Lymphatic vessel markers

The first imaging techniques of the lymphatic vessels involved injection of dyes that arespecifically taken up by the lymphatic vessels. Dyes, such as Patent Blue and fluorescentconjugates of high molecular weight material, including FITC-dextran, are still used both inpatient and animal work. Until recently, immunohistochemical identification of the lymphaticvessels has been somewhat complicated. The small lymphatic vessels lack a continuous basallamina and based on this finding lymphatic capillaries have been identified by usingantibodies that stain basement membranes, including antibodies against type IV collagen orlaminin (Barsky, 1983). The lymphatic endothelium also contains a specific types of ECadhering junction. One component in these junctions is desmoplakin, a feature that can beused to identify lymphatic vessels (Schmelz and Franke, 1993). Furthermore, 5’nucleotidaseactivity of the lymphatic endothelium has been used in several histochemical studies (Kato,1990; Shimoda, 2001). In frozen sections of human tissues, double staining for blood vesselspecific marker PAL-E and some panendothelial cell marker can also be used to define thelymphatics (Schlingemann, 1985).

VEGFR-3 was the first lymphatic endothelial cell (LEC) marker found, but more recentlyother LEC markers have also been characterized (Table 1). The transcription factor Prox1has been shown to be required for the programming of LEC differentiation duringembryogenesis (Wigle, 2002; Wigle and Oliver, 1999). Prox1 is expressed in a subpopulation ofthe ECs that are budding and sprouting from the embryonic veins to give rise to lymphaticsacs, Prox1 deficient mice are devoid of lymphatic vasculature (Wigle, 2002; Wigle and Oliver,1999). Prox1 is expressed in a variety of different cell types but among endothelial cells itsexpression is restricted to the lymphatic endothelium (Wigle and Oliver, 1999).

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The lymphatic endothelial hyaluronan receptor (LYVE-1) is a CD44 homologue that wasidentified as a cell surface protein specific for LECs and activated tissue macrophages(Banerji, 1999; Jackson, 2001). However, LYVE-1 is also expressed by sinusoidal ECs in theliver and spleen and some blood capillary ECs in the lung (Carreira, 2001; T. Partanen, D.Jackson personal communication). LYVE-1 binds hyaluronan (HA), an abundant tissueglycosaminoglycan, that plays a role in the maintenance of tissue integrity and in cellmigration (Jackson, 2001). In the lymphatic vessels, LYVE-1 seems to play a role intransporting HA across the lymphatic vessel wall (Jackson, 2001). Further studies shouldreveal whether LYVE-1-HA interactions are involved in leukocyte migration and tumormetastasis.

Another recently described novel marker for the lymphatic endothelium is podoplanin(Breiteneder-Geleff, 1999). In addition to the lymphatic endothelium, this surface glycoproteinis expressed in several other cell types including kidney podocytes, osteoblastic cells andlung alveolar cells (Wetterwald, 1996). The function of podoplanin in the lymphaticendothelium is not known. Detailed comparison of the expression patterns of Prox1, VEGFR-3, LYVE-1 and podoplanin in different endothelia and in the tumor vasculature requiresfurther study.

Table 1. Lymphatic vessel markers (Adapted from Jussila 2002)Marker Protein class Biological effectVEGFR-3 Receptor tyrosine kinase on ECs Lymphangiogenesis

Survival of LECs

LYVE-1 Receptor for extracellular matrixglycosaminoglycan

Transport of hyaluronan fromtissues to lymph nodes(?)

Podoplanin Integral membrane mucoprotein unknown

Prox1 Homoebox transcription factor Involved in the budding andsprouting of lymphatic vesselsduring development

β-chemokinereceptor D6

Chemokine receptor in the afferentlymphatics

Leukocyte recirculation

Macrophagemannose receptor

Receptor in macrophages, lymphoidorgands, lymphatic endothelial cells,perivascular microglia and glomerularmesangial cells

Phagocytosis of microbes, viralendocytosis

Desmoplakin Component of intercellular adherentjunctions

Cell-cell adhesion of LECs

Other markers of LECs include the macrophage mannose receptor (MR) and β-chemokinereceptor D6 (Irjala, 2001; Linehan, 1999; Nibbs, 2001). Besides being present in the lymphaticendothelium the mannose receptor is also expressed in several non-endothelial cell types

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(Linehan, 1999). In the lymphatic vessels the interaction between MR and L-selectin seems tomediate lymphocyte binding (Irjala, 2001). The β-chemokine receptor D6 is expressed in asubset of lymphatic vessels. In lymph nodes, D6 immunoreactivity is present on the afferentlymphatic vessels suggesting that it may influence the chemokine-driven recirculation ofleukocytes through the lymphatic vessels (Nibbs, 2001).

In addition, there are also several other molecules which have been reported to be importantin lymphatic development, such as the transcription factor Net, integrin α9β1 and Ang-2(Ayadi, 2001b; Huang, 2000; Gale, 2002). Recently, methods to isolate and culture LECsseparately from the blood vascular endothelial cells (BECs) have been published (Kriehuber,2001; Mäkinen, 2001b). Further studies of gene and protein expression patterns of these twoisolated cell populations should result in discovery of new lymphatic specific markers.

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Diseases associated with lymphatic vessel function

Impairment of lymphatic function is involved in various diseases, characterized byinadequate transport of interstitial fluid, edema, impaired immunity and fibrosis (Rockson,2001). On the other hand, abnormal proliferation of lymphatic endothelial cells takes place inlymphangiomas, lymphangiosarcomas and possibly in Kaposi’s sarcoma (Witte, 1997).Lymphatic vessels also serve as an important route for tumor metastasis. Very little wasknown about the molecular mechanisms behind lymphatic diseases until very recently. Thefirst gene mutations causing human lymphedema have now been found and several mousemodels have facilitated the development of new therapeutic applications for lymphedema.Animal tumor models have also been used to analyse mechanisms of tumor metastasis and totest strategies for the inhibition of metastasis.

Lymphedema

Lymphatic vessels play a key role in the immune response to various antigens and inmaintaining fluid homeostasis in the body. Blockage of lymphatic drainage or an abnormaldevelopment of the superficial lymphatic vessels leads to lymphedema, which ischaracterized by a disfiguring and disabling swelling of the extremities (Witte, 1997).Lymphedemas can etiologically be divided into two main categories. Primary lymphedemasare rare developmental disorders whereas more common secondary lymphedema syndromesare caused by infections, surgery or trauma. Secondary lymphedema can develop as the resultof inflammatory or neoplastic obstruction of the draining lymphatic vessels or for exampleafter breast cancer surgery. Approximately 35% of primary lymphedema patients have afamily history of the disease and it has been estimated that 1:6000 newborns develop primarylymphedema, with a sex ratio one male to three females (Dale, 1985). Secondary lymphedemais a relatively common disorder and it has been estimated that there are 3 to 5 million patientswith secondary lymphedema in the US. Lymphatic filariasis is the second leading cause ofpermanent and long-term disability globally. Lymphatic filariasis is caused by a parasiticinfection of the lymphatic vessels and may lead to massive edema and deformation of thelimbs or genitals (Witte, 1997). According to the World Health Organization (WHO), over120 million people suffer from filarial lymphedema worldwide. In all its forms, lymphedemais a chronic disease in which persistent dysfunction of the lymphatic vessels gradually resultsin dermal fibrosis, thickening of the skin and accumulation of adipose tissue.

Genetic alterations in lymphedema

The molecular pathogenesis of various lymphedema phenotypes has been unclear, but recentreports indicate several chromosomal regions and genes which are involved in thedevelopment of lymphedema. Congenital hereditary lymphedema (Milroy’s disease) waslinked to the VEGFR3 region on the distal chromosome 5q and missense mutations thatinactivate VEGFR-3 were found to be involved in the disease (Evans, 1999; Ferrell, 1998;Irrthum, 2000; Karkkainen, 2000; Witte, 1998). While mutations which inhibit the biologicalactivity of VEGFR-3 are one cause of primary lymphedema, there are several families with

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Milroy’s disease and other lymphedema syndromes, which involve other genetic loci (Table2). It is estimated that 5% of patients with primary lymphedema carry a mutation in theVEGFR3 gene (D. Finegold, R. Ferrel, personal communication). For example, FOXC2 genemutations result in the rare hereditary lymphedema-distichiasis syndrome (Fang, 2000).Characterization of other genes involved in the development of lymphedema syndromes willgive us more insight into the molecular mechanisms of lymphedema.

Table 2. Genetic alterations in lymphedema syndromesLymphedemasyndrome

Age at onset Gene loci Gene References

Milroy’s disease congenital 5q34-q35 VEGFR3 (Ferrell, 1998)(Evans, 1999; Irrthum,2000; Karkkainen, 2000;Witte, 1998)

Lymphedema-distichiasis

puberty 16q24.3 FOXC2 (Bell, 2001; Fang, 2000;Finegold, 2001)

Cholestasis-lymphedemasyndrome

puberty 15q Not known (Bull, 2000)

Turner syndrome congenital Xp11.2-p22.1

Not known (Zinn, 1998)

Noonan syndrome congenital 12q24.1 PTPN11(SHP-2)

(Tartaglia, 2001; White,1984; Witt, 1987)

(http://www3.ncbi.nlm.nih.gov/Omim/)

Lymphedema mouse models

Several experimental models of secondary lymphedema have been described includinglymphedema in the mouse tail (Swartz, 1996), rat hindlimb (Kriedman, 2002; Lee-Donaldson,1999), or rabbit ear (Casley-Smith, 1977; Piller, 1978; S. Rockson, personal communication). Inaddition, two existing mouse strains show phenotypes of primary lymphedema. In the Chymouse model, an inactivating VEGFR-3 mutation results in persistent hypoplasia of thesuperficial lymphatic vessels. The subserosal lymphatic vessels are enlarged in these mice,and this leads to formation of chylous ascites shortly after birth (Karkkainen, 2001). In anothermouse model, overexpression of the soluble extracellular domain of VEGFR-3 in mouse skincompetes for VEGF-C/VEGF-D binding with the endogenous receptor, leading to regressionof developing lymphatic vessels in several organs and resulting in lymphedema (Mäkinen,2001a). However the lymphatic vessels regenerate during later postnatal development in mostorgans, except in the skin. As in human lymphedema patients, both these mouse models showswelling of the limbs due to hypoplastic/aplastic cutaneous lymphatic vessel network. Thus,these animal models provide us with tools to develop and test new therapies for lymphaticdysfunction.

Prolymphangiogenic gene therapy

Development of strategies for local and controlled induction of lymphangiogenesis couldbenefit the development of treatment for both primary and secondary lymphedema. Thediscovery of specific genes and signalling cascades involved in regulation of lymphaticvessel growth and in pathogenesis of lymphatic dysfunction have established a basis for the

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development of new targeted treatments

Previously, proangiogenic gene therapy in humans has shown promise in the treatment ofcardiovascular ischemic diseases (Blau and Banfi, 2001; Isner, 2002; Ylä-Herttuala, 2001).Angiogenesis has been stimulated by overexpression of VEGF or various fibroblast growthfactors, using several different gene transfer vectors (Asahara et al., 1995; Ferrara and Alitalo,1999). However, while VEGF is a potent inducer of angiogenesis, the vessels it helps tocreate are immature, tortuous and leaky, often lacking perivascular support structures(Carmeliet, 2000; Epstein, 2001). In addition, only a fraction of the blood vessels induced inresponse to VEGF in the dermis and in subcutaneous fat tissue are stabilized and functionalin the skin (Pettersson, 2000; Sundberg, 2001b), and intramuscular vessels develop into anangioma-like proliferation or regress (Pettersson, 2000; Springer, 1998). Furthermore, edemainduced by VEGF overexpression complicates VEGF-mediated neovascularization, althoughrecent evidence suggests that it can be avoided by providing angiopoietin-1 for vesselstabilization (Thurston, 2000; Thurston, 1999).

Vectors for gene therapy

When specific gene mutations of diseases are known, approaches to treat these diseases bytargeted gene therapy may be developed. Gene therapy is defined as the introduction ofgenetic material into cells in order to achieve a therapeutic effect. Gene therapy can be usedin single gene disorders, such as cystic fibrosis, to replace the function of the mutated gene(Flotte, 2001), or in cancer, to deliver a suicide gene to tumor cells (Alavi, 2001). There arecurrently two types of vector systems used for gene therapy, viral and non-viral. In addition,gene transfer can also be done ex vivo, by modifying cells from the patient in vitro and thentransplanting cells back to the target tissue.

Both viral and non-viral gene transfer vectors have been used in gene therapy trials in man.For the most part, viral vectors are more effective than non-viral vectors for achieving high-efficiency gene transfer. Non-viral vectors include liposome complexes and DNA conjugates.These are both easy to produce, non-pathogenic and have been used in cardiovascular genetherapy approaches because in this group of diseases the target tissue can be easily reachedthrough the vasculature (Isner, 2002; Ylä-Herttuala, 2001).

The most commonly used viral vectors include retroviruses, adenoviruses, adeno associatedviruses, lentiviruses and herpesviruses. Retroviruses can lead to stable integration of thetransfected gene into the host genome and produce long-lasting gene expression (Miller,1988). However, retroviruses can deliver the transgene only to proliferating cells, which limitstheir use (Miller, 1990). Currently retroviral vectors derived from the group of lentiviruses(such as HIV-1) are being developed. The property of lentiviruses also facilitates retrovirallymediated gene transfer to quiescent cells (Zufferey, 1997).

Recombinant adenoviruses are efficient and commonly used gene transfer vectors.Adenoviruses can infect a wide variety of different cell types including both quiescent anddividing cells (Berkner, 1988). In the first generation of recombinant adenoviruses the viralE1 gene region, that is crucial for the expression of other viral genes, is deleted, whichmakes virus replication deficient (Bett, 1993). Adenoviruses enter the cytoplasm by binding to

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specific coxsackie/adenovirus receptors (CAR) and by secondary interaction with membersof the intergrin family, which leads to the internalization of the virus (Roelvink, 1999; Stewart,1997; Tomko, 1997; Wickham, 1993). Inside the cell, adenoviral proteins destroy the lysosomeand transgenes are transported to the nucleus. However, adenoviral transgenes remainextrachromosomal in the cell and transgene expression is lost within a month, due to theimmune response directed towards the remaining viral proteins of the vector (Yang, 1996).

Current research with adenovirus vectors is focusing on strategies to circumvent the hostimmune response to gain long term persistent transgene expression. Second generationadenoviral vectors contain less viral genes and are therefore less immunogenic to the infectedhost. Replication-competent adenoviruses are also being developed for gene therapy. Forexample, the E1A gene can be inserted into a first generation recombinant adenovirus codinga suicide gene under the regulation of a tumor-specific promoter (Miller, 1996). In theory,when this kind of virus is inserted to tumor tissue, it could replicate specifically in tumorcells and destroy the tumor (Miller, 1996).

Whereas the adenoviral gene transfer only provides short term expression, adeno associatedviruses (AAVs) provide transgene expression that may last for over a year (Daly, 2001).AAVs are non-pathogenic human viruses, which do not elicit an inflammatory reaction or acytotoxic immune response, and they infect both dividing and non-dividing cells of severalorgans (Reviewed in Monahan and Samulski, 2000). Viral entry to the cells is mediated byseveral cell surface receptors, including heparan sulfates, αvβ5 integrins and FGFR-1(Qing,1999; Summerford, 1999; Summerford and Samulski, 1998). AAVs are naturally replicationincompetent and they require additional genes from the helper viruses (adeno or herpesviruses) for their replication (Monahan and Samulski, 2000). In addition, in the recombinantAAVs viral rep and cap elements needed for virus production are deleted and therefore, inorder to replicate, recombinant AAV requires co-infection with both the helpervirus and thewildtype AAV(Bordignon, 1995). One of the major limitations of AAV vectors is the limitedinsert capacity of approximately 4.7 kb (Kremer, 1995). The wild type AAV integrates to hostchromosome 19. The integration of the recombinant AAVs is not known but there is possibleconcern of insertional mutagenesis (Monahan and Samulski, 2000). Despite this, recombinantAAV encoded Factor IX and CFTR (cystic fibrosis transmembrane regulator) gene transfershave been successfully used to treat hemophilia B and cystic fibrosis, respectively (Kay, 2000;Wagner, 1999). Studies have confirmed that AAV also gives long-term expression in man.

Tumorigenesis and metastasis

Tumorigenesis and tumor metastasis are multistep processes, and accumulation of severalgenetic mutations are required for both processes. For a tumor cell to metastasize it mustpass through several barriers and finally survive and grow in the target tissue. First tumorcells must enter the vasculature of the primary tumor. VEGF and bFGF, secreted by thetumor cells, induce the expression of plasminogen activators and collagenases, contributingto the degradation of basement membranes (Kalebic, 1983; Nagy, 1989). Because lymphaticvessels start out as thin-walled, blind-ended sacs in extracellular tissue, in general, they canbe more easily penetrated by tumor cells than the blood vessels. However, tumor bloodvessels are abnormal, with fragmented and leaky basement membranes and according to

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some reports a considerable percentage of tumor blood flow is in direct contact with thetumor cells (Hashizume, 2000). After overcoming the first vascular barrier the tumor cellshave to survive the blood or lymphatic vessel circulation and attach to the new target tissue(lymph node or other target organ). Furthermore, in order to form a macrometastasis,micrometastatic cells must also be able to induce angiogenesis in the target tissue. Thereforemany factors, such as proteolytic and migratory activity, the expression of adhesionmolecules and the deposition of the ECM surrounding the stromal and tumor cells contributeto tumor growth and metastasis.

Most human tumors have their own characteristic way of metastasizing via lymphatic orblood vessels to specific target tissues. The mechanisms determining these characteristics indifferent tumor types remain poorly understood. In addition, certain tumor types rarelymetastasize. While angiogenesis is required for tumor growth (Folkman, 1971), it is not yetclear to what extent active lymphangiogenesis occurs in human tumors. In tumors,identification of lymphatic vessels is difficult. Of the known lymphatic specific markers, atleast VEGFR-3 and LYVE-1 have been shown to be expressed in some tumor blood vessels(Niki, 2001; Partanen , 1999; Valtola, 1999, Padera, 2002). According to some analyses,functional lymphatic vessels are absent from the interior of solid tumors, possibly due tocollapse of the vessels caused by the interstitial pressure induced by growing cancer cells(Leu, 2000; Padera, 2002). Functional, enlarged lymphatics are, however, often detected in thetumor periphery (Padera, 2002). In principle, tumor cells can either directly invade pre-existing lymphatic vessels or new lymphatic vessels formed at the tumor periphery by tumorinduced lymphangiogenesis. As discussed in the previous chapter, tumor cells may use thesame trafficking pathways, chemokines and adhesion molecules as lymphocytes and antigenpresenting cells in order to gain access to the lymphatic vessels (Muller, 2001; Viley, 2001).

Role of VEGF-C and VEGF-D in tumors

Recent studies using experimental cancer metastasis models have characterized the possibleroles of VEGF-C and VEGF-D in tumor biology. To characterize the role of the VEGF-C intumor metastasis, mice overexpressing VEGF-C under the rat insulin promoter (Rip) weregenerated. These mice were then mated with mice that spontaneously develop pancreatic betacell tumors as a consequence of SV40 large T antigen oncogene expression driven by thesame Rip promoter (Mandriota, 2001). The tumors of Rip1Tag2 mice are capable of localinvasion, but do not induce lymphangiogenesis nor do they form metastases (Hanahan, 1985).In the double transgenic model VEGF-C induced excessive lymphangiogenesis around thepancreatic beta-cell tumors and this resulted in metastatic spread of tumor cells to pancreaticand regional lymph nodes (Mandriota, 2001). These data suggest that the VEGF-C-inducedincrease in peritumoral lymphatic vessels makes the lymphatics more accessible to the tumorcells. Similarly, human breast cancer cells expressing ectopic VEGF-C were shown to inducelymphangiogenesis in and around the implanted tumor cells resulting in enhanced tumormetastasis to regional lymph nodes (Karpanen, 2001; Skobe, 2001a), and to the lung (Skobe,2001a). Importantly, in the breast cancer model, VEGF-C-induced lymphangiogenesis andintralymphatic tumor growth were inhibited by adenoviral expression of the soluble VEGFR-3 protein (Karpanen, 2001). In the breast cancer models however VEGF-C did not have asignificant effect on tumor angiogenesis (Karpanen, 2001; Skobe, 2001a), whereas in a human

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malignant melanoma xenoplant model overexpression of VEGF-C resulted in both tumorlymphangiogenesis and angiogenesis (Skobe, 2001b). VEGF-D has also been shown topromote the metastatic spread of tumor cells via the lymphatic vessels (Stacker, 2001). Inaddition, VEGF-D secreting tumors had an increased growth rate and tumor angiogenesis.The increased tumor angiogenesis, tumor growth and lymphatic metastasis were inhibited byneutralizing antibodies against VEGF-D. The differences between tumor angiogenicproperties of VEGF-C and -D in different studies may be related to differences in proteolyticprocessing of these growth factors in different tumor types.

More recent tumor animal models have shown that VEGF-C overexpressing tumors exhibit anincrease in lymphatic metastasis but no increase in the (hematogenous?) lung metastasis (He,2002; Padera, 2002). Similarly, blocking of VEGFR-3 signalling by the soluble receptor bodysuppressed tumor lymphangiogenesis and lymph node metastasis, but it did not have an effecton lung metastasis ( He, 2002). Thus mechanisms of lymphatic and lung metastasis seem todiffer, at least in some tumor types. In addition, VEGF-C overexpression did not significantlyalter migration of the tumor cell lines in vitro, nor could it convert a low metastatic N15human lung carcinoma cell line to an aggressive metastatic phenotype (Padera, 2002; He, 2002).Tumor growth and metastasis are multistep processes and overexpression of VEGF-C seemsto be necessary but not sufficient for tumor metastasis.

Several recent reports have suggested a correlation between VEGF-C expression andlymphatic metastasis in human tumors (Akagi, 2000; Bunone, 1999; Kitadai, 2001;Kurebayashi, 1999; Niki, 2000; Ohta , 1999; Tsurusaki, 1999; Yonemura, 2001). Less isknown about the presense of VEGF-D in human tumors (Achen, 2001; Kurebayashi, 1999,White, 2002). However, it is still unknown whether VEGF-C and VEGF-D expression canpromote lymphangiogenesis in human tumors and if this increase could then translate into ahigher rate of metastasis. Activation of the endothelium of lymphatic vessels in the tumorperiphery by growth factors or cytokines secreted by the tumor cells may also promote theinteraction of tumor cells with the lymphatic vessels and thus facilitate tumor metastasis.Furthermore, both VEGF-C and VEGF-D increase vascular permeability and the resultingincreased interstitial pressure could facilitate tumor cell entry to both blood and lymphaticvessels. Although the preliminary results from animal models and patient studies havesuggested that VEGF-C and VEGF-D are involved in tumor metastasis, further studies arerequired to evaluate the role of lymphatic endothelial growth factors in human tumors.

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AIMS OF THE STUDY

Previous studies have shown that VEGFR-3 is expressed specifically in lymphaticendothelial cells and that its ligands VEGF-C and VEGF-D induce lymphangionesis in vivoin transgenic mouse models. In human lymphedema patients, heterozygous inactivation ofVEGFR-3 has been shown to lead to primary lymphedema due to defective lymphaticdrainage in the limbs. The study presented here was carried out to characterize the expressionpatterns of VEGF-C, VEGF-D and VEGFR-3 in human tissues and to analyse in vivo effectsof different VEGF-C forms in blood and lymphatic vessels when applied locally by viralgene transfer.

The specic aims of the studies are listed here:

I Expression of VEGF-C and its receptor VEGFR-3 in the nasal respiratory mucosaand in nasopharyngeal tumors

II Expression patterns of VEGF-C, VEGF-D and VEGFR-3 in other human tissues

III In vivo effects of viral VEGF-C on blood and lymphatic vessels in the skin andmucous membranes

IV Comparison of the effects of the native VEGF-C and the VEGFR-3 specific mutantform of VEGF-C, VEGF-C156S

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MATERIALS AND METHODS

I,II: Analysis of VEGF-C, VEGF-D and VEGFR-3 expression in thehuman tissues and human nasopharyngeal tumors

In order to characterize the expression patterns of VEGF-C, VEGF-D and VEGFR-3 inhuman tissues and in nasopharyngeal tumors, fetal and adult patient samples were collectedand analysed by immunohistochemistry with different antibodies. Some of the results wereconfirmed by comparative analysis of murine tissues.

Tissue samples

Fetal tissues were obtained from legal abortions of healthy women induced by prostaglandinsand the gestational age was estimated from the foot length (Munsick, 1984). Adult patientsamples were obtained from surgical specimens directly from the Department of Oto-rhino-laryngology and from the Department of Pathology. All the studies were approved by theEthical Committee of the Helsinki University Central Hospital. The tissues were either fixedin 4% paraformaldehyde or frozen in liquid nitrogen. The number of samples in each grouphas been mentioned in publications I and II.

mRNA expression

Total RNA was isolated from frozen nasal and nasopharyngeal normal and tumor tissuesamples and Northern analysis was performed using probes for VEGF-C. Human endocrinesystem Northern blot (Clontech) was used to compare VEGF-C and VEGF-D mRNA levels invarious endocrine organs.

Immunohistochemistry

Expression of VEGF-C, VEGF-D and VEGFR-3 was studied in the samples usingimmunohistochemical analysis with several antibodies mentioned in publications I and II. Bothparaffin embedded and frozen sections were used. VEGF-C and VEGF-D antibodies werevalidated by immunofluorescence staining of VEGF-C and VEGF-D transfected 293 EBNAcells.

Confirming the VEGF-C and VEGFR-3 expression patterns in the nasal mucosa by InSitu Hybridization and by ββββ-Galactosidase staining of mouse embryos with the markergene.

In Situ Hybridisation of sections from E16.5 mouse embryos using probes for mouse VEGF-Cand VEGFR-3 was performed as described (Kaipainen, 1995). We also characterized theexpression pattern of VEGFR-3 in the nasal mucosa using mouse embryos in which oneVEGFR-3 allele is replaced by LacZ marker gene by knock-in strategy (Dumont, 1998).Pregnant mice were sacrificed at E16.5 and the embryos were dissected and stained for β-Galactosidase.

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III, IV: In vivo effects of native VEGF-C and VEGFR-3 specific form ofVEGF-C.

After analysis of the expression patterns of VEGF-C and VEGFR-3 in human tissues, westudied the in vivo effects of the full length VEGF-C on blood and lymphatic vessels in the skinand respiratory tract. We compared the effects of adeno- and adeno associated virus (AAV)mediated gene transfer of the full length VEGF-C, combination of VEGF-C and Ang-1 or acontrol virus in the mouse (III ). After this study we wanted to compare the effects of differentVEGF-C forms in viral and transgenic animal models (IV ). We compared the effects of thenative full length VEGF-C and the VEGFR-3 specific mutant form of VEGF-C, called theVEGF-C156S. In this mutant Cys156 of the native full length VEGF-C is replaced by Serresidue by mutagenesis (Joukov, 1998). This single point mutation causes loss of VEGFR-2binding making the VEGF-C156S a specific agonist for VEGFR-3.

Viral gene transfer

For the adenovirus constructs, the full length human VEGF-C, VEGF-C156S and Ang1cDNAs were cloned under the CMV-promoter. Replication-deficient E1-E3 deletedadenoviruses were produced in 293 cells and concentrated by ultracentrifugation. As acontrol virus we used adenovirus encoding nuclear targeted LacZ. For the AAV construct,the full length human VEGF-C and VEGF-C156S were cloned under the CMV-promoter andthe rAAVs (AAV serotype 2) were produced. AAV encoding enhanced green fluorescentprotein (EGFP) was used as a control.

Animal models

Immunodeficient athymic nu/nu mice were used in order to study the in vivo effects ofdifferent VEGF-C forms and the combination of VEGF-C and Ang1 in the skin andrespiratory mucosa. 1x108 – 9x108 pfus of adenoviruses or 5x109-1x1011 pfus of AAV wereinjected either intradermally to the ear or to the nasal cavities (only adenoviruses) of themice. The analysis of the infected nu/nu mice was performed 2 days to 10 weeks afterinfection. By using the Chy lymphedema mouse model our group had previously shown thatnative VEGF-C gene transfer could be used as pro-lymphangiogenic gene therapy(Karkkainen, 2001). In common with certain patients with Milroy’s disease, the Chylymphedema mice have a germline inactivating mutation of VEGFR-3 and they are lackingthe superficial lymphatic vessels of the skin (Karkkainen, 2001). The Chy mice do howeverhave one functional VEGFR-3 allele left, making VEGF-C gene therapy feasible (basic ideaof the VEGF-C gene therapy is explained in the Figure 6). In order to analyze the therapeuticpotential of the VEGFR-3 specific mutant form of VEGF-C (VEGF-C156S) we comparedthe pro-lymphangiogenic effects of the native VEGF-C and VEGF-C156S in the Chylymphedema mice skin using AAV-vectors.

Analysis of the blood and lymphatic vessels

The blood vessels of the mouse skin and nasal cavity were visualized either by staining lectinperfused ears or by whole mount immunohistochemistry using antibodies against PECAM-1.

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To visualize the lymphatic vessels we performed whole mount immunohistochemistry usingbiotinylated polyclonal VEGFR-3 antibodies. The function of the lymphatic vessels in the skinwas analyzed by fluorescent microlymphangiography. We confirmed the results of the wholemount analysis by staining the histological sections with antibodies against the mousepodoplanin, LYVE-1, VEGFR-3 and PECAM-1, that can be used as lymphatic and bloodvessel markers.

The effects on permeability of VEGF-C and VEGF-C156S and the combination of VEGF-Cand Ang1 was studied by comparing the leakage of the Evans blue dye in ear skin wheninjected in the tail vein of mice. Statistical analysis of the results of the permeability assaywas carried out by the Students t-test.

Transgenic approach

To confirm the result of the effects of the viral VEGF-C and VEGF-C156S we also comparedthe effects of these factors using transgenic mouse models that overexpress VEGF-C andVEGF-C156S under the K14-promoter in the skin keratinocytes (Jeltsch et al., 1997; Veikkola etal., 2001). The lymphatic and blood vessels of these mice were analysed by mating them withthe heterozygous VEGFR-3/LacZ and VEGFR-2/LacZ mice in which one allele of theVEGFR-3 or -2 gene is replaced by the LacZ marker gene. This allowed us to visualize theVEGFR-3 and -2 positive vessels by β-galactosidase staining. The blood vessels of the adultmice were visualized by lectin perfusion.

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RESULTS AND DISCUSSION

I Expression of VEGF-C and its receptor VEGFR-3 in nasal mucosa andin nasopharyngeal tumors

Both blood and lymphatic vessels of the upper respiratory tract play important roles inpathological conditions, such as infections and tumors. In the nasal mucosa unlike in otherparts of the respiratory tract, the permeability of the capillaries and small venules is very highand their endothelium is typically fenestrated. The permeability of these vessels is furtherregulated by controlling their thickness and amount of fenestrations. In the first study wewanted to analyse the expression of VEGF-C and its receptor VEGFR-3 in the upperrespiratory tract. Previously, VEGFR-3 was reported to be a specific marker for thelymphatic endothelium in adult tissues (Jussila, 1998; Kaipainen, 1995).

Our results demonstrate that unlike in most other adult tissues, in the nasal mucosa VEGFR-3is expressed in some veins and capillaries. Expression of VEGF-C was detected in therespiratory epithelial cells of the nasal mucosa. VEGF is more potent than histamine inincreasing capillary permeability and VEGF has previously been shown to inducefenestrations in adrenal cortex capillary endothelial cells in culture (Esser, 1998; Roberts, 1997).Inhibition of VEGF activity by specific monoclonal antibodies reduces vascular permeability(Dvorak, 1995; Mesiano, 1998). VEGF mediates most of its blood vascular effects via VEGFR-2. Thus, it is possible that VEGF-C, expressed by the nasal respiratory epithelial cellsregulates the permeablity and fenestration of the VEGFR-3 and VEGFR-2 positive bloodvessels in the respiratory mucosa. VEGF-C expression is upregulated by inflammatorycytokines and recently expression of VEGF-C was reported to induce recruitment ofVEGFR-3 positive macrophages to the skin in an experimental melanoma model (Ristimaki,1998; Skobe, 2001b). VEGF-C, expressed by the nasal respiratory epithelial cells, maytherefore contribute to the inflammary response of the mucous membrane by regulating thepermeability of the blood vessels and by inducing the recruitment of the inflammatory cells.

VEGF-C expression was also detected in nasal and nasopharyngeal tumors, which weresurrounded by VEGFR-3 positive angiogenic blood vessels. Several investigators havereported VEGFR-3 expression in tumor blood vessels (Niki, 2001; Partanen, 1999; Valtola, 1999;Beasley, 2002). Thus, VEGF-C may have a role as an adjunctive growth factor inducing tumorangiogenesis. Recently, LYVE-1 was also reported to be expressed in some tumor bloodvessels in melanoma and fibrosarcoma models (Padera, 2002). Tumor blood vessels areimmature and consist of a heterogenous group of ECs (Ruoslahti and Rajotte, 2000).Identification of tumor lymphatic and blood vessels is difficult and thus several vesselspecific markers should be used.

II Expression of VEGF-C, VEGF-D and VEGFR-3 in normal humantissues

In the second study we wanted to characterize in detail the expression pattern of VEGFR-3 indifferent human tissues and to analyse which cell types express VEGF-C and VEGF-D. During

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murine embryogenesis, VEGFR-3 is initially expressed in angioblasts of head mesenchyme,dorsal aorta, cardinal vein and allantois (Kaipainen, 1995; Dumont, 1998). At mouse embryonicday 12.5 VEGFR-3 is expressed both in developing venous and presumptive lymphaticendothelia, but later during development VEGFR-3 expression becomes largely restricted tolymphatic endothelium (Kaipainen, 1995). VEGF-C mRNA is expressed in mesenchymal cells,particularly in the regions where the lymphatic vessels undergo sprouting from the embryonicveins (Kukk, 1996). High expression of VEGF-C is also detected in the murine mesenterium,lung, hearth and kidney (Kukk, 1996).

Our results show that besides the lymphatic endothelium, VEGFR-3 was expressed in certainfenestrated and discontinuous blood vessel endothelia. In most human tissues that havecontinuous capillary endothelium, VEGFR-3 expression was restricted to the lymphaticendothelia. Fenestrated capillary endothelia are found in the tissues where highly activetransport/exchange of molecules across the vessel wall is necessary. Such processes includeendocrine gland hormone secretion and the filtration of urine in the kidney glomeruli.Discontinuous or sinusoidal capillary endothelia can be found in the liver, spleen and bonemarrow. All these tissues also participate in hematopoiesis and in blood cell trafficking.VEGFR-3 may therefore play a role in regulating the translocation of the hematopoietic cellsacross the endothelial wall. Interestingly, another lymphatic endothelial cell marker, LYVE-1has also been reported to be expressed in the liver sinusoidal blood vascular endothelium(Carreira, 2001). VEGFR-3 expression was also detected in the blood vascular channels of thevertebral bodies. Non-endothelial expression of VEGFR-3 was detected in the notochordalcells of 5 week old human embryos. This finding was consistent with previous findings inavian embryos (Wilting, 1997). However, in avian embryos VEGFR-3 expression has also beendetected in the podocytes and in a subpopulation of bile duct epithelial cells (Wilting, 1997). Therole of VEGFR-3 in these cell types during embryonic development is not understood. As asummary we can say that the expression pattern of VEGFR-3 differs depending on the tissueand developmental stage, but in most adult human tissues VEGFR-3 expression is restricted toLECs.

VEGF-C and VEGF-D, ligands of VEGFR-3, were found to be expressed in vascular smoothmuscle cells. In addition, intense cytoplasmic staining for VEGF-C was detected inneuroendocrine cells, such as the α-cells of the islets of Langerhans, prolactin secreting cells inthe anterior pituitary, adrenal medullary cells and neuroendocrine cells of the gastrointestinaltract. VEGF-D expression was detected in the innermost zone of the adrenal cortex and incertain dispersed neuroendocrine cells. Like VEGF, both VEGF-C and VEGF-D are potentvascular permeability factors and they may regulate the permeability and number offenestrations of the VEGFR-2 and/or VEGFR-3 positive the blood vessels in different organs.Expression of VEGFR-3 in certain fenestrated and sinusoidal capillary endothelia maypotentiate the permeability regulation effects of VEGF-C and -D in some organs.

III In vivo effect of VEGF-C on blood and lymphatic vesselsFollowing the results of the expression analysis we next characterized the in vivo effects ofVEGF-C in blood and lymphatic vessels. We overexpressed VEGF-C via adenovirus- and

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adeno associated virus -mediated transfection in the skin and in the respiratory tract ofathymic nude mice. In the previous studies, VEGF-C had been shown to induce hyperplasiaof cutaneous lymphatic vessels in both transgenic and adenoviral gene transduction models,while causing very little or no angiogenesis (Enholm, 2001; Jeltsch, 1997; Veikkola, 2001).However, our results showed that in addition to lymphangiogenesis viral VEGF-C alsoinduced enlargement, tortuosity and leakiness of veins and venules both in skin and in nasalmucosa in a dose dependent manner. We also showed that in the skin VEGFR-2 wasexpressed in the veins and in the collecting lymphatic vessels, whereas arteries were negativefor VEGFR-2. Smaller concentrations or longer periods (14-21 days) of adenoviral VEGF-Cexpression resulted in a weaker blood vascular response while the lymphangiogenic responsewas maintained. This is consistent with previous studies in which VEGF-C was reported tobe mainly lymphangiogenic in a transgenic mouse model and following adenoviral genetransfer (Jeltsch, 1997; Enholm, 2001). The proteolytically processed 21kDa form of VEGF-C isable to bind and activate VEGFR-2 (Joukov, 1997). However, the binding affinity of the 21kDa form of VEGF-C to VEGFR-2 is 4-5 fold weaker than its affinity for VEGFR-3(Mäkinen, 2001b). This may explain the strong dose-dependency of the blood vascular effectsof VEGF-C. Neutralizing VEGFR-2 antibodies have been shown to block the vascularpermeability effect of VEGF-C in tumor models, which further confirms that VEGF-Cinduces vascular permeability via VEGFR-2 signalling (Kadambi, 2001). Our results alsodemonstrated that VEGF-C is proteolytically processed in the skin. In the nasal mucosa theblood vascular effect may also be mediated via VEGFR-3 which is expressed in thefenestrated blood capillary endothelium.

Angiogenic sprouting of new blood vessels was not observed in response to AdVEGF-C orAAV-VEGF-C. The enlargement of blood vessels in response to both AdVEGF-C andAdVEGF in the skin may in part result from receptor-mediated vasodilation as the veins andvenules of the skin were shown to express VEGFR-2. VEGF-C has been shown to stimulaterelease of endothelial nitric oxide, which is a potent mediator of vasodilation and whichcould also contribute to enhanced vascular permeability (Witzenbichler, 1998). However, theproliferation of the endothelial cells (intercalated or circumferential growth) could alsocontribute significantly to the enlargement of the vessels. Consistent with this, some ECs ofthe blood vessels were stained for the cell proliferation marker PCNA one week afterinfection with the highest concentrations of AdVEGF-C. However, despite some proliferationresponse in the ECs, new sprouting angiogenic blood vessels were not seen in AdVEGF-Cinfected tissues unlike in the AdVEGF infected tissues. AdVEGF-mediated sproutingangiogenesis has been reported to involve the detachment of VEGFR-1 expressing pericytes,which are considered to stabilize blood vessels (Sundberg, 2001a). Failure of VEGF-C toactivate VEGFR-1 might thus contribute to the lack of sprouting angiogenesis in VEGF-Coverexpressing tissues, although enlargement of blood vessels can be induced by highconcentrations of VEGF-C.

VEGF-D is a close homologue of VEGF-C. The proteolytically processed short form ofhuman VEGF-D binds to and activates VEGFR-2 and VEGFR-3 (Achen, 1998). However,unlike human VEGF-D, murine VEGF-D does not bind to mouse VEGFR-2, suggesting thatthe biological functions of VEGF-D may differ in different species or that VEGF-Dsignalling via VEGFR-2 is not significant for normal development and physiology (Baldwin,

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2001). Overexpression of the human VEGF-D in a mouse tumor xenocraft model did notincrease blood vessel permeability, even though it resulted in angiogenic andlymphangiogenic responses (Stacker, 2001). In the the rat cremaster muscle, adenoviraldelivery of the short form of human VEGF-D induced predominantly angiogenic responsewhereas it induced both lymphangiogenesis and angiogenesis in the rat skin (Byzova, 2002).In the rabbit hindlimb ischemia model viral human VEGF-D was a very potent inducer ofblood vessel permeability and angiogenesis, whereas human VEGF-C had only marginaleffects on the blood vessels (J. Markkanen, T. Rissanen, S. Ylä-Herttuala, personal communication).In an other study VEGF-C was reported to induce angiogenesis in the rabbit hindlimbischemia model (Witzenbichler, 1998). Thus, the in vivo effects of VEGF-C and VEGF-D seemto differ in different animal models and tissues. Rabbit VEGF receptors have not been clonedand we do not know to which rabbit receptor human VEGF-C and VEGF-D bind.Differences in the proteolytic processing, availability of the receptors and vessel types maymodulate the biological effects of VEGF-C and VEGF-D. Furthermore, VEGF is know to beupregulated normally during ischemia whereas VEGF-C is not (Plate, 1992; Shweiki, 1992;Enholm, 1997). Therefore in the ischemic tissues VEGF-C may act as a adjunctive pro-angiogenic growth factor by enhancing VEGF mediated angiogenesis.

Although VEGF was unable to induce the growth of new lymphatic vessels, it induced theirenlargement. This might be related to the VEGF-induced increased permeability and tissueedema resulting in lymphatic vessel volume overload. However, VEGFR-2 was found to beexpressed in the deep collecting lymphatic vessels, and also weakly in the lymphaticcapillaries. Therefore VEGF induced enlargement of the lymphatic vessels might also berelated to intercalated growth of these vessels. Recent studies have suggested that adenoviralVEGF can induce enlargement and proliferation of the lymphatic vessels (H.F. Dvorak,personal communication). However, enhanced expression of VEGF by epidermal keratinocytesin a transgenic mouse model did not cause changes in the skin lymphatic vasculature, despitesubstantial edema (Detmar, 1998; Thurston, 1999).

Expression of angiopoietin-1 blocked the increased leakiness of blood vessels induced byVEGF-C, while vessel enlargement and lymphangiogenesis were not affected. Previouslyboth transgenic and adenovirally administred Ang-1 have been reported to protect adult bloodvasculature against the plasma leakage induced by VEGF or inflammatory mediators(Thurston, 2000; Thurston, 1999). In the transgenic model Ang-1 had effects on blood vesselmorphology, but adenovirally mediated expression of Ang-1 did not change the morphologyof the blood vessels in the skin. The data from several mouse models suggests that Ang-1signalling via Tie-2 is needed for the stabilization of the vessels and for the maintainance ofthe interactions between the ECs and the surrounding ECM and mesenchyme (Sato, 1995;Suri, 1997; Suri, 1998). Recent study of the Ang-2 null mouse has revealed that angiopoietinsare likely to play complex roles in the lymphatic system, which to date are poorlyunderstood. Mice lacking Ang-2 have lymphatic defects, but expression of Ang-1 in the Ang-2 locus was sufficient to rescue the lymphatic phenotype (Gale, 2002). Tie-2 expressionpattern in the blood and lymphatic vessels is poorly characterized. As in angiogenesis,angiopoietins may contribute to the remodelling and stabilization of the newly formedlymphatic vessels. Further studies are needed to characterize the role of angiopoietins inlymphatic vessel development.

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IV Characterization of the VEGFR-3 specific mutant form of VEGF-C(VEGF-C156S) as a lymphangiogenic factor

In the previous study (III ) we had shown that high levels of native VEGF-C induce bloodvascular effects, including blood vessel enlargement and increased leakiness. In order todevelop a lymphatic specific gene therapy approach without the unwanted blood vascularside effects, we studied the potential of a VEGFR-3 specific mutant form of VEGF-C(VEGF-C156S) as a therapeutic agent in lymphedema. We compared the effects of nativeVEGF-C and VEGF-C156S using transgenic mice that overexpress these growth factorsunder the K14-promoter in the skin. In addition, we compared the effects of a virallyproduced VEGF-C and VEGF-C156S in the skin. The results showed that overexpression ofVEGF-C156S potently induces lymphangiogenesis both during embryonic development andin adult skin. Recombinant VEGF-C156S viruses were capable of inducing growth oflymphatic vessels and the formation of new lymphatic sprouts, although this was lesspronounced than with native VEGF-C. In addition, we showed that VEGF-C156S hadminimal effects on blood vessels, in contrast to VEGF-C.

Previously VEGFR-3 signalling has been shown to be important for lymphatic endothelialcell survival in vitro (Mäkinen, 2001a; Mäkinen, 2001b). Our results showed that a majority ofthe lymphatic vessels formed in response to adenoviral VEGF-C expression regress when theadenovirus was no longer active. In contrast, the AAV-mediated gene transfer resulted intolong-lasting transgene expression and therefore the VEGF-C induced lymphatic vessels weresustained.

Whole mount images of ongoing embryonic and adult lymphangiogenesis suggested that themechanisms of lymphangiogenesis are very similar to those of angiogenesis (Risau, 1997) inthat lymphangiogenesis also appears to proceed via vessel enlargement, sprouting andsplitting (Figure 6). A previous study of the mechanisms of lymphangiogenesis in ratjejunum also suggested that lymphangiogenesis occurs via sprouting and splitting (Shimoda,2001). Although VEGF-C156S and VEGF-C both induced an increase in lymphaticvascularity, their lymphangiogenic mechanisms seemed to differ. The difference between thecapacities of VEGF-C156S and VEGF-C to induce new lymphatic sprouts was most obviousin the transgenic skin model. VEGF-C156S induced lymphatic vessel hyperplasia, whereasVEGF-C induced a strong increase in the number of lymphatic vessels. This suggests thatactivation of VEGFR-2 is required for the induction of lymphatic sprouting during embryonicdevelopment. VEGFR-3 activation by VEGF-C156S seems sufficient for the induction oflymphatic sprouting in adult tissues but a more robust growth signal may also requireVEGFR-2 signalling. Signalling via VEGFR-2 and VEGFR-3 receptor heterodimers may berequired for efficient formation of vessel sprouts. On the other hand, it is also possible thatVEGF-C is more effective in recruiting other effector cells and factors needed forlymphangiogenic sprouting.

We also compared the effects of native VEGF-C and VEGF-C156S in Chy lymphedemamice. Similar to certain patients with Milroy’s disease, the Chy lymphedema mice have agermline inactivating mutation of VEGFR-3, which results in hypoplasia of cutaneous

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lymphatic vessels (Karkkainen, 2000; Karkkainen, 2001). Yet the mice have a normal deeplymphatic network. We had previously analysed the effects of adenoviral and AAV-mediatedVEGF-C overexpression in Chy mice using microlymphangiography andimmunohistochemistry of the sections 2 weeks to 7 weeks after gene transfer (Karkkainen,2001). The data showed that VEGF-C was sufficient for inducing growth of lymphatic vesselsin the Chy mouse model (Karkkainen, 2001). However, in the last study we wanted to comparethe effects of the different VEGF-C forms, to analyse the structure and morpholgy of thevessel network in more detail and to characterize the longer follow-up effects of AAV-mediated VEGF-C gene therapy.

Figure 6. VEGFR-3 whole mount staining of the lymphatic vessels in the skin. Ongoinglymphangiogenesis in the AAV-VEGF-C infected skin. Enlargement of the lymphatic vessels,sprouting (arrow) and splitting (arrowhead) of the vessels.

For the VEGF-C156S and VEGF-C gene transfer in the Chy mice we used AAV vectors thatresult in long-lasting transgene expression (up to a year). Normally the Chy mice lackfunctional superficial lymphatic vessels of the skin. However, fluorescentmicrolymphangiography revealed that functional, normal-looking, but slightly dilatedlymphatic vessels had formed in the AAV-VEGF-C156S infected mice during the two-monthfollow-up period and we also observed that in the AAV-VEGF-C infected Chy mice suchvessels persisted at least for 8 months. To characterize the morphology of the lymphaticvessels in treated and untreated Chy mice, we visualized the lymphatic vessels of the skin byVEGFR-3 whole-mount staining. In the AAV-EGFP treated or unreated Chy mice we coulddetect only a few large sack-like vessel structures (Figure 7). AAV mediated gene expressionbegins approximately 1 week after gene transfer. Three to five weeks after infection withAAV-VEGF-C156S/VEGF-C we could detect sprouting of new VEGFR-3 posive vesselsfrom these lymphatic remnants. From 2 months after VEGF-C156S or VEGF-C gene transferbranched lymphatic vessel network was detected in the skin (Figure 7). However, in mostcases the lymphatic vessels that formed in the skin of the Chy mice in response to thelymphangiogenic gene therapy appeared slightly hyperplastic in comparison with those ofwild type mouse skin.

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Figure 7. Schematic illustration of the efficacy of VEGF-C gene therapy in primary lymphedema.In the Chy lymphedema mice AAV mediated VEGF-C gene transfer is sufficient for inducing theformation of a functional lymphatic vessel network in the skin. VEGFR-3 whole mount staining wasused to visualize the lymphatic vessels in treated and untreated Chy mice skin. The magnification is thesame in histological figures. Only few abnormal lymphatic remnant structures can be seen in theuntreated Chy mouse skin, whereas in the VEGF-C treated Chy mouse lymphatic vessels form ahierarchic vessel network.

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The idea of VEGF-C gene therapy in primary lymphedema has been summarized in Figure 7.VEGF-C therapy could probably also be used in the secondary lymphedema. In regionallymphedema the treatment would be easier because local gene or protein transfer could beused. Angiogenic gene therapy has raised concerns regarding the potential stimulation ofdormant tumor growth due to increased tumor angiogenesis in response to elevated systemicVEGF levels. As tumor induced lymphangiogenesis has been associated with enhancedmetastasis to the lymph nodes (Mandriotta, 2001; Skobe, 2001; Stacker, 2001; Karpanen, 2001; He,2002, Mattila, 2002), the risk of enhanced growth and spread of dormant metastases duringVEGF-C/D therapy needs to be carefully evaluated. Secondary lymphedema is common afterlymphadenectomy for the treatment of cancer. One possibility to improve the safety of theVEGF-C/D therapy in these patients could be to combine the growth factor therapy withmicrosurgical treatment. Surgeons have performed lymphatic-venous anastomoses in order toreconstruct lymphatic drainage in patients with obstructive lymphedema (Campisi, 2002). Bycombining this treatment with VEGF-C gene therapy one may reduce the extent of thegrowth factor therapy and thus increase the safety of the treatment. The half-life of VEGF-Cin the blood circulation is short (Veikkola, 2001) and local VEGF-C therapy is thus likely tofunction without systemic effects.

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CONCLUSIONS

Understanding the molecular mechanisms of different forms of lymphedemas and discoveryof lymphangiogenic growth factors and markers now allows us to develop targeted therapiesfor disease involving lymphatic vessel dysfunction. The first lymphangiogenic growthfactors, VEGF-C and VEGF-D have been known for some years. Other molecules that havebeen reported to be necessary for normal lymphatic development include the transcriptionfactor Prox-1, integrin α9 and Ang-2 (Huang, 2000; Wigle and Oliver, 1999; Gale, 2002). Forboth pro- and anti-lymphangiogenic therapy approaches it is important to know the normalphysiological role of the growth factors that target the lymphatic vessels.

In the present study we have characterized expression patterns of VEGF-C and VEGF-D andtheir receptors in normal human tissues. We have confirmed that VEGF-C/D receptorVEGFR-3 is specific for lymphatic vessels in most human tissues, but it is also expressed insome fenestrated capillary endothelia. VEGF-C was shown to be expressed by theneuroendocrine cells and by the nasal respiratory epithelial cells and thus VEGF-C mayregulate the permeability of the nearby VEGFR-3 and VEGFR-2 positive fenestrated bloodvessels in these tissues. In addition we and others have shown that VEGFR-3 is upregulatedin some human tumor blood vessels and therefore VEGF-C may act as an adjunctiveangiogenic factor in tumors. Using viral vectors in animal models we have described that inaddition to lymphangiogenesis native full length VEGF-C can induce enlargement andleakiness of blood vessels via VEGFR-2. Importantly, Ang1 was shown to block thepermeability effect but not the lymphangiogenic effect of VEGF-C. Finally we have shownthat the VEGFR-3 specific mutant form of VEGF-C (VEGF-C156S) lacks the blood vasculareffects of native VEGF-C. The new lymphatic vessels formed in response to AAV-mediatedVEGF-C gene transfer remained functional during the 8 month follow-up.

VEGF-C gene therapy may be a powerful tool in the treatment of various forms of humanlymphedema, once the problems with gene delivery can be solved. Patients with localsecondary lymphedema, without malignancies, would be the most suitable group for pro-lymphangiogenic therapy. Treatment of late stage lymphedema may be difficult becauselong-term lymphedema results in accumulation of secondary changes in the tissues, such asdermal fibrosis and fat deposition. However, advanced diagnostic methods, such aslymphoscintigraphy, allow lymphedema diagnosis at an early stage. The treatment ofsecondary lymphedema in patients with a history of a malignant disease may pose problems.Whether VEGF-C or VEGF-D can enhance growth or lymphatic metastasis of dormanttumors in these patients needs to be carefully considered. However, the half life of VEGF-Cin the systemic blood circulation is short and local VEGF-C gene therapy is not likely tocause systemic effects (Veikkola, 2001).

Although the basic mechanisms of lymphangiogenic growth factors are now better known,the molecular control of the formation of a patterned hierarchy of lymphatic vessels is stillunclear. Since both capillaries and collecting vessels are needed for lymphatic drainage, moreattention should be paid to the development of strategies to specifically target the differenttypes of lymphatic vessels. The blood vessels, formed in response to VEGF are immature,tortuous and leaky and they often lack perivascular support structures (Carmeliet, 2000). This

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may also apply to the newly formed collecting lymphatic vessels. Characteristics andfunction of the new lymphatic vessels formed in response to VEGF-C should be studied indetail. Growth factor combinations could be utilized to create a better-functioning hierarchiclymphatic network. The identification of other growth factors involved in lymphatic vesselformation and characterization of their specific roles in the process are needed. Cell-cell andcell-matrix interactions possibly contribute to patterning and function of the lymphatic vesselnetwork, but the specific signalling cascades mediating these interactions are unknown.Current data from our and other studies suggest that lymphangiogenesis in adults occurs byoutgrowth of new lymphatic vessels from the pre-existing vessels (Paavonen, 2000). However,the potential role of lymphangioblasts in adult lymphagiogenesis requires further studies.

In the future, narrowing the target-cell range of the vector mediated gene expression would beadvantageous for all gene therapy approaches. Therapeutic applications may include targetingof either gene expression or viral entry to the desired endothelial cells of the specific tissue(Ruoslahti and Rajotte, 2000). Gene therapy vectors have been modified with homing peptidesor antibodies. Importantly, now that we have lymphatic and blood endothelial cell or pericytespecific markers we could use these molecules for homing (Ruoslahti and Rajotte, 2000).Alternatively, endothelial cell specific promoters could be used to achieve endothelialexpression of the therapeutic gene. The lymphatic endothelial cell specific promoters, such asVEGFR-3 gene regulatory region could also be used to target the lymphatic endothelium (Iljin,2001). Combining growth factor or gene therapy with lymphatic microsurgery may also bebeneficial. Future studies will show the therapeutic potential and relevance of the pro-lymphangiogenic and anti-lymphangiogenic therapies in human diseases.

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ACKNOWLEDGEMENTS

I began the study presented here at the University of Helsinki, Molecular/ Cancer Biology laboratory, inthe autumn 1998. I wish to express my deepest gratitude to my supervisor Professor Kari Alitalo for hissupport and commitment for science, for providing me great facilities for work and for opportunity tovisit all those interesting places. I also wish to thank Professor Eero Saksela, the head of theDepartment of Pathology for the excellent working environment.

I want to thank all my collaborators. I thank Taina Partanen and Johanna Arola for excellent guidancein histology and immunochemistry. I thank Maija Hytönen for her kind instructions when I started myfirst own project. My special thanks to Tanja Veikkola for the crucial collaboration during the last 9months. Without our teamwork this would still be an ongoing project. Working with you has beengreat!

I want to thank all the members of M/CBL for great company and for good collaboration. I especiallywant to thank Marika for her true friendship, collaboration and encouragement. Our discussions aboutlife in general (and science) have been a great support for me. I want to thank Marko and Joni for theirsupport and for always having time for me. I thank Lotta for guiding me to the lab and for herfriendship. I thank Tuomas for helping me with the experiments and for his enthusiasm for lab work. Ithank Terhi for company, collaboration and skiing lessions. I also thank Marja for being such a niceperson, and Nikke and Tapio for being the generators of our team spirit.

My warm thanks to Sanna Karttunen, Tapio Tainola, Mari Helanterä, Pipsa Ylikantola, KaisaMakkonen, Paula Hyvärinen, Riikka Kivirikko, Eija Koivunen, Mari Elemo, Paula Turkkelin, MiiaPutkinen, Tarja Pitkänen, Marja-Leena Saastamoinen, Seija Kajander and many other people for yourhelp. I want to thank Karri Paavonen, Tuomas Tammela and Alun Parson for checking the Englishlanguage.

I am most grateful to my family: my mother Helena, my father Reino and my dear sister Leena foralways being there for me. My parents’ dedication to their children has made it all possible for me andLeena. I want to thank my godparents Marja, Matti and Martti and my more recent family membersVellu, Salme, Arvo, Anu, Mika, Sirkka, Aati, Helvi, Irma and Benjamin for all your support. Warmthanks to my grandparents Sylvi, Matti, Esteri and Heikki. I also wish to thank Anita and Pentti fortheir special interest for my work. I am ever grateful to all my dear friends for still being around afterthese tough years.

Finally, my dearest thanks to Janne for his care, understanding and love over the last 10 years. Youhave greatly contributed to this work with your support and genuine interest for science and my work.

I have been financially supported by the Finnish Cancer Organizations, the Finnish CulturalFoundation, the Finnish Medical Foundation Duodecim, the Ida Montin Foundation, the FinnishAcademy, the Helsinki University Central Hospital and Astra Zeneca.

In Helsinki, July 2002

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REFERENCES

Aase, K., von Euler, G., Li, X., Ponten, A., Thoren, P., Cao, R., Cao, Y., Olofsson, B., Gebre-Medhin, S., Pekny, M.,Alitalo, K., Betsholtz, C. and Eriksson, U. (2001) Vascular endothelial growth factor-B-deficient mice displayan atrial conduction defect. Circulation, 104, 358-364.

Achen, M.G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, A.F., Alitalo, K. and Stacker, S.A. (1998)Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) andVEGF receptor 3 (Flt4). Proc. Natl Acad. Sci. USA, 95, 548-553.

Achen, M.G., Williams, R.A., Minekus, M.P., Thornton, G.E., Stenvers, K., Rogers, P.A., Lederman, F., Roufail, S.and Stacker, S.A. (2001) Localization of vascular endothelial growth factor-D in malignant melanoma suggestsa role in tumour angiogenesis. J. Pathol., 193, 147-154.

Adams, R.H., Wilkinson, G.A., Weiss, C., Diella, F., Gale, N.W., Deutsch, U., Risau, W. and Klein, R. (1999) Rolesof ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains,vascular morphogenesis, and sprouting angiogenesis. Genes Dev., 13, 295-306.

Akagi, K., Ikeda, Y., Miyazaki, M., Abe, T., Kinoshita, J., Maehara, Y. and Sugimachi, K. (2000) Vascularendothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. Br. J. Cancer, 83, 887-891.

Alavi, J.B., Eck, S.L. (2001) Gene therapy for high grade gliomas. Expert Opin. Biol. Ther., 1, 239-252.Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J. and Keshet, E. (1995) Vascular endothelial growth factor acts as a

survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med, 1,1024-1028.

Aprelikova, O., Pajusola, K., Partanen, J., Armstrong, E., Alitalo, R., Bailey, S.K., McMahon, J., Wasmuth, J.,Huebner, K. and Alitalo, K. (1992) FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter.Cancer Res., 52, 746-748.

Asahara, T., Bauters, C., Zheng, L.P., Takeshita, S., Bunting, S., Ferrara, N., Symes, J.F. and Isner, J.M. (1995)Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis invivo. Circulation, 92, II365-11371.

Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman, G. andIsner, J.M. (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science, 275, 964-967.

Ayadi, A., Zheng, H., Sobieszczuk, P., Buchwalter, G., Moerman, P., Alitalo, K. and Wasylyk, B. (2001b) Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1. EMBO J., 20, 5139-5152.

Baldwin, M.E., Catimel, B., Nice, E.C., Roufail, S., Hall, N.E., Stenvers, K.L., Karkkainen, M.J., Alitalo, K.,Stacker, S.A. and Achen, M.G. (2001) The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man. J. Biol. Chem., 276, 19166-19171.

Banerji, S., Ni, J., Wang, S.-X., Clasper, S., Su, J., Tammi, R., Jones, M. and Jackson, D.G. (1999) LYVE-1, a newhomologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol., 144, 789-801.

Barleon, B., Hauser, S., Schollmann, C., Weindel, K., Marme, D., Yayon, A. and Weich, H.A. (1994) Differentialexpression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells. J Cell Biochem,54, 56-66.

Barsky, S.H., Baker, M.W., Siegal, G.P., Togo, S., Liotta, L.A. (1983) Use of anti-basement membrane antibodies todistinguish blood vessel capillaries from lymphatic capillaries. Am. J. Surg. Pathol., 7, 667-677.

Beasley, N.J., Prevo, R., Banerjii, S., Leek, R., Moore, L.J., von Trappen, P., Cox, G., Harris, A.L., Jackson, D.G.(2002) Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res. 62,1315-1320.

Bell, R., Brice, G., Child, A.H., Murday, V.A., Mansour, S., Sandy, C.J., Collin, J.R.O., Brady, A.F., Callen, D.F.,Burnand, K., Mortimer, P. and Jeffery, S. (2001) Analysis of lymphoedema-distichiasis families for FOXC2mutations reveals small insertions and deletions throughout the gene. Hum. Genet., 108, 546-551.

Bellomo, D., Headrick, J.P., Silins, G.U., Paterson, C.A., Thomas, P.S., Gartside, M., Mould, A., Cahill, M.M.,Tonks, I.D., Grimmond, S.M., Townson, S., Wells, C., Little, M., Cummings, M.C., Hayward, N.K. and Kay,G.F. (2000) Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts,dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ. Res., 86, E29-E35.

Benjamin, L.E., Hemo, I. and Keshet, E. (1998) A plasticity window for blood vessel remodelling is defined bypericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development,125, 1591-1598.

Benjamin, L.E. and Keshet, E. (1997) Conditional switching of vascular endothelial growth factor (VEGF)expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by

46

VEGF withdrawal. Proc. Natl Acad. Sci. USA, 94, 8761-8766.Berens von Rautenfield, D. (1993) Grundlagen der vergleichenden Lymphologie. G. Fisher, Stuttgart.Berkner, K.L. (1988) Development of adenovirus vectors for the expression of heterologous genes. BioTechniques,

6, 616-629.Bett, A.J., Prevec, L., Granham, F.L. (1993) Packaging capasity and stability of human adenovirus type 5 vectors. J.

Virol., 67, 5911-5921.Blau, H.M. and Banfi, A. (2001) The well-tempered vessel. Nat. Med., 7, 532-534.Bordignon, C., Notarangelo, L.D., Nobili, N. (1995) Gene therapy in periferal blood lymphocytes and bone marrow

for ADA- immunodeficient patients. Science, 270, 470-475.Breiteneder-Geleff, S., Soleiman, A., Kowalski, H., Horvat, R., Amann, G., Kriehuber, E., Diem, K., Weninger, W.,

Tschachler, E., Alitalo, K. and Kerjaschki, D. (1999) Angiosarcomas express mixed endothelial phenotypes ofblood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am. J. Pathol.,154, 385-394.

Bruce, J.N., Criscuolo, G.R., Merrill, M.J., Moquin, R.R., Blacklock, J.B., Oldfield, E.H. (1987) Vascularpermeability induced by protein product of malignant tumors:inhibition by dexametasone. J. Neurosurgery, 67,880-884.

Bull, L.N., Roche, E., Song, E.J., Pedersen, J., Knisely, A.S., van der Hagen, C.B., Eiklid, K., Aagenaes, O.,Freimer, N.B. (2000) Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a6.6-cM interval on chromosome 15q. Am. J. Hum. Genet., 67, 994-999.

Bunone, G., Vigneri, P., Mariani, L., Buto, S., Collini, P., Pilotti, S., Pierotti, M.A. and Bongarzone, I. (1999)Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinicalpathological features. Am. J. Pathol., 155, 1967-1976.

Butcher, E.C., Williams, M., Youngman, K., Rott, L., Briskin, M. (1999) Lymphocyte trafficking and regionalimmunity. Adv. Immunol., 72, 209-253.

Campisi, C., Boccardo, F. (2002) Lymphedema and microsurgery. Microsurgery, 22, 74-80.Cao, Y., Chen, H., Zhou, L., Chiang, M.-K., Anand-Apte, B., Weatherbee, J.A., Wang, Y., Fang, F., Flanagan, J.G.

and Tsang, M.L.-S. (1996) Heterodimers of placenta growth factor/vascular endothelial growth factor. J. Biol.Chem., 271, 3154-3162.

Cao, Y., Ji, W.R., Qi, P., Rosin, A. and Cao, Y. (1997) Placenta growth factor: identification and characterization ofa novel isoform generated by RNA alternative splicing. Biochemical & Biophysical Research Communications,235, 493-498.

Cao, Y., Linden, P., Farnebo, J., Cao, R., Eriksson, A., Kumar, V., Qi, J.-H., Claesson-Welsh, L. and Alitalo, K.(1998) Vascular endothelial growth factor C induces angiogenesis in vivo. Proc. Natl Acad. Sci. USA, 95,14389-14394.

Carmeliet, P. (2000) VEGF gene therapy: stimulating angiogenesis or angioma-genesis? Nat. Med., 6, 1102-1103.Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A.,

Harpal, K., Ebenhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W. and Nagy, A. (1996)Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 380, 435-439.

Carmeliet, P., Moons, L., Luttun, A., Vincenti, V., Compernolle, V., De Mol, M., Wu, Y., Bon, F., Devy, L., Beck,H., Scholz, D., Acker, T., DiPalma, T., Dewerchin, M., Noel, A., Stalmans, I., Barra, A., Blacher, S.,Vandendriessche, T., Ponten, A., Eriksson, U., Plate, K.H., Foidart, J.M., Schaper, W., Charnock-Jones, D.S.and et al. (2001) Synergism between vascular endothelial growth factor and placental growth factor contributesto angiogenesis and plasma extravasation in pathological conditions. Nat. Med., 7, 575-583.

Carreira, C.M., Nasser, S.M., di Tomaso, E., Padera, T.P., Boucher, Y., Tomarev, S.I., Jain, R.K. (2001) LYVE-1 isnot restricted to the lymph vessels: Expression in normal liver blood sinusoids and down-regulation in humanliver in cancer and cirrhosis. Cancer Res., 61, 8079-8084.

Casley-Smith, J.R., Clodius, L., Foldi, M. (1977) Experimental blood vascular and lymphatic occlusion in the rabbitear and the effect of benzopyrones. Azneimittelforschung, 27, 379-382.

Casley-Smith, J.R. (1980) The fine structure and functioning of tissue channels and lymphatics. Lymphology, 12,177-183.

Charnock-Jones, D.S., Boocock, C.A., Ahmed, A., Plevin, R., Ferrara, N., Smith, S.K. (1994) Vascular endothelialgrowth factor receptor localization and activation in human trofoblast and choriocarcinoma cells. Biol. Reprod.,51, 524-530.

Chen, H., Bagri, A., Zupicich, J.A., Zou, Y., Stoeckli, E., Pleasure, S.J., Lowenstein, D.H., Skarnes, W.C., Chedotal,A. and Tessier-Lavigne, M. (2000) Neuropilin-2 regulates the development of selective cranial and sensorynerves and hippocampal mossy fiber projections. Neuron, 25, 43-56.

Clark, E.L. (1912) General observations on early superficial lymphatics in living chick embryo. Anat. Rec., 6, 247-

47

251.Clauss, M., Weich, H., Breier, G., Knies, U., Rockl, W., Waltenberger, J., Risau, W. (1996) The vascular endothelial

growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growthfactor in monocyte activation and chemotaxis. J. Biol. Chem., 271, 17629-17634.

Clauss, M., Gerlach, M., Gerlach, H., Brett, J., Wang, F., Familetti, P.C., Pan, Y.-C.E., Olander, J.V., Connolly, D.T.and Stern, D. (1990) Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell andmonocyte procoagulant activity and promotes monocyte migration. J. Exp. Med., 172, 1535-1545.

Conn, G., Bayne, M.L., Soderman, D.D., Kwok, P.W., Sullivan, K.A., Palisi, T.M., Hope, D.A. and Thomas, K.A.(1990) Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor. Proc. Natl. Acad. Sci. USA., 87, 2628-2632.

Connolly, D.T., Heuvelman, D.M., Nelson, R., Olander, J.V. and Eppley, B.L. (1989) Tumor vascular permeabilityfactor stimulates endothelial cell growth and angiogenesis. J. Clin. Invest., 84, 1478-1489.

Cotran, R.S., Kumar, V., Tucker, C. (1999) Robbins Pathologic Basis of Diseases. Philadelphia, PA: Saunders.Cyster, J.G. (1999) Chemokines and the Homing of Dendritic Cells to the T Cell Areas of Lymphoid Organs. J. Exp.

Med., 189, 447-450.Dale RF. (1985) The inheritance of primary lymphoedema. J. Med. Genet., 22, 274-278.Daly, T.M., Ohlemiller, K.K., Roberts, M.S., Vogler, C.A. and Sands, M.S. (2001) Prevention of systemic clinical

disease in MPS VII mice following AAV-mediated neonatal gene transfer. Gene Ther., 8, 1291-1298.Davis, S., Aldrich, T.H., Jones, P.F., Acheson, A., Compton, D.L., Jain, V., Ryan, T.E., Bruno, J., Raziejewski, C.,

Maisonpierre, P.C. and Yancopoulos, G.D. (1997) Isolation of Angiopoietin-1, a Ligand for the TIE2 receptor,by Secretion-Trap Expression Cloning. Cell, 87, 1161-1169.

De Vries, C., Escobedo, J.A., Ueno, H., Houck, K., Ferrara, N. and Williams, L.T. (1992) The fms-like tyrosinekinase, a receptor for vascular endothelial growth factor. Science, 255, 989-991.

Dejana, E., Corada, M., Lampugnani, M.G. (1995) Endothelial cell-to-cell junctions. Faseb J., 9, 910-918.Detmar, M., Brown, L.F., Schön, M.P., Elicker, B.M., Velasco, P., Richard, L., Fukamura, D., Monsky, W., Claffey,

K.P., Jain, R.K. (1998) Increased microvascular density and enhanced leukocyte rolling and adhesion in the skinof VEGF transgenic mice. J. Invest. Dermatol.,. 111, 1-6.

DiSalvo, J., Bayne, M.L., Conn, G., Kwok, P.W., Trivedi, P.G., Soderman, D.D., Palisi, P.M., Sullivan, K.A. andThomas, K.A. (1995) Purification and characterisation of a naturally occurring vascular endothelial growthfactor.placenta growth factor heterodimer. J. Biol. Chem., 270, 7717-7723.

Dumont, D.J., Gradwohl, G., Fong, G.-H., Puri, M.C., Gertsenstein, M., Auerbach, A. and Breitman, M.L. (1994)Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a criticalrole in vasculogenesis of the embryo. Genes Dev., 8, 1897-1909.

Dumont, D.J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, T., Pajusola, K., Breitman, M. and Alitalo, K.(1998) Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science, 282, 946-949.

Dvorak, H.F., Brown, L.F., Detmar, M. and Dvorak, A.M. (1995) Vascular permeability factor/vascular endothelialgrowth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Pathol., 146, 1029-1039.

Enholm, B., Karpanen, T., Jeltsch, M., Kubo, H., Stenback, F., Prevo, R., Jackson, D.G., Yla-Herttuala, S. andAlitalo, K. (2001) Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis inthe skin. Circ. Res., 88, 623-629.

Enholm, B., Paavonen, K., Ristimaki, A., Kumar, V., Gunji, Y., Klefstrom, J., Kivinen, L., Laiho, M., Olofsson, B.,Joukov, V., Eriksson, U. and Alitalo, K. (1997) Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNAregulation by serum, growth factors, oncoproteins and hypoxia. Oncogene, 14, 2475-2483.

Epstein, S.E., Kornowski, R., Fuchs, S. and Dvorak, H.F. (2001) Angiogenesis therapy: amidst the hype, theneglected potential for serious side effects. Circulation, 104, 115-119.

Esser, S., Wolburg, K., Wolburg, H., Breier, G., Kurzchalia, T., Risau, W. (1998) Vascular endothelial growth factorinduces endothelial fenestrations in vitro. J. Cell Biol., 140, 947-959.

Evans, A.L., Brice, G., Sotirova, V., Mortimer, P., Beninson, J., Burnand, K., Rosbotham, J., Child, A. and Sarfarazi,M. (1999) Mapping of primary congenital lymphedema to the 5q35.3 region. Am. J. Hum. Genet., 64, 547-555.

Ezaki, T., Matsuno, K., Fujii, H., Hayashi, N., Miyakawa, K., Ohmori, J., Kotani, M. (1990) A new approach foridentification of rat lymphatic capillaries using monoclonal antibodies. Arch. Histol. Cytol. Suppl., 53, 77-86.

Fang, J., Dagenais, S.L., Erickson, R.P., Arlt, M.F., Glynn, M.W., Gorski, J.L., Seaver, L.H. and Glover, T.W.(2000) Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditarylymphedema-distichiasis syndrome. Am. J. Hum. Genet., 67, 1382-1388.

Ferrara, N. (1999a) Molecular and biological properties of vascular endothelial growth factor. J. Mol. Med., 77, 527-543.

Ferrara, N. (1999b) Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int., 56,794-814.

48

Ferrara, N. and Alitalo, K. (1999) Clinical applications of angiogenic growth factors and their inhibitors. Nat. Med.,5, 1359-1364.

Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O'Shea, K.S., Powell-Braxton, L., Hilan, K.J. andMoore, M.W. (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature, 380, 438-442.

Ferrara, N. and Davis-Smyth, T. (1997) The biology of vascular endothelial growth factor. Endocrine Reviews, 18,4-25.

Ferrara, N. and Henzel, W.J. (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specificfor vascular endothelial cells. Biochem. Biophys. Res. Commun., 161, 851-855.

Ferrell, R.E., Levinson, K.L., Esman, J.H., Kimak, M.A., Lawrence, E.C., Barmada, M.M. and Finegold, D.N.(1998) Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum. Mol. Genet., 7, 2073-2078.

Finegold, D.N., Kimak, M.A., Lawrence, E.C., Levinson, K.L., Cherniske, E.M., Pober, B.R., Dunlap, J.W. andFerrell, R.E. (2001) Truncating mutations in FOXC2 cause multiple lymphedema syndromes. Hum. Mol. Genet.,10, 1185-1189.

Flotte, T., Laube, BL. (2001) Gene therapy in cystic fibrosis. Chest, 120, 124S-131S.Folkman, J. (1971) Tumour angiogenesis: therapeutic implications. N. Engl. J. Med., 285, 1182-1186.Folkman, J. (1995) Angiogenesis in carcer, vascular, rheumatoid and other disease. Nat. Med., 1, 27-31.Fong, G.-H., Rossant, J., Gertsenstein, M. and Breitman, M.L. (1995) Role of the Flt-1 receptor tyrosine kinase in

regulating the assembly of vascular endothelium. Nature, 376, 66-70.Fong, G.-H., Zhang, L., Bryce, D.-M. and Peng, J. (1999) Increased hemangioblast commitment, not vascular

disorganization, is the primary defect in flt-1 knock-out mice. Development, 126, 3015-3025.Fujisawa, H., Kitsukawa, T. (1998) Receptors for collapsin/semaphorins. Curr. Opin. Neurobiol., 8, 587-592.Fujisawa, H., Kitsukawa, T., Kawakami, A., Takagi, S., Shimizu, M. and Hirata, T. (1997) Roles of a neuronal cell-

surface molecule, neuropilin, in nerve fiber fasciculation and guidance. Cell Tissue Res., 290, 465-470.Gale, N.W., G. Thurston, S.F. Hackett, R. Renard, Q. Wang, J. McClain, C. Martin, C. Witte, M.H. Witte, D.

Jackson, C. Suri, P.A. Campochiaro, S.J. Wiegand, and G.Y. Yancopoulos. (2002) Angiopoietin-2 is requiredfor postnatal angiogenesis and lymphatic patterning and only the latter role is rescued by angiopoietin-1. Dev.Cell . In press.

Galland, F., Karamysheva, A., Pebusque, M.-J., Borg, J.-P., Rottapel, R., Dubreuil, P., Rosnet, O. and Birnbaum, D.(1993) The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growthfactor receptor. Oncogene, 8, 1233-1240.

Gerber, H.P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B.A., Dixit, V. and Ferrara, N. (1998) Vascularendothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Aktsignal transduction pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem., 273, 30336-30343.

Gerber, H.B., Malik, A.K., Solar, G.P., Sherman, D., Liang, X.H., Meng, G., Hong, K., Marsters, J.C., Ferrara, N.(2002) VEGF regulates hematopoietic stem cell survival by an internal autocrine loop mechanisms. Nature, 417,954-958.

Gerety, S.S., Wang, H.U., Chen, Z.-F. and J., A.D. (1999) Symmetrical Mutant Phenotypes of the Receptor EphB4and Its Specific Transmembrane Ligand ephrin-B2 in Cardiovascular Development. Mol. Cell, 4, 403-414.

Giger, R.J., Cloutier, J.F., Sahay, A., Prinjha, R.K., Levengood, D.V., Moore, S.E., Pickering, S., Simmons, D.,Rastan, S., Walsh, F.S., Kolodkin, A.L., Ginty, D.D. and Geppert, M. (2000) Neuropilin-2 is required in vivo forselective axon guidance responses to secreted semaphorins. Neuron, 25, 29-41.

Gille, H., Kowalski, J., Li, B., LeCouter, J., Moffat, B., Zioncheck, T.F., Pelletier, N. and Ferrara, N. (2001)Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2) - Areassessment using novel receptor-specific vascular endothelial growth factor mutants. J. Biol. Chem., 276,3222-3230.

Gleadle, J.M., Ratcliffe, P.J. (1998) Hypoxia and the regulation of gene expression. Mol. Med. Today, 4, 122-129.Gluzman-Poltorak, Z., Cohen, T., Shibuya, M., Neufeld, G. (2001) Vascular endothelial growth factor receptor-1

and neuropilin-2 form complexes. J. Biol. Chem., 276, 18688-18694.Gluzman-Poltorak, Z., Cohen, T., Herzog, Y. and Neufeld, G. (2000) Neuropilin-2 and neuropilin-1 are receptors for

the 165-amino acid form of vascular endothelial growth factor (VEGF) and of placenta growth factor-2, but onlyneuropilin-2 functions as a receptor for the 145-amino acid form of VEGF. J. Biol. Chem., 275, 18040-18045.

Gnarra, J.R., Zhou, S., Merrill, M.J., Wagner, J.R., Krumm, A., Papavassiliou, E., Oldfield, E.H., Klausner, R.D. andLinehan, W.M. (1996) Post-transcriptional regulation of vascular endothelial growth factor mRNA by theproduct of the VHL tumor suppressor gene. Proc. Natl. Acad. Sci. USA, 93, 10589-10594.

Gospodarowicz, D., Abraham, J.A. and Schilling, J. (1989) Isolation and characterization of a vascular endothelialcell mitogen produced by pituitary-derived folliculo stellate cells. Proc. Natl. Acad. Sci. USA, 86, 7311-7315.

49

Grunstein, J., Masbad, J.J., Hickley, R., Giardano, F., Johnson, R.S. (2000) Isoforms of vascular endothelial growthfactor act in coordinated fashion to recruit and expand tumor vasculature. Mol. Cell. Biol., 20, 7282-7291.

Gunn, M.D., Tangemann, K., Tam, C., Cyster, J.G., Rosen, S.D., Williams, L.T. (1998) A chemokine expressed inlymphoid high endothial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. Natl.Acad. Sci. USA, 95, 258-263.

Hanahan, D. (1985) Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinantinsulin/simian virus 40 oncogenes. Nature, 315, 115-122.

Hanahan, D., Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell, 86, 353-364.

Hashizume, H., Baluk, P., Morikawa S., McLean, J.W., Thurston, G., Roberge, S., Jain, R., McDonald, D.M. (2000)Openings between defective endothelial cells explain tumor vessel leakiness. Am. J. Pathol., 156, 1363-1380.

Hattori, K., Heissig, B., Wu, Y., Hicklin, D., Zhu, Z., Bohlen, P., Witte, L., Ferris, B., Dias, S., Hendriks, J., Hacket,N.R., Crystal, R.G., Moore, M., Zerb, Z., Lynden, D., Rafii, S. (2002) Placental growth factor reconstituteshematopoiesis by recruiting VEGFR-1+ stem cells from bone marrow microenvironment. Nat. Med. In press.

He, Y., Kozaki, K., Karpanen, T., Koshikawa, K., Yla-Herttuala, S, Takahashi, T., Alitalo, K. (2002) Suppression oftumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3signalling. J. Natl. Cancer Inst., 94,819-825.

Herzog, Y., Kalcheim, C., Kahane, N., Reshef, R., Neufeld, G. (2001) Differential expression of neuropilin-1 andneuropilin-2 in arteries and veins. Mechanisms of Development, 109, 115-119.

Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. and Shibuya, M. (1998) Flt-1 lacking the tyrosine kinase domain issufficient for normal development and angiogenesis in mice. Proc. Natl Acad. Sci. USA, 95, 9349-9354.

Holash, J., Maisonpierre, P.C., Compton, D., Boland, P., Alexander, C.R., Zagzag, D., Yancopoulos, G.D. andWiegand, S.J. (1999) Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.Science, 284, 1994-1998.

Holder, N., Klein, R. (1999) Eph receptors and ephrins: effectors of morphogenesis. Development, 126, 2033-2044.Houck, K., Leung, D., Rowland, A., Winer, J. and Ferrara, N. (1992) Dual regulation of vascular endothelial growth

factor bioavailability by genetic and proteolytic mechanisms. J. Biol. Chem., 267, 26031-26037.Houck, K.A., Ferrara, N., Winer, J., Cachianes, G., Li, B. and Leung, D.W. (1991) The vascular endothelial growth

factor family - identification of a fourth molecular species and characterization of alternative splicing of RNA.Mol. Endocrinol., 5, 1806-1814.

Huang, X.Z., Wu, J.F., Ferrando, R., Lee, J.H., Wang, Y.L., Farese, R.V., Jr. and Sheppard, D. (2000) Fatal bilateralchylothorax in mice lacking the integrin α9β1. Mol. Cell. Biol., 20, 5208-5215.

Huntington, G.S. and McClure, C.F.W. (1908) The anatomy and development of the jugular lymph sac in thedomestic cat (Felis domestica). Anat. Rec., 2, 1-19.

Hyder, S.M., Chiappetta, C. and Stancel, G.M. (2000) Induction of the angiogenic factor VEGF in the uterus by theantiprogestin onapristone. Cancer Lett., 156, 101-107.

Iljin, K., Karkkainen, M.J., Lawrence, E.C., Kimak, M.A., Uutela, M., Taipale, J., Pajusola, K., Alhonen, L.,Halmekytö, M., Finegold, D.N., Ferrell, R.E. and Alitalo, K. (2001) VEGFR3 gene structure, regulatory regionand sequence polymorphisms. FASEB J., 15, 1028-1036.

Irjala, H., Johansson, E.L., Grenman, R., Alanen, K., Salmi, M., Jalkanen, S. (2001) Mannose receptor is a novelligand for L-selectin and mediates lymphocyte binding to lymphatic endothelium. J. Exp. Med, 194, 1033-1042.

Irrthum, A., Karkkainen, M.J., Devriendt, K., Alitalo, K. and Vikkula, M. (2000) Congenital hereditary lymphedemacaused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet., 67, 295-301.

Isner, J.M. (2002) Myocardial gene therapy. Nature, 415, 234-243.Jackson, D.G., Prevo, R., Clasper, S. and Banerji, S. (2001) LYVE-1, the lymphatic system and tumor

lymphangiogenesis. Trends Immunol., 22, 317-321.Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D., Jain, R.K. and

Alitalo, K. (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science, 276, 1423-1425.Jingjing, L., Xue, Y., Agarwal, N., Roque, R.S. (1999) Muller cells express VEGF183, a novel spliced variant of

vascular endothelial growth factor. Invest. Ophthalmol. Vis. Sci., 40, 752-759.Joukov, V., Kumar, V., Sorsa, T., Arighi, E., Weich, H., Saksela, O. and Alitalo, K. (1998) A recombinant mutant

vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding,activation, and vascular permeability activities. J. Biol. Chem., 273, 6599-6602.

Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N. and Alitalo,K. (1996) A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR(VEGFR-2) receptor tyrosine kinases. EMBO J., 15, 290-298.

Joukov, V., Sorsa, T., Kumar, V., Jeltsch, M., Claesson-Welsh, L., Cao, Y., Saksela, O., Kalkkinen, N. and Alitalo,K. (1997) Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J., 16, 3898-

50

3911.Jussila, L., Valtola, R., Partanen, T.A., Salven, P., Heikkila, P., Matikainen, M.T., Renkonen, R., Kaipainen, A.,

Detmar, M., Tschachler, E., Alitalo, R. and Alitalo, K. (1998) Lymphatic endothelium and Kaposi's sarcomaspindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res., 58,1599-1604.

Kadambi, A., Carreira, C.M., Yun, C., Padera, T.P., Dolmans, D., Carmeliet, P., Fukumura, D. and Jain, R.K. (2001)Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyterecruitment: Role of VEGF-receptor 2 and host VEGF-A. Cancer Res., 61, 2404-2408.

Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V.W.M., Fang, G.-H., Dumont, D., Breitman, M. andAlitalo, K. (1995) Expression of the fms-like tyrosine kinase FLT4 gene becomes restricted to lymphaticendothelium during development. Proc. Natl Acad. Sci. USA, 92, 3566-3570.

Kalebic, T., Garbisa, S., Glaser, B., Liotta, L.A.(1983) Basement membrane collagen: degradation by migratingendothelial cells. Science, 221, 281-283.

Karkkainen, M.J., Ferrell, R.E., Lawrence, E.C., Kimak, M.A., Levinson, K.L., McTigue, M.A., Alitalo, K. andFinegold, D.N. (2000) Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat.Genet., 25, 153-159.

Karkkainen, M.J., Saaristo, A., Jussila, L., Karila, K.A., Lawrence, E.C., Pajusola, K., Bueler, H., Eichmann, A.,Kauppinen, R., Kettunen, M.I., Ylä-Herttuala, S., Finegold, D.N., Ferrell, R.E. and Alitalo, K. (2001) A modelfor gene therapy of human hereditary lymphedema. Proc. Natl Acad. Sci. USA, 98, 12677-12682.

Karpanen, T., Egeblad, M., Karkkainen, M.J., Kubo, H., Jackson, D.G., Ylä-Herttuala, S., Jäättelä, M. and Alitalo,K. (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumorgrowth. Cancer Res., 61, 1786-1790.

Kato, S. (1990) Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanningelectron microscopy. Stain Technology, 65, 131-137.

Katoh, O., Tauchi, H., Kawaishi, K., Kimura, A., Satow, Y. (1995) Expression of the vascular endothelial growthfactor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitort effect of VEGF on apoptotic cell deathcaused by ionizing radiation. Cancer Res., 55, 5687-5692.

Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T. and Fujisawa, H. (1999) A requirementfor neuropilin-1 in embryonic vessel formation. Development, 126, 4895-4902.

Kay, M.A., Manno, C.S., Ragni, M.V., Larson, P.J., Couto, L.B., McClelland, A., Glader, B., Chew, A.J., Tai, S.J.,Herzog, R.W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A.W. and High, K.A. (2000) Evidencefor gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet.,24, 257-261.

Keck, P.J., Hauser, S.D., Krivi, G., Sanzo, K., Warren, T., Feder, J. and Connolly, D.T. (1989) Vascularpermeability factor, an endothelial cell mitogen related to PDGF. Science, 246, 1309-1312.

Kerbel, R.S., Viloria-Petit, A., Okada, F., Rak, J. (1998) Establishing a link between oncogenes and tumorangiogenesis. Molecular Med., 4, 286-295.

Keyt, B.A., Nguyen, H.V., Berleau, L.T., Duarte, C.M., Park, J., Chen, H. and Ferrara, N. (1996) Identification ofvascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. J. Biol. Chem., 271,5638-5646.

Kim, I., Kwak, H.J., Ahn, J.E., So, J.N., Liu, M., Koh, K.N. and Koh, G.Y. (1999) Molecular cloning andcharacterization of a novel angiopoietin family protein, angiopoietin-3. FEBS Lett., 443, 353-356.

Kitadai, Y., Amioka, T., Haruma, K., Tanaka, S., Yoshihara, M., Sumii, K., Matsutani, N., Yasui, W. and Chayama,K. (2001) Clinicopathological significance of vascular endothelial growth factor (VEGF)-C in humanesophageal squamous cell carcinomas. Int. J. Cancer, 93, 662-666.

Kremer, E.J., Perricaudet, M. (1995) Adenovirus and adeno associated virus-mediated gene transfer. British Med.Bull., 51, 31-44.

Kriedman, B., Myloyde, T., Bernas, M., Lee-Donaldson, L., Preciado, S., Lynch, M., Stea, B., Summers, P., Witte,C., Witte, M. (2002) Limb volume reduction after physiological treatment by compression and/or massage inrodent model of periferal lymphedema. Lymphology, 35, 23-27.

Kriehuber, E., Breiteneder-Geleff, S., Groeger, M., Soleiman, A., Schoppmann, S.F., Stingl, G., Kerjaschki, D. andMaurer, D. (2001) Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stableand functionally specialized cell lineages. J. Exp. Med., 194, 797-808.

Kukk, E., Lymbussaki, A., Taira, S., Kaipainen, A., Jeltsch, M., Joukov, V., Alitalo, K. (1996) VEGF-C receptorbinding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development.Development, 122, 3829-3837.

Kunkel, E., Butcher, E.C. (2002) Chemokines and tissue-specific migration of lymphocytes. Immunology, 16, 1-4.Kurebayashi, J., Otsuki, T., Kunisue, H., Mikami, Y., Tanaka, K., Yamamoto, S. and Sonoo, H. (1999) Expression

51

of vascular endothelial growth factor (VEGF) family members in breast cancer. Jpn. J. Cancer Res., 90, 977-981.

Lawson, N.D., Scheer, N., Pham, V.N., Kim, C.H., Chitnis, A.B., Campos-Ortega, J.A., Weinstein, B.M. (2001)Notch signalling is required for arterial-venous differentiation during embryonic development. Development,128, 3675-3683.

Lee-Donaldson, L., Witte, M., Bernas, M., Witte, C.L., Way, D., Stea, B. (1999) Refinement of a rodent model ofperiferal lymphedema. Lymphology, 32, 111-117.

Leu, A.J., Berk, D.A., Lymboussaki, A., Alitalo, K. and Jain, R.K. (2000) Absence of functional lymphatics within amurine sarcoma: a molecular and functional evaluation. Cancer Res., 60, 4324-4327.

Leung, D.W., Cachianes, G., Kuang, W.-J., Goeddel, D.V. and Ferrara, N. (1989) Vascular endothelial growth factoris a secreted angiogenic mitogen. Science, 246, 1306-1309.

Li, X., Aase, K., Li, H., von Euler, G., Eriksson, U. (2001) Isoform-specific expression of VEGF-B in normal tissuesand tumors. Growth Factors, 19, 49-59.

Linehan, S.A., Martinez-Pomares, L., Stahl, P.D. and Gordon, S. (1999) Mannose receptor and its putative ligands innormal murine lymphoid and nonlymphoid organs: In situ expression of mannose receptor by selectedmacrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells. J. Exp. Med.,189, 1961-1972.

Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., Nagy, J., Hooper, A., Priller, J., DeKlerck, B., Compernolle, V., Daci, E., Bohlen, P., Dewerchin, M., Herbert, J.M., Fava, R., Matthys, P.,Carmeliet, G., Collen, D., Dvorak, H., Hicklin, D.J., Carmeliet, P. (2002) Revascularization of ischemic tissuesby PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt-1 antibody.Nat. Med. In press.

Maglione, D., Guerriero, V., Viglietto, G., Delli-Bovi, P. and Persico, M.G. (1991) Isolation of a human placentacDNA coding for a protein related to the vascular permeability factor. Proc. Natl Acad. Sci. USA, 88, 9267-9271.

Maglione, D., Guerriero, V., Viglietto, G., Ferraro, M.G., Aprelikova, O., Alitalo, K., Del Vecchio, S., Lei, K.-J.,Chou, J.Y. and Persico, M.G. (1993) Two alternative mRNAs coding for the angiogenic factor, placenta growthfactor (PlGF), are transcribed from a single gene of chromosome 14. Oncogene, 8, 925-931.

Maisonpierre, P.C., Suri, C., Jones, P.F., Bartunkova, S., Wiegand, S.J., Radziejewski, C., Compton, D., McClain, J.,Aldrich, T.H., Papadopoulos, N., Daly, T.J., Davis, S., Sato, T.N. and Yancopoulos, G.D. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science, 277, 55-60.

Mandriota, S.J., Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R.,Jackson, D.G., Orci, L., Alitalo, K., Christofori, G. and Pepper, M.S. (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J., 20, 672-682.

Matthews, W., Jordan, C.T., Gavin, M., Jenkins, N.A., Copeland, N.G. and Lemischka, I.R. (1991) A receptortyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting closegenetic linkage to c-kit. Proc. Natl Acad. Sci. USA, 88, 9026-9030.

Maxwell, P.H., Weisener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R., Ratcliffe, P.J. (1999) The tumor suppressor protein VHL targets hypoxia-inducible factors foroxygen-dependent proteolysis. Nature, 399, 271-275.

Mesiano, S., Ferrara, N., Jaffe, RB. (1998) Role of vascular endothelial growth factor in ovarian cancer. Am. J.Pathol., 153, 1249-1256.

Meyer, M., Clauss, M., Lepple-Wienhues, A., Waltenberger, J., Augustin, H.G., Ziche, M., Lanz, C., Böttner, M.,Rziha, H.-J. and Dehio, C. (1999) A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E,mediates angiogenesis via signaling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosinekinases. EMBO J., 18, 363-374.

Migdal, M., Huppertz, B., Tessler, S., Comforti, A., Shibuya, M., Reich, R., Baumann, H. and Neufeld, G. (1998)Neuropilin-1 is a placenta growth factor-2 receptor. J. Biol. Chem., 273, 22272-22278.

Millauer, B., Wizigmann-Voos, S., Schnürch, H., Martinez, R., Moller, N.P., Risau, W. and Ullrich, A. (1993) Highaffinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis andangiogenesis. Cell, 72, 835-846.

Miller, A.D., Bender, M.A., Harris, E.A.S., Kaleko, M., Gelinas, R.E. (1988) Design of retrovirus vectors for genetransfer and expression of the human b-globin gene. J. Virol., 62, 4337-4345.

Miller, D.B., Adam, M.A., Miller, A.D. (1990) Gene transfer by retrovirus vectors occurs only in cells that areactively replicating at the time of infection. Mol. Cell Biol., 10, 4239-4242.

Miller, R., Curiel, D.T. (1996) Towards the use of replicative adenoviral vectors for cancer gene therapy. GeneTher., 3, 557-559.

Monahan, P.E. and Samulski, R.J. (2000) Adeno-associated virus vectors for gene therapy: more pros than cons?

52

Mol. Med. Today, 6, 433-440.Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M.E., McClanahan, T., Murphy, E., Yuan, W.,

Wagner, S.N., Barrera, J.L., Mohar, A., Verastegui, E. and Zlotnik, A. (2001) Involvement of chemokinereceptors in breast cancer metastasis. Nature, 410, 50-56.

Munsick, R.A. (1984) Human fetal extremity lengths in the interval from 9 to 21 menstrual weeks of pregnancy. Am.J. Obstet. Gynecol., 149, 883-887.

Mäkinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M.I., Pulkkanen, K.J., Kauppinen, R., Jackson, D.G.,Kubo, H., Nishikawa, S.-I., Ylä-Herttuala, S. and Alitalo, K. (2001a) Inhibition of lymphangiogenesis withresulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med., 7, 199-205.

Mäkinen, T., Olofsson, B., Karpanen, T., Hellman, U., Soker, S., Klagsbrun, M., Eriksson, U. and Alitalo, K. (1999)Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1. J.Biol. Chem., 274, 21217-21222.

Mäkinen, T., Veikkola, T., Mustjoki, S., Karpanen, T., Catimel, B., Nice, E.C., Kowalski, H., Kerjaschki, D.,Stacker, S.A., Achen, M.G. and Alitalo, K. (2001b) Isolated lymphatic endothelial cells transduce growth,survival and migratory signals via the VEGF-C receptor VEGFR-3. EMBO J., 20, 4762-4773.

Mattila, M.M., Ruohola, J.K., Karpanen, T., Jackson, D., Alitalo, K., Harkonen, P.L. (2002) VEGF-C inducedlymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int. J. Cancer, 98,946-951.

Nagy, J.A., Brown, L.F., Senger, D.R. (1989) Pathogenesis of tumor stroma generation: a critical role for leakyblood vessels and fibrin deposition. Biochem Biophys Acta, 948, 305-326.

Nibbs, R.J.B., Kriehuber, E., Ponath, P.D., Parent, D., Qin, S.X., Campbell, J.D.M., Henderson, A., Kerjaschki, D.,Maurer, D., Graham, G.J. and Rot, A. (2001) The β-chemokine receptor D6 is expressed by lymphaticendothelium and a subset of vascular tumors. Am. J. Pathol., 158, 867-877.

Niki, T., Iba, S., Tokunou, M., Yamada, T., Matsuno, Y. and Hirohashi, S. (2000) Expression of vascular endothelialgrowth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. Clin.Cancer Res., 6, 2431-2439.

Niki, T., Iba, S., Yamada, T., Matsuno, Y., Enholm, B. and Hirohashi, S. (2001) Expression of vascular endothelialgrowth factor receptor 3 in blood and lymphatic vessels of lung adenocarcinoma. J. Pathol., 193, 450-457.

Oh, S.-J., Jeltsch, M.M., Birkenhager, R., McCarthy, J.E., Weich, H.A., Christ, B., Alitalo, K. and Wilting, J. (1997)VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avianchorioallantoic membrane. Dev. Biol., 188, 96-109.

Ohta, Y., Shridhar, V., Bright, R.K., Kalemkerian, G.P., Du, W., Carbone, M., Watanabe, Y. and Pass, H.I. (1999)VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignantmesothelioma tumours. Br. J. Cancer, 81, 54-61.

Okada, F., Rak, J.W., Croix, B.S., Lieubeau, B., Kaya, M., Roncari, L., Shirasawa, S., Sasazuki, T., Kerbel, R.S.(1998) Impact of oncogenes on tumor angiogenesis: mutant k-ras upregulation of VEGF/VPF is necessary butnot sufficient for tumorigenity of human colorectal carcinoma cells. Proc. Natl. Acad. Sci. USA, 95, 3609-3614.

Olofsson, B., Korpelainen, E., Pepper, M.S., Mandriota, S.J., Aase, K., Kumar, V., Gunji, Y., Jeltsch, M.M.,Shibuya, M., Alitalo, K. and Eriksson, U. (1998) Vascular endothelial growth factor B (VEGF-B) binds toVEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc. Natl Acad. Sci. USA,95, 11709-11714.

Olofsson, B., Pajusola, K., Kaipainen, A., von Euler, G., Joukov, V., Saksela, O., Orpana, A., Pettersson, R.F.,Alitalo, K. and Eriksson, U. (1996) Vascular endothelial growth factor B, a novel growth factor for endothelialcells. Proc. Natl Acad. Sci. USA, 93, 2576-2581.

Paavonen, K., Puolakkainen, P., Jussila, L., Jahkola, T., Alitalo, K. (2000) Vascular endothelial growth factorreceptor-3 in lymphangiogenesis in wound healing. Am. J. Pathol., 156, 1499-1504.

Pajusola, K., Aprelikova, O., Korhonen, J., Kaipainen, A., Pertovaara, L., Alitalo, R. and Alitalo, K. (1992) FLT4receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissuesand cell lines. Cancer Res., 52, 5738-5743.

Padera, T.P., Kadambi, A., Tomaso, E., Carreira, C.M., Brown, E.B., Boucher, Y., Choi, N.C., Mathisen, D., Wain,J., Mark, E.J., Munn, L.J., Jain, R. (2002) Lymphatic metastasis in the absense of functional intratumorlymphatics. Science, 296, 1883-1886.

Park, J.E., Chen, H.H., Winer, J., Houck, K.A. and Ferrara, N. (1994) Placenta growth factor. Potentiation ofvascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not toFlk-1/KDR. J. Biol. Chem., 269, 25646-25654.

Park, J.E., Keller, G.-A. and Ferrara, N. (1993) The vascular endothelial growth factor (VEGF) isoforms:Differential deposition into the subepithelial extracellular matrix-bound VEGF. Mol. Biol. Cell., 4, 1317-1326.

Partanen, T.A., Alitalo, K. and Miettinen, M. (1999) Lack of lymphatic vascular specificity of vascular endothelial

53

growth factor receptor 3 in 185 vascular tumors. Cancer, 86, 2406-2412.Pettersson, A., Nagy, J.A., Brown, L.F., Sundberg, C., Morgan, E., Jungles, S., Carter, R., Krieger, J.E., Manseau,

E.J., Harvey, V.S., Eckelhoefer, I.A., Feng, D., Dvorak, A.M., Mulligan, R.C. and Dvorak, H.F. (2000)Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeabilityfactor/vascular endothelial growth factor. Lab. Invest., 80, 99-115.

Piller, N.B., Clodius, L. (1978) Lymphoedema of rabbit ear following partial and complete lymphatic blockade; itseffects on fibrotic development, enzyme types and their activity levels. Br. J. Exp. Pathol., 59, 319-326.

Plate, K.H., Breier, G., Weich, H.A. and Risau, W. (1992) Vascular endothelial growth factor is a potential tumourangiogenesis factor in human gliomas in vivo. Nature, 359, 845-848.

Plouet, J., Schilling, J. and Gospodarowicz, D. (1989) Isolation and characterization of a newly identified endothelialcell mitogen produced by AtT-20 cells. EMBO J., 8, 3801-3806.

Poltorak, Z., Cohen, T., Sivan, R., Kandelis, Y., Spira, G., Vlodavsky, I., Keshet, E. and Neufeld, G. (1997)VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J. Biol.Chem., 272, 7151-7158.

Puri, M.C., Rossant, J., Alitalo, K., Bernstein, A. and Partanen, J. (1995) The receptor tyrosine kinase TIE isrequired for integrity and survival of vascular endothelial cells. EMBO J., 14, 5884-5891.

Qing, K., Mah, C., Hansen, J., Zhou, S., Dwarki, V. and Srivastava, A. (1999) Human fibroblast growth factorreceptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat. Med., 5, 71-77.

Quinn, T., Peters, K., DeVries, C., Ferrara, N. and Williams, L. (1993) Fetal liver kinase 1 is a receptor for vascularendothelial growth factor and is selectively expressed in vascular endothelium. Proc.Natl.Acad.Sci.USA, 90,7533-7537.

Risau, W. (1997) Mechanisms of angiogenesis. Nature, 386, 671-674.Risau, W. and Flamme, I. (1995) Vasculogenesis. Ann. Rev .Cell. Dev. Biol,. 11, 73-91.Ristimaki, A., Narko, K., Enholm, B., Joukov, V. and Alitalo, K. (1998) Proinflammatory cytokines regulate

expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C. J. Biol. Chem., 273,8413-8418.

Roberts, W.G., Palade, G.E. (1997) Neovasculature induced by vascular endothelial growth factor is fenestrated.Cancer Res., 57, 765-772.

Roberts, W.G. and Palade, G.E. (1995) Increased microvascular permeability and endothelial fenestrations inducedby vascular endothelial growth factor. J. Cell. Sci., 108, 2369-2379.

Rockson, S.G. (2001) Lymphedema. Am. J. Med., 110, 288-295.Roelvink, P.W., Mi Lee, G., Einfeld, D.A., Kovesdi, I. and Wickham, T.J. (1999) Identification of a conserved

receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science, 286, 1568-1571.Rosen, S.D. (1999) Endothelial ligands for L-selectin: From lymphocyte recirculation to allocraft rejection. Am. J.

Pathol., 155, 1013-1020.Ross, M.H., Romrell, L.J., Kaye, G.I. (1995) Histology, a text and atlas. Wiliams&Wilkins, Baltimore.Ruoslahti, E. and Rajotte, D. (2000) An address system in the vasculature of normal tissues and tumors. Ann. Rev.

Immunol., 18, 813-827.Sabin, F.R. (1902) On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am. J. Anat., 1, 367-391.Schlingemann, R.O., Dingjam, G.M., Emeis, J.J., Blok, J., Warnaar, S.O., Ruiter, D.J. (1985) Monoclonal antibody

PAL-E specific for endothelium. Lab. Invest., 52, 71-76.Schmelz, M. and Franke, W.W. (1993) Complexus adhaerentes, a new group of desmoplakin-containing junctions in

endothelial cells: the syndesmos connecting retothelial cells of lymph nodes. Eur. J. Cell Biol., 61, 274-289.Schmucker, D. and Zipursky, S.L. (2001) Signaling downstream of Eph receptors and ephrin ligands. Cell, 105, 701-

704.Schneider, M., Othman-Hassan, K., Christ, B. and Wilting, J. (1999) Lymphangioblasts in the avian wing bud. Dev.

Dyn., 216, 311-319.Seetharam, L., Gotoh, N., Maru, Y., Neufeld, G., Yamaguchi, S. and Shibuya, M. (1995) A unique signal

transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene, 10,135-147.

Senger, D.R., Galli, S.J., Dvorak, A.M., Perruzzi, C.A., Harvey, V.S. and Dvorak, H.F. (1983) Tumor cells secrete avascular permeability factor that promotes accumulation of ascites fluid. Science, 219, 983-985.

Shalaby, F., Ho, J., Stanford, W.L., Fischer, K.D., Schuh, A.C., Schwartz, L., Bernstein, A. and Rossant, J. (1997) Arequirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell, 89, 981-990.

Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Breitman, M.L. and Schuh, A.C. (1995)Failure of blood island formation and vasculogenesis in Flk-1-deficient mice. Nature, 376, 62-66.

Shi, Q., Rafii, S., Wu, M.H., Wijelath, E.S., Yu, C., Ishida, A., Fujita, Y., Kothari, S., Mohle, R., Sauvage, L.R.,

54

Moore, M.A., Storb, R.F. and Hammond, W.P. (1998) Evidence for circulating bone marrow-derivedendothelial cells. Blood, 92, 362-367.

Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H. and Sato, M. (1990) Nucleotidesequence and expression of a novel human receptor type tyrosine kinase gene (flt) closely related to the fmsfamily. Oncogene, 5, 519-524.

Shimoda, H., Takahashi, Y., Kato, S. (2001) Development of the lymphatic network in muscle coat of the ratjejunum as revealed by enzyme histochemistry. Arch. Histol. Cytol., 64, 523-533.

Shunichi, M., Baluk, P., Kaidoh, T., Haskell, A., Jain, R.K., McDonald, D.M. (2002) Abnormalities in pericytes onblood vessels and endothelial sprouts in tumors. Am. J. Pathol., 160, 985-1000.

Shweiki, D., Itin, A., Soffer, D. and Keshet, E. (1992) Vascular endothelial growth factor induced by hypoxia maymediate hypoxia-initiated angiogenesis. Nature, 359, 843-848.

Siemeister, G., Weindel, K., Mohrs, K., Barleon, B., Martiny-Baron, G., Marme, D. (1996) Reversion of deregulatedexpression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumorsuppressor protein. Cancer Res, 56, 2299-2301.

Skobe, M., Hamberg, L.M., Hawighorst, T., Schirner, M., Wolf, G.L., Alitalo, K. and Detmar, M. (2001b)Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelialgrowth factor-C in melanoma. Am. J. Pathol., 159, 893-903.

Skobe, M., Hawighorst, T., Jackson, D.G., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K. andDetmar, M. (2001a) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat.Med., 7, 192-198.

Soker, S., Fidder, H., Neufeld, G. and Klagsbrun, M. (1996) Characterization of novel vascular endothelial growthfactor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. J. Biol. Chem., 271,5761-5767.

Soker, S., Takashima, S., Miao, H.Q., Neufeld, G. and Klagsbrun, M. (1998) Neuropilin-1 is expressed byendothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell, 92, 735-745.

Springer, M.L., Chen, A.S., Kraft, P.E., Bednarski, M. and Blau, H.M. (1998) VEGF gene delivery to muscle:potential role for vasculogenesis in adults. Mol. Cell, 2, 549-558.

Stacker, S.A., Caesar, C., Baldwin, M.E., Thornton, G.E., Williams, R.A., Prevo, R., Jackson, D.G., Nishikawa, S.,Kubo, H. and Achen, M.G. (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat. Med., 7, 186-191.

Stacker, S.A., Stenvers, K., Caesar, C., Vitali, A., Domagala, T., Nice, E., Roufail, S., Simpson, R.J., Moritz, R.,Karpanen, T., Alitalo, K. and Achen, M.G. (1999) Biosynthesis of vascular endothelial growth factor-D involvesproteolytic processing which generates non-covalent homodimers. J. Biol. Chem., 274, 32127-32136.

Stalmans, I., Ng, Y.S., Rohan, R., Fruttiger, M., Bouche, A., Yuce, A., Fujisawa, H., Hermans, B., Shani, M.,Jansen, S., Hicklin, D., Anderson, D.J., Gardiner, T., Hammes, H.P., Moons, L., Dewerchin, M., Collen, D.,Carmeliet, P., D'Amore, P.A. (2002) Arteriolar and venular patterning in retinas of mice selectively expressingVEGF isoforms. J. Clin. Invest., 109, 327-336.

Stewart, P.L., Chiu, C.Y., Huang, S., Muir, T., Zhao, Y., Chait, B., Mathias, P. and Nemerow, G.R. (1997) Cryo-EMvisualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization. EMBO J., 16,1189-1198.

Summerford, C., Bartlett, J.S. and Samulski, R.J. (1999) αVβ5 integrin: a co-receptor for adeno-associated virustype 2 infection. Nat. Med., 5, 78-82.

Summerford, C. and Samulski, R.J. (1998) Membrane-associated heparan sulfate proteoglycan is a receptor foradeno-associated virus type 2 virions. J. Virol., 72, 1438-1445.

Sundberg, C., Nagy, J.A., Brown, L.F., Feng, D., Eckelhoefer, I.A., Manseau, E.J., Dvorak, A.M. and Dvorak, H.F.(2001a) Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascularendothelial growth factor-164 gene delivery. Am. J. Pathol., 158, 1145-1160.

Sundberg, C., Nagy, J.A., Brown, L.F., Feng, D., Eckelhoefer, I.A., Manseau, E.J., Dvorak, A.M. and Dvorak, H.F.(2001b) Glomeruloid microvascular proliferation follows adenoviral VPF/VEGF 164 gene delivery. Am. J.Pathol., 158, 1145-1160.

Suri, C., Jones, P.F., Patan, S., Bartunkova, S., Maisonpierre, P.C., Davis, S., Sato, T.N. and Yancopoulos, G.D.(1996) Requisite role of Angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell,87, 1161-1169.

Swartz, M.A., Berk, D.A. and Jain, R.K. (1996) Transport in lymphatic capillaries. I. Macroscopic measurementsusing residence time distribution theory. Am. J. Physiol., 270, 324-329.

Tartaglia, M., Mehler, E.L., Goldberg, R., Zampino, G., Brunner, H.G., Kremer, H., van der Burgt, I., Crosby, A.H.,Ion, A., Jeffery, S., Kalidas, K., Patton, M.A., Kucherlapati, R.S. and Gelb, B.D. (2001) Mutations in PTPN11,

55

encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat. Genet., 29, 465 - 468.Tedder, T.F., Steeber, D.A., Chen, A., Engel, P. (1995) The selectins: vascular adhesion molecules. Faseb J., 9, 866-

873.Terman, B.I., Carrion, M.E., Kovacs, E., Rasmussen, B.A., Eddy, R.L. and Shows, T.B. (1991) Identification of a

new endothelial cell growth factor receptor tyrosine kinase. Oncogene, 6, 1677-1683.Terman, B.I., Dougher-Vermazen, M., Carrion, M.E., Dimitrov, D., Armellino, D.C., Gospodarowicz, D. and

Böhlen, P. (1992) Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growthfactor. Biochem. Biophys. Res. Comm., 187, 1579-1586.

Thurston, G., Rudge, J.S., Ioffe, E., Zhou, H., Ross, L., Croll, S.D., Glazer, N., Holash, J., McDonald, D.M. andYancopoulos, G.D. (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat. Med., 6,460-463.

Thurston, G., Suri, C., Smith, K., McClain, J., Sato, T.N., Yancopoulos, G.D. and McDonald, D.M. (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science, 286, 2511-2514.

Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J.C. and Abraham, J.A. (1991) Thehuman gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exonsplicing. J. Biol. Chem., 266, 11947-11954.

Tomko, R.P., Xu, R. and Philipson, L. (1997) HCAR and MCAR: the human and mouse cellular receptors forsubgroup C adenoviruses and group B coxsackieviruses. Proc. Natl Acad. Sci. USA, 94, 3352-3356.

Tsurusaki, T., Kanda, S., Sakai, H., Kanetake, H., Saito, Y., Alitalo, K. and Koji, T. (1999) Vascular endothelialgrowth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br. J.Cancer, 80, 309-313.

Valenzuela, D.M., Griffiths, J.A., Rojas, J., Aldrich, T.H., Jones, P.F., Zhou, H., McClain, J., Copeland, N.G.,Gilbert, DJ., Jenkins, N.A, Huang, T., Papadopoulous, N., Maisonpierre, P.C., Davis, S., Yancopoulos, G.D.(1999) Angiopoietin 3 and 4: diverging gene counterparts in mice and humans. Proc. Natl., Acad., Sci., USA, 96,1904-1909.

Valtola, R., Salven, P., Heikkila, P., Taipale, J., Joensuu, H., Rehn, M., Pihlajaniemi, T., Weich, H., deWaal, R. andAlitalo, K. (1999) VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J.Pathol., 154, 1381-1390.

van der Putte, S.C.J. (1975) The development of the lymphatic system in man. Adv. Anat. Embryol. Cell. Biol., 51, 3-60.

Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Petrova, T.V., Kubo, H., Thurston, G., McDonald,D.M., Achen, M.G., Stacker, S.A. and Alitalo, K. (2001) Signalling via vascular endothelial growth factorreceptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J., 20, 1223-1231.

Viley, H.E., Gonzales, E.B., Maki, W., Wu, M.T., Hwang, S.T. (2001) Expression of CC chemokine receptor-7 andregional lymph node metastasis of B16 murine melanoma. J. Natl. Cancer Inst., 93, 1638-1643.

Viloria-Petit, A.M., Rak, J., Hung, M.-C., Rockwell, P., Goldstein, N., Kerbel, R.S. (1997) Neutralizing antibodiesagainst EGF and ErbB-2/neu receptor tyrosine kinases down-regulate VEGF production by tumor cells in vitroand in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am. J. Pathol., 151, 1523-1530.

Wagner, J.A., Messner, A.H., Moran, M.L., Daifuku, R., Kouyama, K., Desch, J.K., Manley, S., Norbash, A.M.,Conrad, C.K., Friborg, S., Reynolds, T., Guggino, W.B., Moss, R.B., Carter, B.J., Wine, J.J., Flotte, T.R. andGardner, P. (1999) Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosistransmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope, 109, 266-274.

Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. and Heldin, C.H. (1994) Different signaltransduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem.,269, 26988-26995.

Wang, H.U., Chen, Z.F. and Anderson, D.J. (1998) Molecular distinction and angiogenic interaction betweenembryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell, 93, 741-753.

Wetterwald, A., Hoffstetter, W., Cecchini, M.G., Lanske, B., Wagner, C., Fleisch, H. and Atkinson, M. (1996)Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. Bone, 18,125-132.

White, S.W. (1984) Lymphedema in Noonan's syndrome. Int. J. Dermatol., 23, 656-657.White, J.D., Hewett, P.W., Kosoge, D., McCulloch, T., Enholm, B., Carmichael, J., Murray, J.C. (2002) Vascular

endothelial growth factor-D expression is an independent prognostic marker for survival in colorectalcarcinoma. Cancer Res., 62, 1669-1675.

Wickham, T.J., Mathias, P., Cheresh, D.A. and Nemerow, G.R. (1993) Integrins and promote adenovirusinternalization but not virus attachment. Cell, 73, 309–319.

Wigle, J.T., Harvey, N., Detmar, M., Lagutina, I., Grosveld, G., Gunn, M.D., Jackson, D.G., Oliver, G. (2002) An

56

essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J., 21, 1505-1513.Wigle, J.T. and Oliver, G. (1999) Prox1 function is required for the development of the murine lymphatic system.

Cell, 98, 769-778.Wilkinson, D.G. (2001) Multiple roles of EPH receptors and ephrins in neural development. Nat. Rev. Neurosci., 2,

155-164.Wilting, J., Eichmann, A., Christ, B. (1997) Expression of the avian VEGF receptor homologues Quek1 and Quek2

in blood-vascular and lymphatic endothelium and non-endothelial cells during quial embryonic development.Cell&Tissue Res., 288, 207-223.

Wilting, J., Neeff, H. and Christ, B. (1999) Embryonic lymphangiogenesis. Cell Tissue Res., 297, 1-11.Wilting, J., Schneider, M., Papoutsi, M., Alitalo, K. and Christ, B. (2000) An avian model for studies of embryonic

lymphangiogenesis. Lymphology, 33, 81-94.Wise, L.M., Veikkola, T., Mercer, A.A., Savory, L.J., Fleming, S.B., Caesar, C., Vitali, A., Makinen, T., Alitalo, K.

and Stacker, S.A. (1999) Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds toVEGFR2 and neuropilin-1. Proc. Natl Acad. Sci. USA, 96, 3071-3076.

Witt, D.R., Hoyme, H.E., Zonana, J., Manchester, D.K., Fryns, J.P., Stevenson, J.G., Curry, C.J., Hall, J.G. (1987)Lymphedema in Noonan syndrome: clues to pathogenesis and prenatal diagnosis and review of the literature.Am. J. Med. Genet., 27, 841-856.

Witte, M.H., Erickson, R., Bernas, M., Andrade, M., Reiser, F., Conlon, W., Hoyme, H.E. and Witte, C.L. (1998)Phenotypic and genotypic heterogeneity in familial Milroy lymphedema. Lymphology, 31, 145-155.

Witte, M.H., Way, D.L., Witte, C.L. and Bernas, M. (1997) Lymphangiogenesis: Mechanisms, significance andclinical implications. In Goldberg, I.D. and Rosen, E.M. (eds.), Regulation of angiogenesis. Birkhäuser Verlag,Basel, Switzerland, pp. 65-112.

Witzenbichler, B., Asahara, T., Murohara, T., Silver, M., Spyridopoulos, I., Magner, M., Principe, N., Kearney, M.,Hu, J.-S. and Isner, J.M. (1998) Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotesangiogenesis in the setting of tissue ischemia. Am. J. Pathol., 153, 381-394.

Yamada, Y., Takakura, N., Yasue, H., Ogawa, H., Fujisawa, H. and Suda, T. (2001) Exogenous clustered neuropilin1 enhances vasculogenesis and angiogenesis. Blood, 97, 1671-1678.

Yancopoulos, G.D., Davis, S., Gale, N.W., Rudge, J.S., Wiegand, S.J. and Holash, J. (2000) Vascular-specificgrowth factors and blood vessel formation. Nature, 407, 242-248.

Yancopoulos, G.D., Klagsbrun, M. and Folkman, J. (1998) Vasculogenesis, angiogenesis, and growth factors:ephrins enter the fray at the border [comment]. Cell, 93, 661-664.

Yang, Y., Jooss, K.U., Su, Q., Ertl, H.C.J., Wilson, J.M. (1996) Immune response to viral antigens versus transgeneproduct in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther., 3, 137-144.

Ylikorkala, A., Rossi, D.J., Korsisaari, N., Luukko, K., Alitalo, K., Henkemeyer, M., Mäkelä, T. (2001) Vascularabnormalities and degranulation of VEGF in Lkbl-deficient mice. Science, 293, 1323-1326.

Ylä-Herttuala, S. (2001) Gene therapy for coronary heart disease. J. Intern. Med., 250, 367-368.Yonemura, Y., Fushida, S., Bando, E., Kinoshita, K., Miwa, K., Endo, Y., Sugiyama, K., Partanen, T., Yamamoto,

H. and Sasaki, T. (2001) Lymphangiogenesis and the vascular endothelial growth factor receptor (VEGFR)-3 ingastric cancer. Eur. J. Cancer, 37, 918-923.

Yuan, F., Chen, Y., Dellian, M., Safabakhsh, N., Ferrara, N., Jain, R.K. (1996) Time-dependent vascular regressionand permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growthfactor/ vascular permeability factor antibody. Proc. Natl. Acad. Sci. USA, 93, 14765-14770.

Ziegler, B.L., Valtieri, M., Porada, G.A., De Maria, R., Muller, R., Masella, B., Gabbianelli, M., Casella, I., Pelosi,E., Bock, T., Zanjani, E.D., Peschle, C. (1999) KDR receptor: a key marker defining hematopoietic stem cells.Science, 285, 1553-1558.

Ziegler, S.F., Bird, T.A., Schneringer, J.A., Schooley, K.A. and Baum, P.R. (1993) Molecular cloning andcharacterization of a novel receptor protein tyrosine kinase from human placenta. Oncogene, 8, 663-670.

Zinn, A.R., Tonk, V.S., Chen, Z., Flejter, W.L., Gardner, H.A., Guerra, R., Kushner, H., Schwartz, S., Sybert, V.P.,Van Dyke, D.L. and Ross, J.L. (1998) Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1. Am. J.Hum. Genet., 63, 1757-1766.

Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., Trono, D. (1997) Multiply attenuated lentiviral vectors achieves

efficient gene delivery in vivo. Nat. Biotech., 15, 871-875.