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

Correspondence to: T V Petrova, Molecular 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 (Flt4) are endothelial specific receptor tyrosine kinases, regulated by members of the vascular endothelial growth factor family. VEGFRs are indispensable for embryonic vascular development, and are involved in the regulation of many aspects of physiological and pathological angiogenesis. VEGF-C and VEGF-D, as ligands for VEGFR-3 are also capable of stimulating lymphangiogenesis and at least VEGF-C can enhance lymphatic metastasis. Recent studies have shown that missense mutations within the VEGFR-3 tyrosine kinase domain are associated with human hereditary lymphedema, suggesting an important role for this receptor in the development of the lymphatic vasculature. Oncogene (2000) 19, 5598-5605.

angiogenesis; endothelial cells; receptor tyrosine kinases; signal transduction; VEGF receptors; VEGF

eNOS, endothelial nitric oxide synthase; FAK, focal adhesion kinase; HUVE, human umbilical vein endothelial; IP3, inositol-3-phosphate; MAPK, mitogen activated protein kinase; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PlGF, placenta growth factor; RTK, receptor tyrosine kinase; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor

Introduction

Angiogenesis or the formation of new blood vessels by sprouting from the pre-existing vasculature is of central importance for embryonic development and organogenesis. In adults, physiological angiogenesis occurs during the female reproductive cycle and in wound healing. Abnormally enhanced neovascularization is observed in rheumatoid arthritis, psoriasis, diabetic retinopathy and during tumor development (Folkman, 1995), whereas insufficient vascular proliferation is implicated in the pathophysiology of e.g. myocardial and limb ischemia (Rivard and Isner, 1998).

So-called sprouting angiogenesis is a complex multistage process. Endothelial cells undergo a transition from a quiescent to a proliferative state, loosen the cell-cell contacts within the vascular wall, detach from the parent vessels and migrate directionally. Vasodilation, mediated by nitric oxide, and increased vascular permeability, which allows extravasation of plasma proteins serving as a provisional scaffold for migrating endothelial cells, are initial components of the angiogenic program. Further steps in the formation of functional vessels include lumen formation and vessel stabilization (for a recent review see Carmeliet, 2000). In conjunction with other endothelial specific signaling systems such as angiopoietins and Tie receptors, VE-cadherin/-catenin and _v3 integrins, vascular endothelial growth factor receptors (VEGFRs) relay signals for at least five processes essential in stimulation of vessel growth: vasorelaxation, induction of vascular permeability, endothelial cell migration, proliferation and survival. The present review focuses on the last three functions of the VEGFR's and, more specifically, on the signaling events initiated in endothelial cells upon activation of the VEGFRs.

The VEGFR family includes VEGFR-1 (Flt-1), VEGFR-2 (KDR) and VEGFR-3 (Flt4), which belong to the platelet derived growth factor receptor subfamily of receptor tyrosine kinases, characterized by the presence of seven extracellular immunoglobulin homology domains and a split tyrosine kinase intracellular domain. VEGFR tyrosine kinase activity is stimulated by specific ligands of the six-member VEGF family: VEGF, placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and the viral homologues, collectively called VEGF-E (reviewed in Eriksson and Alitalo, 1999; Olofsson et al., 1999; Ferrara, 1999). Among a number of different factors implicated in the regulation of the angiogenic response, VEGF, an endothelial cell specific mitogen and a potent inducer of vascular permeability, is perhaps the most important player. Inactivation of one of the two VEGF alleles in the DNA of mouse embryos leads to embryonic lethality due to severe defects in vascular development, whereas increased expression of VEGF seems to be pre-requisite for tumor growth beyond the microscopic stage (for recent reviews, see Veikkola and Alitalo, 1999; Neufeld et al., 1999). The more detailed questions on VEGF and related ligands are discussed in recent reviews (Olofsson et al., 1999; Ferrara, 1999) and will not be addressed here. However, it should be noted that the existence of different alternatively spliced isoforms (reported so far for VEGF, VEGF-B and PlGF), proteolytic processing and heterodimerization contribute an additional dimension into the complexity of regulation of VEGFR signaling in endothelial cells.

VEGFs belong to the cysteine knot growth factor family and they all contain an approximately 100 amino acid VEGF homology domain characterized by the precise spacing of eight cysteine residues. PlGF and VEGF-B bind to VEGFR-1, whereas VEGF interacts with both VEGFR-1 and VEGFR-2. VEGF-C and VEGF-D bind VEGFR-2 and VEGFR-3 and the viral homologue VEGF-E binds and activates only VEGFR-2 (Figure 1). In addition, neuropilin-1, a transmembrane protein involved in the regulation of axonal guidance in neurons, was described as a co-receptor for 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 many RTKs, binding of the ligand leads to VEGFR dimerization and transphosphorylation. Further recruitment of diverse adaptor and signaling molecules activates a complex cascade of intracellular pathways resulting in the activation of the angiogenic program by endothelial cells.

Roles of VEGFR-2 and VEGFR-1 in endothelial cell proliferation, migration and survival

VEGFR-1

VEGFR-1 binds VEGF with a 10-fold higher affinity than VEGFR-2 (Terman et al., 1992; de Vries et al., 1992). However, in contrast to VEGFR-2, VEGFR-1 autophosphorylation upon ligand binding is rather difficult to detect, and in many instances, the effects of VEGFR-2 in endothelial cells, such as cell survival and proliferation, cannot be induced by treatment with VEGFR-1 specific ligands nor can they be observed in VEGFR-1 overexpressing cells lacking VEGFR-2 (Seetharam et al., 1995; Kroll and Waltenberger, 1997; Gerber et al., 1998b).

Two major and two minor phosphorylation sites of VEGFR-1 have been identified as Tyr1213/Tyr1242 and Tyr1327/Tyr1333, respectively (Ito et al., 1998). They are located in the C-terminal tail domain and may serve as docking sites for signaling molecules such as PLC, Nck, Crk, SHP-1/2, or the p85 subunit of PI3 kinase, as shown by yeast two hybrid assays and by in vitro binding studies (Cunningham et al., 1995; Igarashi et al., 1998a). Activation of PLC and RasGAP has been reported in VEGFR-1 transfected fibroblasts (Seetharam et al., 1995).

Mice expressing a truncated form of VEGFR-1, which lacks the tyrosine kinase domain, develop 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 disorganized embryonic and extraembryonic vasculature (Fong et al., 1995). Taken together with the weak signaling abilities of VEGFR-1, these data suggest that endothelial VEGFR-1 might act as a negative regulator of angiogenesis by sequestering VEGF and thus by preventing VEGFR-2 activation by VEGF. Nevertheless, there is still a considerable controversy regarding the role of VEGFR-1 in endothelial cells. For example, it has been reported that in VEGFR-1-transfected endothelial cells PlGF but not VEGF could activate the MAPK pathway to induce a mitogenic response and plasminogen activator production (Landgren et al., 1998). Another report suggests that VEGFR-1 might be important for endothelial cell migration. Indeed, VEGFR-1 blocking antibodies prevented migration but not proliferation of human umbilical vein endothelial (HUVE) cells in response to VEGF (Kanno et al., 2000). In the same study the VEGFR-1-mediated signal appeared to modulate preferentially actin reorganization via the p38 MAP kinase, whereas VEGFR-2 contributed to the reorganization of the cytoskeleton by phosphorylating FAK and paxillin, thus suggesting a differential contribution of the two receptors to the chemotactic response. VEGF-induced actin stress fiber formation, activation of p38 MAPK and endothelial cell migration migration were documented also in earlier studies using HUVE cells (Abedi and Zachary, 1997; Rousseau et al., 1997), along with phosphorylation of FAK, related focal adhesion kinase RAFTK and paxillin (Liu et al., 1997b; Abedi and Zachary, 1997). In line with the suggested role of VEGFR-1 in endothelial cell migration, monocytes/macrophages, which express VEGFR-1 but not VEGFR-2, respond to the stimulation with PlGF and VEGF with increased intracellular calcium levels and enhanced migration (Clauss et al., 1990, 1996; Shen et al., 1993; Barleon et al., 1996), and this response is suppressed in monocytes from mice with the kinase-deleted VEGFR-1 (Hiratsuka et al., 1998).

VEGFR-2

Cultured primary endothelial cells express both VEGFR-1 and VEGFR-2 and, in some cases, also VEGFR-3, making it difficult to evaluate the contribution of individual receptors to the responses elicited by the VEGFs. However, based on studies of cell lines expressing individual receptors or employing ligands specific for only one receptor type, the current view is that VEGFR-2 is a major receptor transducing the effects of VEGF into endothelial cells (see Figure 2). For example, similar to VEGF, viral homologues VEGF-E that bind and activate only VEGFR-2, can stimulate the release of tissue factor, proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo (Meyer et al., 1999; Wise et al., 1999) and VEGFR-2 blocking antibodies prevent DNA synthesis in response to VEGF (Kanno et al., 2000). In several tumor models, angiogenesis is prevented by expression of a dominant negative mutant VEGFR-2 or by use of VEGFR-2 blocking antibodies (Millauer et al., 1994; Skobe et al., 1997). Targeted inactivation of VEGFR-2 results in early embryonic lethality due to defects in the differentiation of endothelial and primitive hematopoietic cells, with an inhibition of blood vessel formation (Shalaby et al., 1995).

Major autophosphorylation sites of VEGFR-2 are located in the kinase insert domain (Tyr951/996) and in the tyrosine kinase catalytic domain (Tyr1054/1059) (Dougher-Vermazen et al., 1994). In endothelial cells overexpressing VEGFR-2, activation of the receptor leads to a rapid recruitment of the adaptor proteins Shc, Grb2 and Nck, and protein tyrosine phosphatases SHP-1 and SHP-2 (Kroll and Waltenberger, 1997). The Shc homologue Sck has also been shown to interact with VEGFR-2 via its SH2 domain in yeast two-hybrid assay (Igarashi et al., 1998b; Warner et al., 2000). On the other hand, in VEGF-stimulated endothelial cells phosphorylation of Shc was barely detectable in spite of activation of the guanine nucleotide exchange factor Sos and stimulation of a mitogenic response (Seetharam et al., 1995). Recently, two other signaling molecules interacting with phosphorylated VEGFR-2 have been identified in yeast two-hybrid assays. The first is a low molecular weight protein tyrosine phosphatase HCPTPA, which, similar to SHP-1 and SHP-2, may serve as a negative regulator of VEGFR-2 activity (Huang et al., 1999). The second is a new SH2-containing proline rich adaptor protein which binds constitutively to PLC and PI3-kinase and becomes recruited to VEGFR-2 upon receptor autophosphorylation (Wu et al., 2000).

Activation of the p42/44 MAP kinase pathway by VEGF and subsequent cell proliferation has been documented for many types of endothelial cells (Seetharam et al., 1995; D'Angelo et al., 1995; Rousseau et al., 1997; Kroll and Waltenberger, 1997; Takahashi and Shibuya, 1997; Landgren et al., 1998; Takahashi et al., 1999). Association of VEGFR-2 with Grb2 suggests that activated Ras might be involved in the regulation of the VEGF-induced mitogenic response. However, it was reported recently that in liver sinusoidal endothelial cells the VEGF growth signal is transduced mostly through PLC-PKC and not via Ras (Takahashi et al., 1999). Phosphorylated VEGFR-2 rapidly recruits and activates PLC (Waltenberger et al., 1994; D'Angelo et al., 1995; Guo et al., 1995; Xia et al., 1996; Takahashi and Shibuya, 1997) which then hydrolyses a cell membrane phospholipid, phosphatidyl inositol 4,5-bisphosphate, and releases sn-1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). These two second messengers are responsible for direct activation of certain PKC isoforms and for the release of Ca²⁺ from intracellular stores, respectively (Criscuolo et al., 1989; Brock et al., 1991; de Vries et al., 1992). VEGF has been shown to selectively activate the Ca²⁺ - sensitive PKC isoforms and 2 in bovine aortic endothelial cells (Xia et al., 1996; Takahashi et al., 1999) and PKC in HUVE cells (Wu et al., 2000). In the former case the mitogenic effect of VEGF in endothelial cells was suppressed by PKC- and -- selective inhibitors.

VEGF stimulates nitric oxide release from endothelial cells, which results from the IP3 induced rise in intracellular calcium concentration which enhances the activity of the endothelial nitric oxide synthase (eNOS) directly, and from the activation of the Akt/PKB serine/threonine kinase. Akt can phosphorylate and activate eNOS in a calcium-independent manner (Dimmeler et al., 1999; Fulton et al., 1999). A subsequent increase in intracellular levels of cGMP may contribute to the activation of the MAPK cascade and endothelial cell proliferation, since these responses can be partially prevented in HUVE cells by treatment with nitric oxide synthase inhibitors (Parenti et al., 1998).

In addition to stimulation of endothelial cell migration and proliferation, VEGF also provides a cell survival signal. VEGF protects endothelial cells against tumor necrosis factor or serum-starvation induced apoptosis (Spyridopoulos et al., 1997; Gerber et al., 1998b). Increased survival is observed in endothelial cells treated with a VEGFR-2 selective mutant of VEGF but not with a VEGFR-1 selective mutant or PlGF. This VEGFR-2 mediated survival effect is transduced via the phosphatidylinositol 3-kinase (PI3-K), which further activates Akt (Gerber et al., 1998b). VEGFR-2, PI3-K, -catenin and the endothelial adherens junction protein VE-cadherin participate in the formation of a mutimeric signaling protein complex. Targeted inactivation or truncation of VE-cadherin (which suppresses the interaction with -catenin) prevented activation of PI3-K and Akt in response to VEGF, and abolished VEGF-induced cell survival, demonstrating that VE-cadherin and -catenin lie upstream of PI3-K and Akt in the regulation of endothelial cell survival (Carmeliet et al., 1999). Akt can counteract apoptotic signaling by phosphorylating and inactivating caspase-9, pro-apoptotic protein Bad and Forkhead transcription factors involved in the expression of proapoptotic proteins (reviewed in Datta et al., 1999). In addition, expression of antiapoptotic molecules such as Bcl-2, A1 and the caspase-3 inhibitor survivin can contribute to the increased survival of endothelial cells in the presence of VEGF (Gerber et al., 1998a; Tran et al., 1999). Interestingly, it appears that PI3-K and Akt do not relay the mitogenic effect of VEGF since inhibition of PI3-K does not impair the proliferative responses of endothelial cells (Xia et al., 1996).

Similar to many cell types, also endothelial cell survival and proliferation require adhesion to the pericellular matrix, which is mediated by various members of the heterodimeric transmembrane proteins called intergins. More specifically, the _vb₃ integrin which is expressed by angiogenic endothelia (Brooks et al., 1994; Stromblad et al., 1996; Scatena et al., 1998; Soldi et al., 1999), forms a complex with VEGFR-2 and enhance VEGFR-2 phosphorylation, PI3-K activation and cell proliferation. A co-operative signaling of a vascular integrin and VEGFR-2 may thus be necessary for the execution of the angiogenic program (Soldi et al., 1999).

Lymphangiogenic role of VEGFR-3

The lymphatic vessels transport tissue fluids, extravasated plasma proteins and cells back into the blood circulation, and they also make an important part of the body's immunological surveillance system. In many tumor types, lymphatic vasculature serves as a major route for tumor metastasis. VEGFR-3 is one of the rare proteins that have been shown to control the development and growth of the lymphatic system. This receptor is essential in the formation of the primary cardiovascular network before the emergence of the lymphatic vessels, as VEGFR-3 knockout embryos die early in development because of cardiovascular failure (Dumont et al., 1998). Later in development, the VEGFR-3 expression becomes restricted mainly to lymphatic vessels (Kaipainen et al., 1995; Kukk et al., 1996). Recently, it has also been shown, that in addition to lymphatic endothelium, VEGFR-3 is expressed in tumor blood vessels during neovascularization (Valtola et al., 1999) and in some fenestrated endothelia (Partanen et al., 2000). The VEGFR-3 ligand VEGF-C is mitogenic towards lymphatic endothelial cells and it showed a selective lymphangiogenic response in differentiated avian chorioallantoic membrane (Oh et al., 1997). When VEGF-C was over-expressed in mouse skin as a transgene, it induced a hyperplastic lymphatic vessel network in the dermis (Jeltsch et al., 1997). Expression of VEGF-C under the control of rat insulin promoter II (Rip) led to the formation of extensive lymphatic vasculature around islets of Langherans. Moreover, the RipVEGF-C/RipTag double transgenic mice developed highly metastatic -cell tumors, in contrast to the parental RipTag mice which served as a model of non-metastatic -cell carcinogenesis (S Mandriota et al., manuscript submitted). Taken together, these results suggest that a specific lymphangiogenic response is mediated by VEGF-C/VEGFR-3 signaling, and that VEGF-C/VEGFR-3-induced lymphangiogenesis has a direct role in the metastatic dissemination of tumor cells.

In humans, two isoforms of the VEGFR-3 protein occur, designated VEGFR-3s (short) and VEGFR-31 (long), differing in their carboxyl termini as a result of alternative mRNA splicing (Pajusola et al., 1993; Galland et al., 1993). The long form is the predominant one in the tissues. It contains three additional tyrosyl residues, of which Tyr1337 serves as an important autophosphorylation site in the receptor (Pajusola et al., 1993; Fournier et al., 1995). Only VEGFR-31 was able to mediate anchorage-independent growth in soft agar and tumorigenicity in nude mice (Pajusola et al., 1994; Fournier et al., 1995; Borg et al., 1995). VEGF-C and VEGF-D bind to and induce tyrosine phosphorylation of VEGFR-3 (Lee et al., 1996; Joukov et al., 1996; Achen et al., 1998). Ligand stimulation of VEGFR-3 induced rapid tyrosine phosphorylation of Shc and activation of MAPK, increased cell motility, actin reorganization and proliferation, thus suggesting that VEGFR-2 and VEGFR-3 may activate similar or overlapping signaling pathways (Cao et al., 1998; Joukov et al., 1998). In addition, in a human erythroleukemia cell line that expresses high levels of the VEGFR-3 protein, VEGF-C stimulation induced recruitment of the signaling molecules Shc, Grb2 and Sos to the activated receptor and a cell growth response (Wang et al., 1997). The binding of VEGFR-3 to Grb2 is mediated by the Grb2 SH2 domain (Pajusola et al., 1994; Fournier et al., 1995), and the PTB domain of Shc is required for Shc tyrosine phosphorylation by VEGFR-3 (Fournier et al., 1999). Mutations in Shc phosphorylation sites increased VEGFR-3 transforming activity in the soft agar assay, suggesting that Shc has an inhibitory role in VEGFR-3 mediated growth response. In human erythroleukemia cell line, VEGF-C also induces phosphorylation of paxillin by related adhesion focal tyrosine kinase (RAFTK), a recently identified member of the focal adhesion kinase family (Liu et al., 1997a).

The importance of VEGFR-3 for the development of the lymphatic vasculature has been shown recently, when early onset primary lymphedema was linked to the VEGFR3 locus in distal chromosome 5q (Ferrell et al., 1998; Witte et al., 1998; Evans et al., 1999). Primary lymphedema is an inherited swelling of the limbs, that can be present either at birth (Milroy's disease) or can develop at the onset of puberty (Meige's disease). Primary lymphedema generally shows an autosomal dominant pattern of inheritance with reduced penetrance, variable expression, and variable age at onset. In primary lymphedema, the superficial lymphatic vessels are usually hypoplastic or aplastic, and they fail to transport the lymphatic fluid back into the venous circulation, resulting in disfiguring swelling of the extremities. The disease is further complicated by a variety of secondary conditions, such as fibrosis, adipose degeneration and infections.

The lymphedema-linked mutations in the VEGFR-3 tyrosine kinase domain (G857R, H1035R, R1041P, L1044P and P1114L), indicate that disturbed VEGFR-3 signaling may play a role in the development of this disease. In VEGFR-3 activation, ligand binding results receptor dimerization and intracellular tyrosyl transphosphorylation, whereas all lymphedema-associated mutant receptors were incapable of transphosphorylation (Irrthum et al., 2000; Karkkainen et al., 2000). The kinase inactive VEGFR-3 proteins were also poor activators of the downstream signaling cascades, suggesting that they fail to transduce VEGF-C/VEGF-D signals into the endothelial cells. The kinase inactive receptors were degraded at slower rate than the wild-type receptors, and were thus more stable on the cell surface. This probably contributes to the development of lymphedema by reducing the relative amount of 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 VEGFR-3 kinase and interfere with its signaling function, lead to the dominant-negative type of response and development of lymphedema (Figure 3). Also other lymphedema genes exist (Mangion et al., 1999) and they may code for proteins involved in e.g. VEGFR-3 downstream signaling pathways. The elucidation of molecular components of these pathways could be beneficial both in terms of diagnosis and possible therapy of lymphedema with VEGFR-3 as a target.

Conclusions

VEGFRs control many aspects of vascular growth both during embryonic development and physiological and pathological angiogenic responses in adults. While VEGFR-1 and VEGFR-2 act as universal regulators of endothelial cell functions, VEGF-C/VEGFR-3 signaling controls mainly the growth of the lymphatic vasculature. A significant progress has been made in the elucidation of molecular events initiated in endothelial cells upon activation of VEGFRs. However, many questions remain unanswered, such as the existence of endothelial specific signaling pathways, the precise role of VEGFR-1 in modulation of the angiogenic responses, differences between the signaling properties of VEGFR-2 and VEGFR-3, and, most importantly, the question of how the VEGFR signaling network is integrated and coordinated with other receptor tyrosine kinase systems in endothelial cells, such as angiopoietins/Ties and ephrins/Ephs (Gale and Yancopoulos, 1999; Holash et al., 1999). Answering these questions will provide better understanding of molecular processes implicated in regulation of vascular growth, and consequently, will contribute to the design of better treatment strategies of angiogenesis related disorders.

Acknowledgements

We thank Dr Kari Alitalo for support and critical comments. The studies in the authors' laboratory were supported by the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cancer Organizations, the Finnish Cultural Foundation, the Ida Montini Foundation, the Emil Aaltonen foundation, the Finnish State Technology Development Centre, the Helsinki University Hospital Research Fund, the Novo Nordisk Foundation and the European Union Biomed Program.

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Figure 1 Receptor binding specificity of VEGFs. VEGFs are marked in green, VEGFR-1, VEGFR-2 and VEGFR-3 in blue and red and neuroplilin-1 in yellow. The different 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 first 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 fifth immunoglobulin-like domain, but the resulting 120 and 75 kDa chains remain linked by disulfide bonds (Pajusola et al., 1993)

Figure 2 Simplified 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 -catenin, and with the _v3integrin

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 fluid mainly in the lower extremities. Mutant residues identified so far are indicated in the boxes