Oncogene (2000) 19, 5598 ± 5605 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis

Marika J Karkkainen1 and Tatiana V Petrova*,1

1Molecular Cancer Biology Laboratory, and the Ludwig Institute for Cancer Research, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland

VEGFR-1 (Flt-1), VEGFR-2 (KDR) and VEGFR-3 stimulation of vessel growth: vasorelaxation, induction (Flt4) are endothelial speci®c receptor tyrosine kinases, of vascular permeability, endothelial cell migration, regulated by members of the vascular endothelial growth proliferation and survival. The present review focuses factor family. VEGFRs are indispensable for embryonic on the last three functions of the VEGFR's and, more vascular development, and are involved in the regulation speci®cally, on the signaling events initiated in of many aspects of physiological and pathological endothelial cells upon activation of the VEGFRs. angiogenesis. VEGF-C and VEGF-D, as ligands for The VEGFR family includes VEGFR-1 (Flt-1), VEGFR-3 are also capable of stimulating lymphangio- VEGFR-2 (KDR) and VEGFR-3 (Flt4), which belong genesis and at least VEGF-C can enhance lymphatic to the platelet derived growth factor receptor sub- metastasis. Recent studies have shown that missense family of receptor tyrosine kinases, characterized by mutations within the VEGFR-3 tyrosine kinase domain the presence of seven extracellular immunoglobulin are associated with human hereditary lymphedema, homology domains and a split tyrosine kinase suggesting an important role for this receptor in the intracellular domain. VEGFR tyrosine kinase activity development of the lymphatic vasculature. Oncogene is stimulated by speci®c ligands of the six-member (2000) 19, 5598 ± 5605. VEGF family: VEGF, placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and the viral homo- Keywords: angiogenesis; endothelial cells; receptor logues, collectively called VEGF-E (reviewed in tyrosine kinases; signal transduction; VEGF receptors; Eriksson and Alitalo, 1999; Olofsson et al., 1999; VEGF Ferrara, 1999). Among a number of di€erent factors implicated in the regulation of the angiogenic response, VEGF, an endothelial cell speci®c mitogen Introduction and a potent inducer of vascular permeability, is perhaps the most important player. Inactivation of Angiogenesis or the formation of new blood vessels by one of the two VEGF alleles in the DNA of mouse sprouting from the pre-existing vasculature is of central embryos leads to embryonic lethality due to severe importance for embryonic development and organo- defects in vascular development, whereas increased genesis. In adults, physiological angiogenesis occurs expression of VEGF seems to be pre-requisite for during the female reproductive cycle and in wound tumor growth beyond the microscopic stage (for healing. Abnormally enhanced neovascularization is recent reviews, see Veikkola and Alitalo, 1999; observed in rheumatoid arthritis, psoriasis, diabetic Neufeld et al., 1999). The more detailed questions retinopathy and during tumor development (Folkman, on VEGF and related ligands are discussed in recent 1995), whereas insucient vascular proliferation is reviews (Olofsson et al., 1999; Ferrara, 1999) and will implicated in the pathophysiology of e.g. myocardial not be addressed here. However, it should be noted and limb ischemia (Rivard and Isner, 1998). that the existence of di€erent alternatively spliced So-called sprouting angiogenesis is a complex multi- isoforms (reported so far for VEGF, VEGF-B and stage process. Endothelial cells undergo a transition PlGF), proteolytic processing and heterodimerization from a quiescent to a proliferative state, loosen the cell- contribute an additional dimension into the complex- cell contacts within the vascular wall, detach from the ity of regulation of VEGFR signaling in endothelial parent vessels and migrate directionally. Vasodilation, cells. mediated by nitric oxide, and increased vascular VEGFs belong to the cysteine knot growth factor permeability, which allows extravasation of plasma family and they all contain an approximately 100 proteins serving as a provisional sca€old for migrating amino acid VEGF homology domain characterized by endothelial cells, are initial components of the the precise spacing of eight cysteine residues. PlGF and angiogenic program. Further steps in the formation VEGF-B bind to VEGFR-1, whereas VEGF interacts of functional vessels include lumen formation and with both VEGFR-1 and VEGFR-2. VEGF-C and vessel stabilization (for a recent review see Carmeliet, VEGF-D bind VEGFR-2 and VEGFR-3 and the viral 2000). In conjunction with other endothelial speci®c homologue VEGF-E binds and activates only VEGFR- signaling systems such as angiopoietins and Tie 2 (Figure 1). In addition, neuropilin-1, a transmem- receptors, VE-cadherin/b-catenin and avb3 integrins, brane protein involved in the regulation of axonal vascular endothelial growth factor receptors (VEGFRs) guidance in neurons, was described as a co-receptor for relay signals for at least ®ve processes essential in VEGF-B, PlGF-2, VEGF-E and the VEGF165 isoform of VEGF (Migdal et al., 1998; Soker et al., 1998; Wise et al., 1999; Makinen et al., 1999). As is the case for *Correspondence: TV Petrova many RTKs, binding of the ligand leads to VEGFR VEGF receptors and signal transduction MJ Karkkainen and TV Petrova 5599

Figure 1 Receptor binding speci®city of VEGFs. VEGFs are marked in green, VEGFR-1, VEGFR-2 and VEGFR-3 in blue and red and neuroplilin-1 in yellow. The di€erent structural elements of the receptors are illustrated as follows: blue circle, immunoglobulin domain; red oval, tyrosine kinase domain. VEGFR-1 is a 180 kDa transmembrane glycoprotein, but alternative splicing can also produce a shorter soluble protein containing only six ®rst extracellular immunoglobulin homology domains followed by 31 unique amino acid residues (Shibuya et al., 1990; Kendall and Thomas, 1993; Kendall et al., 1996). VEGFR-2 is a 230 kDa protein and no splice variants have been reported for this receptor (Terman et al., 1991). After its biosynthesis, the glycosylated 195 kDa VEGFR-3 is proteolytically cleaved in the ®fth immunoglobulin-like domain, but the resulting 120 and 75 kDa chains remain linked by disul®de bonds (Pajusola et al., 1993) dimerization and transphosphorylation. Further re- been reported in VEGFR-1 transfected ®broblasts cruitment of diverse adaptor and signaling molecules (Seetharam et al., 1995). activates a complex cascade of intracellular pathways Mice expressing a truncated form of VEGFR-1, resulting in the activation of the angiogenic program which lacks the tyrosine kinase domain, develop by endothelial cells. normally (Hiratsuka et al., 1998). This is in striking contrast to the VEGFR-1 knock out mice, which die early in their development because of increased production of endothelial progenitors and consequently Roles of VEGFR-2 and VEGFR-1 in endothelial cell disorganized embryonic and extraembryonic vascula- proliferation, migration and survival ture (Fong et al., 1995). Taken together with the weak signaling abilities of VEGFR-1, these data suggest that VEGFR-1 endothelial VEGFR-1 might act as a negative regulator VEGFR-1 binds VEGF with a 10-fold higher anity of angiogenesis by sequestering VEGF and thus by than VEGFR-2 (Terman et al., 1992; de Vries et al., preventing VEGFR-2 activation by VEGF. Never- 1992). However, in contrast to VEGFR-2, VEGFR-1 theless, there is still a considerable controversy autophosphorylation upon ligand binding is rather regarding the role of VEGFR-1 in endothelial cells. dicult to detect, and in many instances, the e€ects of For example, it has been reported that in VEGFR-1- VEGFR-2 in endothelial cells, such as cell survival and transfected endothelial cells PlGF but not VEGF could proliferation, cannot be induced by treatment with activate the MAPK pathway to induce a mitogenic VEGFR-1 speci®c ligands nor can they be observed in response and plasminogen activator production VEGFR-1 overexpressing cells lacking VEGFR-2 (Landgren et al., 1998). Another report suggests that (Seetharam et al., 1995; Kroll and Waltenberger, VEGFR-1 might be important for endothelial cell 1997; Gerber et al., 1998b). migration. Indeed, VEGFR-1 blocking antibodies Two major and two minor phosphorylation sites of prevented migration but not proliferation of human VEGFR-1 have been identi®ed as Tyr1213/Tyr1242 umbilical vein endothelial (HUVE) cells in response to and Tyr1327/Tyr1333, respectively (Ito et al., 1998). VEGF (Kanno et al., 2000). In the same study the They are located in the C-terminal tail domain and VEGFR-1-mediated signal appeared to modulate may serve as docking sites for signaling molecules such preferentially actin reorganization via the p38 MAP as PLCg, Nck, Crk, SHP-1/2, or the p85 subunit of PI3 kinase, whereas VEGFR-2 contributed to the reorga- kinase, as shown by yeast two hybrid assays and by in nization of the cytoskeleton by phosphorylating FAK vitro binding studies (Cunningham et al., 1995; Igarashi and paxillin, thus suggesting a di€erential contribution et al., 1998a). Activation of PLCg and RasGAP has of the two receptors to the chemotactic response.

Oncogene VEGF receptors and signal transduction MJ Karkkainen and TV Petrova 5600 VEGF-induced actin stress ®ber formation, activation chemotaxis and sprouting of cultured vascular en- of p38 MAPK and endothelial cell migration migration dothelial cells in vitro and angiogenesis in vivo (Meyer were documented also in earlier studies using HUVE et al., 1999; Wise et al., 1999) and VEGFR-2 blocking cells (Abedi and Zachary, 1997; Rousseau et al., 1997), antibodies prevent DNA synthesis in response to along with phosphorylation of FAK, related focal VEGF (Kanno et al., 2000). In several tumor models, adhesion kinase RAFTK and paxillin (Liu et al., angiogenesis is prevented by expression of a dominant 1997b; Abedi and Zachary, 1997). In line with the negative mutant VEGFR-2 or by use of VEGFR-2 suggested role of VEGFR-1 in endothelial cell blocking antibodies (Millauer et al., 1994; Skobe et al., migration, monocytes/macrophages, which express 1997). Targeted inactivation of VEGFR-2 results in VEGFR-1 but not VEGFR-2, respond to the stimula- early embryonic lethality due to defects in the tion with PlGF and VEGF with increased intracellular di€erentiation of endothelial and primitive hemato- calcium levels and enhanced migration (Clauss et al., poietic cells, with an inhibition of blood vessel 1990, 1996; Shen et al., 1993; Barleon et al., 1996), and formation (Shalaby et al., 1995). this response is suppressed in monocytes from mice Major autophosphorylation sites of VEGFR-2 are with the kinase-deleted VEGFR-1 (Hiratsuka et al., located in the kinase insert domain (Tyr951/996) and in 1998). the tyrosine kinase catalytic domain (Tyr1054/1059) (Dougher-Vermazen et al., 1994). In endothelial cells overexpressing VEGFR-2, activation of the receptor VEGFR-2 leads to a rapid recruitment of the adaptor proteins Cultured primary endothelial cells express both Shc, Grb2 and Nck, and protein tyrosine phosphatases VEGFR-1 and VEGFR-2 and, in some cases, also SHP-1 and SHP-2 (Kroll and Waltenberger, 1997). The VEGFR-3, making it dicult to evaluate the contribu- Shc homologue Sck has also been shown to interact tion of individual receptors to the responses elicited by with VEGFR-2 via its SH2 domain in yeast two-hybrid the VEGFs. However, based on studies of cell lines assay (Igarashi et al., 1998b; Warner et al., 2000). On expressing individual receptors or employing ligands the other hand, in VEGF-stimulated endothelial cells speci®c for only one receptor type, the current view is phosphorylation of Shc was barely detectable in spite that VEGFR-2 is a major receptor transducing the of activation of the guanine nucleotide exchange factor e€ects of VEGF into endothelial cells (see Figure 2). Sos and stimulation of a mitogenic response (Seethar- For example, similar to VEGF, viral homologues am et al., 1995). Recently, two other signaling VEGF-E that bind and activate only VEGFR-2, can molecules interacting with phosphorylated VEGFR-2 stimulate the release of tissue factor, proliferation, have been identi®ed in yeast two-hybrid assays. The

Figure 2 Simpli®ed scheme of signaling events initiated in endothelial cells upon VEGFR-2 activation. The pathways are discussed in the text. The dashed lines indicate potential signaling pathways. Not shown are the interactions of VEGFR-2 with VE-cadherin and b-catenin, and with the avb3integrin

Oncogene VEGF receptors and signal transduction MJ Karkkainen and TV Petrova 5601 ®rst is a low molecular weight protein tyrosine protein complex. Targeted inactivation or truncation of phosphatase HCPTPA, which, similar to SHP-1 and VE-cadherin (which suppresses the interaction with b- SHP-2, may serve as a negative regulator of VEGFR-2 catenin) prevented activation of PI3-K and Akt in activity (Huang et al., 1999). The second is a new SH2- response to VEGF, and abolished VEGF-induced cell containing proline rich adaptor protein which binds survival, demonstrating that VE-cadherin and b- constitutively to PLCg and PI3-kinase and becomes catenin lie upstream of PI3-K and Akt in the recruited to VEGFR-2 upon receptor autophosphor- regulation of endothelial cell survival (Carmeliet et ylation (Wu et al., 2000). al., 1999). Akt can counteract apoptotic signaling by Activation of the p42/44 MAP kinase pathway by phosphorylating and inactivating caspase-9, pro-apop- VEGF and subsequent cell proliferation has been totic protein Bad and Forkhead transcription factors documented for many types of endothelial cells involved in the expression of proapoptotic proteins (Seetharam et al., 1995; D'Angelo et al., 1995; (reviewed in Datta et al., 1999). In addition, expression Rousseau et al., 1997; Kroll and Waltenberger, 1997; of antiapoptotic molecules such as Bcl-2, A1 and the Takahashi and Shibuya, 1997; Landgren et al., 1998; caspase-3 inhibitor survivin can contribute to the Takahashi et al., 1999). Association of VEGFR-2 with increased survival of endothelial cells in the presence Grb2 suggests that activated Ras might be involved in of VEGF (Gerber et al., 1998a; Tran et al., 1999). the regulation of the VEGF-induced mitogenic re- Interestingly, it appears that PI3-K and Akt do not sponse. However, it was reported recently that in liver relay the mitogenic e€ect of VEGF since inhibition of sinusoidal endothelial cells the VEGF growth signal is PI3-K does not impair the proliferative responses of transduced mostly through PLCg-PKC and not via Ras endothelial cells (Xia et al., 1996). (Takahashi et al., 1999). Phosphorylated VEGFR-2 Similar to many cell types, also endothelial cell rapidly recruits and activates PLCg (Waltenberger et survival and proliferation require adhesion to the al., 1994; D'Angelo et al., 1995; Guo et al., 1995; Xia pericellular matrix, which is mediated by various et al., 1996; Takahashi and Shibuya, 1997) which then members of the heterodimeric transmembrane proteins hydrolyses a cell membrane phospholipid, phosphatidyl called intergins. More speci®cally, the avb3 integrin inositol 4,5-bisphosphate, and releases sn-1,2-diacylgly- which is expressed by angiogenic endothelia (Brooks et cerol (DAG) and inositol 1,4,5-trisphosphate (IP3). al., 1994; Stromblad et al., 1996; Scatena et al., 1998; These two second messengers are responsible for direct Soldi et al., 1999), forms a complex with VEGFR-2 activation of certain PKC isoforms and for the release and enhance VEGFR-2 phosphorylation, PI3-K acti- of Ca2+ from intracellular stores, respectively (Criscuo- vation and cell proliferation. A co-operative signaling lo et al., 1989; Brock et al., 1991; de Vries et al., 1992). of a vascular integrin and VEGFR-2 may thus be VEGF has been shown to selectively activate the Ca2+ - necessary for the execution of the angiogenic program sensitive PKC isoforms g and b2 in bovine aortic (Soldi et al., 1999). endothelial cells (Xia et al., 1996; Takahashi et al., 1999) and PKCe in HUVE cells (Wu et al., 2000). In the former case the mitogenic e€ect of VEGF in Lymphangiogenic role of VEGFR-3 endothelial cells was suppressed by PKC-b and -g- selective inhibitors. The lymphatic vessels transport tissue ¯uids, extra- VEGF stimulates nitric oxide release from endothe- vasated plasma proteins and cells back into the blood lial cells, which results from the IP3 induced rise in circulation, and they also make an important part of the intracellular calcium concentration which enhances the body's immunological surveillance system. In many activity of the endothelial nitric oxide synthase (eNOS) tumor types, lymphatic vasculature serves as a major directly, and from the activation of the Akt/PKB route for tumor metastasis. VEGFR-3 is one of the rare serine/threonine kinase. Akt can phosphorylate and proteins that have been shown to control the develop- activate eNOS in a calcium-independent manner ment and growth of the lymphatic system. This receptor (Dimmeler et al., 1999; Fulton et al., 1999). A is essential in the formation of the primary cardiovas- subsequent increase in intracellular levels of cGMP cular network before the emergence of the lymphatic may contribute to the activation of the MAPK cascade vessels, as VEGFR-3 knockout embryos die early in and endothelial cell proliferation, since these responses development because of cardiovascular failure (Dumont can be partially prevented in HUVE cells by treatment et al., 1998). Later in development, the VEGFR-3 with nitric oxide synthase inhibitors (Parenti et al., expression becomes restricted mainly to lymphatic 1998). vessels (Kaipainen et al., 1995; Kukk et al., 1996). In addition to stimulation of endothelial cell Recently, it has also been shown, that in addition to migration and proliferation, VEGF also provides a lymphatic endothelium, VEGFR-3 is expressed in cell survival signal. VEGF protects endothelial cells tumor blood vessels during neovascularization (Valtola against tumor necrosis factor a or serum-starvation et al., 1999) and in some fenestrated endothelia induced apoptosis (Spyridopoulos et al., 1997; Gerber (Partanen et al., 2000). The VEGFR-3 ligand VEGF- et al., 1998b). Increased survival is observed in C is mitogenic towards lymphatic endothelial cells and endothelial cells treated with a VEGFR-2 selective it showed a selective lymphangiogenic response in mutant of VEGF but not with a VEGFR-1 selective di€erentiated avian chorioallantoic membrane (Oh et mutant or PlGF. This VEGFR-2 mediated survival al., 1997). When VEGF-C was over-expressed in mouse e€ect is transduced via the phosphatidylinositol 3- skin as a transgene, it induced a hyperplastic lymphatic kinase (PI3-K), which further activates Akt (Gerber et vessel network in the dermis (Jeltsch et al., 1997). al., 1998b). VEGFR-2, PI3-K, b-catenin and the Expression of VEGF-C under the control of rat insulin endothelial adherens junction protein VE-cadherin promoter II (Rip) led to the formation of extensive participate in the formation of a mutimeric signaling lymphatic vasculature around islets of Langherans.

Oncogene VEGF receptors and signal transduction MJ Karkkainen and TV Petrova 5602 Moreover, the RipVEGF-C/RipTag double transgenic the VEGFR-3 protein, VEGF-C stimulation induced mice developed highly metastatic b-cell tumors, in recruitment of the signaling molecules Shc, Grb2 and contrast to the parental RipTag mice which served as Sos to the activated receptor and a cell growth response a model of non-metastatic b-cell carcinogenesis (S (Wang et al., 1997). The binding of VEGFR-3 to Grb2 is Mandriota et al., manuscript submitted). Taken mediated by the Grb2 SH2 domain (Pajusola et al., together, these results suggest that a speci®c lymphan- 1994; Fournier et al., 1995), and the PTB domain of Shc giogenic response is mediated by VEGF-C/VEGFR-3 is required for Shc tyrosine phosphorylation by signaling, and that VEGF-C/VEGFR-3-induced lym- VEGFR-3 (Fournier et al., 1999). Mutations in Shc phangiogenesis has a direct role in the metastatic phosphorylation sites increased VEGFR-3 transforming dissemination of tumor cells. activity in the soft agar assay, suggesting that Shc has an In humans, two isoforms of the VEGFR-3 protein inhibitory role in VEGFR-3 mediated growth response. occur, designated VEGFR-3s (short) and VEGFR-31 In human erythroleukemia cell line, VEGF-C also (long), di€ering in their carboxyl termini as a result of induces phosphorylation of paxillin by related adhesion alternative mRNA splicing (Pajusola et al., 1993; focal tyrosine kinase (RAFTK), a recently identi®ed Galland et al., 1993). The long form is the predominant member of the focal adhesion kinase family (Liu et al., one in the tissues. It contains three additional tyrosyl 1997a). residues, of which Tyr1337 serves as an important The importance of VEGFR-3 for the development of autophosphorylation site in the receptor (Pajusola et al., the lymphatic vasculature has been shown recently, when 1993; Fournier et al., 1995). Only VEGFR-31 was able early onset primary lymphedema was linked to the to mediate anchorage-independent growth in soft agar VEGFR3 locus in distal chromosome 5q (Ferrell et al., and tumorigenicity in nude mice (Pajusola et al., 1994; 1998; Witte et al., 1998; Evans et al., 1999). Primary Fournier et al., 1995; Borg et al., 1995). VEGF-C and lymphedema is an inherited swelling of the limbs, that VEGF-D bind to and induce tyrosine phosphorylation can be present either at birth (Milroy's disease) or can of VEGFR-3 (Lee et al., 1996; Joukov et al., 1996; develop at the onset of puberty (Meige's disease). Achen et al., 1998). Ligand stimulation of VEGFR-3 Primary lymphedema generally shows an autosomal induced rapid tyrosine phosphorylation of Shc and dominant pattern of inheritance with reduced pene- activation of MAPK, increased cell motility, actin trance, variable expression, and variable age at onset. In reorganization and proliferation, thus suggesting that primary lymphedema, the super®cial lymphatic vessels VEGFR-2 and VEGFR-3 may activate similar or are usually hypoplastic or aplastic, and they fail to overlapping signaling pathways (Cao et al., 1998; transport the lymphatic ¯uid back into the venous Joukov et al., 1998). In addition, in a human circulation, resulting in dis®guring swelling of the erythroleukemia cell line that expresses high levels of extremities. The disease is further complicated by a

Figure 3 A model of the dominant-negative mode of action of VEGFR-3 in hereditary lymphedema. After its activation, the wild type VEGFR-3 is rapidly internalized and degraded, whereas inactive or weakly active receptor dimers containing mutant VEGFR- 3 remain longer at the cell surface. This results in reduced VEGFR-3 signaling, hypoplastic lymphatic vascular formation and accumulation of the tissue ¯uid mainly in the lower extremities. Mutant residues identi®ed so far are indicated in the boxes

Oncogene VEGF receptors and signal transduction MJ Karkkainen and TV Petrova 5603 variety of secondary conditions, such as ®brosis, adipose ing controls mainly the growth of the lymphatic degeneration and infections. vasculature. A signi®cant progress has been made in The lymphedema-linked mutations in the VEGFR-3 the elucidation of molecular events initiated in tyrosine kinase domain (G857R, H1035R, R1041P, endothelial cells upon activation of VEGFRs. How- L1044P and P1114L), indicate that disturbed VEGFR- ever, many questions remain unanswered, such as the 3 signaling may play a role in the development of this existence of endothelial speci®c signaling pathways, the disease. In VEGFR-3 activation, ligand binding results precise role of VEGFR-1 in modulation of the receptor dimerization and intracellular tyrosyl transpho- angiogenic responses, di€erences between the signaling sphorylation, whereas all lymphedema-associated mu- properties of VEGFR-2 and VEGFR-3, and, most tant receptors were incapable of transphosphorylation importantly, the question of how the VEGFR signaling (Irrthum et al., 2000; Karkkainen et al., 2000). The network is integrated and coordinated with other kinase inactive VEGFR-3 proteins were also poor receptor tyrosine kinase systems in endothelial cells, activators of the downstream signaling cascades, such as angiopoietins/Ties and ephrins/Ephs (Gale and suggesting that they fail to transduce VEGF-C/VEGF- Yancopoulos, 1999; Holash et al., 1999). Answering D signals into the endothelial cells. The kinase inactive these questions will provide better understanding of receptors were degraded at slower rate than the wild- molecular processes implicated in regulation of vas- type receptors, and were thus more stable on the cell cular growth, and consequently, will contribute to the surface. This probably contributes to the development design of better treatment strategies of angiogenesis of lymphedema by reducing the relative amount of related disorders. ligand binding to the wild-type VEGFR-3 and the resulting signaling. This in vitro data suggests that inherited amino acid changes, which inactivate the Abbreviations VEGFR-3 kinase and interfere with its signaling eNOS, endothelial nitric oxide synthase; FAK, focal function, lead to the dominant-negative type of response adhesion kinase; HUVE, human umbilical vein endothelial; and development of lymphedema (Figure 3). Also other IP3, inositol-3-phosphate; MAPK, mitogen activated pro- tein kinase; PI3-K, phosphatidylinositol 3-kinase; PKC, lymphedema genes exist (Mangion et al., 1999) and they protein kinase C; PLC, phospholipase C; PlGF, placenta may code for proteins involved in e.g. VEGFR-3 growth factor; RTK, receptor tyrosine kinase; VEGF, downstream signaling pathways. The elucidation of vascular endothelial growth factor; VEGFR, VEGF re- molecular components of these pathways could be ceptor bene®cial both in terms of diagnosis and possible therapy of lymphedema with VEGFR-3 as a target. Acknowledgments We thank Dr Kari Alitalo for support and critical Conclusions comments. The studies in the authors' laboratory were supported by the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cancer Organizations, the Finnish VEGFRs control many aspects of vascular growth Cultural Foundation, the Ida Montini Foundation, the both during embryonic development and physiological Emil Aaltonen foundation, the Finnish State Technology and pathological angiogenic responses in adults. While Development Centre, the Helsinki University Hospital VEGFR-1 and VEGFR-2 act as universal regulators of Research Fund, the Novo Nordisk Foundation and the endothelial cell functions, VEGF-C/VEGFR-3 signal- European Union Biomed Program.

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