VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.
THE VEGF (VASCULAR ENDOTHELIAL GROWTH FACTOR) FAMILY
VEGF denotes a family of five related mammalian growth factors: VEGFA (the prototype), VEGFB, VEGFC, VEGFD and PlGF (placental growth factor). An alternative designation for VEGFA is VFP (vascular permeability factor) . The VEGFs are homodimeric polypeptides, although naturally occurring heterodimers of VEGFA and PlGF have been described . The complexity of the VEGF family is increased further by alternative splicing of VEGFA, VEGFB and PlGF, and processing of VEGFC and VEGFD . VEGFA can also be processed by matrix metalloproteinases . Splicing and processing regulates the ability of the ligands to bind to VEGF receptors, to HS (heparan sulfate) and NRPs (neuropilins) [5,6]. For example, VEGFA is alternatively spliced to generate VEGFA121, VEGFA145, VEGFA165 and VEGFA189 (the numbers indicating amino acid residues in the human polypeptides). VEGFA121 does not bind to HS and is freely diffusible, whereas VEGFA165 and VEGFA189 bind to HS as well as to NRP1. The restricted diffusion of HS-binding VEGFs affects the temporal and spatial distribution of VEGF receptor signalling and, in addition, it modulates signalling in a qualitative manner, e.g. by recruitment of integrins into the signalling complex . Additional mammalian VEGF variants include a series of VEGFA splice variants denoted VEGFAxxxb, which bind, but fail to efficiently activate, VEGF receptors and have therefore been described as anti-angiogenic . Furthermore, a number of non-vertebrate polypeptides with structural and functional relatedness to the mammalian VEGFs have been identified. These include the parapox virus open reading frame denoted VEGFE  and the snake venom-derived polypeptides denoted VEGFF .
Several of the VEGF family of ligands and receptors, notably VEGFA, are regulated by HIF (hypoxia-inducible factor) , leading to increased expression during tissue growth both in health (wound healing and embryonic development) and disease (cancer). In addition, a wide range of other transcription factors and metabolic regulators, such as the ETS (gene transduced by the leukaemia virus E26) family of transcription factors  and ROS (reactive oxygen species) , modulate the expression levels of VEGFs as well as of the VEGF receptors.
The VEGF ligands, in particular VEGFA, are produced by most parenchymal cells and act in a paracrine manner on adjacent endothelial cells to regulate VEGF receptor signalling and biology. Autocrine VEGFA may be essential for endothelial cell survival as shown in recombinant mice specifically lacking endothelium-produced VEGFA .
THE VEGF RECEPTOR TYROSINE KINASE FAMILY
In humans, mice and other mammals, three structurally related VEGFRs (VEGF receptor tyrosine kinases) have been identified, namely VEGFR1, VEGFR2 and VEGFR3 (Figure 1). In the zebrafish, there are four genes encoding VEGFRs: the Flt (Fms-like tyrosine kinase) 1 orthologue (corresponding to VEGFR1), the Flt4 orthologue (corresponding to VEGFR3) and two genes with highest similarity to VEGFR2 denoted Kdr (kinase insert domain receptor)-like and Kdr . The VEGFRs show a similar organization with an extracellular, ligand-binding, domain composed of immunoglobulin-like loops, a transmembrane domain, a juxtamembrane domain, a split tyrosine kinase domain and a C-terminal tail (Figure 1). There is an overall pattern of VEGFR1 expression in monocytes and macrophages, VEGFR2 in vascular endothelial cells and VEGFR3 in lymphatic endothelial cells. However, with increasing sensitivity and accuracy of reagents and techniques, many exceptions to this basic pattern have been reported.
VEGF binding specificities and VEGFR signalling complexes
VEGFR DIMERIZATION AND ACTIVATION OF THE KINASE
Binding of VEGF occurs to the N-terminal part of the extracellular domain of the receptor (more detailed information is given in each VEGFR section below). Binding of VEGF to its VEGFR can occur in cis, e.g. by freely diffusible VEGF or by presentation of VEGF through co-receptors expressed on the same cell as the VEGFR, or in trans, e.g. by presentation through co-receptors expressed on adjacent cells (see the ‘VEGF co-receptors’ section below). Dimerization of receptors to form homo- or hetero-dimers  is necessary, but not sufficient, for receptor activation [17,18]. Dimerization is stabilized by contact points between receptor extracellular domains in Ig-loop 7 [19,20]. The orientation of the receptor monomers is influenced by the transmembrane domains . The resulting rigid arrangement of two receptor monomers is required for exact positioning of the intracellular kinase domains .
According to the consensus model for activation of receptor tyrosine kinases , dimerization is accompanied by changes in the intracellular domain conformation. Formal proof is missing, however, due to challenges in structural analyses of large transmembrane proteins. The conformational changes in turn lead to exposure of the ATP-binding site in the intracellular kinase domain, followed by binding of ATP and auto- or trans-phosphorylation of tyrosine residues on the receptor dimer itself as well as on downstream signal transducers, often SH2 (Src homology 2)-domain-containing proteins. Whether a particular tyrosine residue will serve as a substrate for the inherent kinase cannot be predicted, and the identification of phosphotyrosine sites in the receptors rely on in vitro analyses. However, as discussed below, studies of mice carrying mutations in VEGFR2 have begun to shed light on in vivo functions of specific phosphorylation sites.
Tyrosine phosphorylation is tightly regulated by internalization and degradation (see below) or by dephosphorylation through PTPs (protein tyrosine phosphatases), such as DEP1 (density-enhanced phosphatase 1), VEPTP (vascular endothelial PTP), SH2-domain PTP and PTP1B .
Structure and expression of VEGFR1
VEGFR1 (also called Flt1 in the mouse) is a 180–185 kDa glycoprotein [23,24] that is activated in response to binding of VEGFA, VEGFB and PlGF (Figure 1). The ligands bind to Ig-loop 2, but loops 1 and 3 are also required for high-affinity binding [25,26]. The crystal structure of the receptor-binding domain of VEGF bound to the second Ig-loop of VEGFR1 shows hydrophobic interactions stabilizing the ligand–receptor dimers . There is a similar pattern for PlGF binding to VEGFR1 .
VEGFR1 is expressed in vascular endothelial cells at relatively high levels throughout development and in the adult [29,30]. In addition, a wide range of non-endothelial cells, such as monocytes and macrophages, human trophoblasts, renal mesangial cells, vascular smooth muscle cells, dendritic cells and different human tumour cell types, express VEGFR1 [31–35]. VEGFR1 expression is regulated by hypoxia through a hypoxia-inducible enhancer element in the VEGFR1 promoter . Alternative splicing of VEGFR1 results in the generation of sVEGFR (soluble VEGFR) 1 [also denoted sFlt1 (soluble Flt1)], encompassing the N-terminal six extracellular Ig-loops . VEGFR1 splicing is controlled by intronic signals , and gives rise to abundant expression of sFlt1 in the placenta (see below). Truncated intracellular variants of VEGFR1 have also been described .
VEGFR1 signal transduction
Although VEGFR1 binds VEGFA with higher affinity than does VEGFR2 (Kd=15 pM compared with 750 pM) , VEGFR1 tyrosine kinase activity is only weakly induced by its ligands; several underlying mechanisms have been suggested. First, Gille et al.  identified a repressor sequence in the juxtamembrane domain of VEGFR1. Secondly, structural properties of the activation loop of VEGFR1 , including the lack of positive regulatory tyrosine residues , contribute to the poor kinase activity. By overexpression of VEGFR1 in insect cells or mammalian cells, several VEGFR1 tyrosine phosphorylation sites have been identified, namely Tyr794, Tyr1169, Tyr1213, Tyr1242, Tyr1327 and Tyr1333 [43–45]. Interestingly, the pattern of tyrosine phosphorylation of VEGFR1 depends on the ligand, i.e. PlGF, but not VEGFA, induces phosphorylation of Tyr1309  (Figure 2).
VEGFR1 tyrosine phosphorylation sites and signal transduction
A range of signalling molecules associate with VEGFR1 phosphorylation sites in vitro. Tyr794  and Tyr1169  are involved in binding and activation of PLC (phospholipase C) γ, resulting in intracellular Ca2+ fluxes and generation of inositol 1,4,5-trisphosphate . Tyr1213 binds different SH2-containing proteins, such as PLCγ, GRB (growth-factor-receptor-bound protein) 2, Nck (non-catalytic region of tyrosine kinase adaptor protein) and SHP-2 (SH2-domain-containing protein tyrosine phosphatase 2) [43,48], and, according to other studies, also the p85 subunit of PI3K (phosphoinositide 3-kinase) [45,49]. Clearly, further analyses, e.g. using in vivo models, are required to determine the biological role of VEGFR1-interacting signal transducers.
The exact role for VEGFR1 in endothelial cells, apart from serving as a reservoir for VEGF, is disputed. Several studies imply that VEGFR1 is dispensable for proliferation or migration of endothelial cells in vitro [50–52]. On the other hand, VEGFR1-neutralizing antibodies have been employed to implicate VEGFR1 in endothelial cell actin reorganization and migration  through RACK1 (receptor for activated C-kinase 1) . Endothelial cell differentiation and organization into vascular tubes may involve VEGFR1-dependent activation of PI3K/Akt .
VEGFR1 may regulate endothelial cells via cross-talk with VEGFR2, through dimerization, through transphosphorylation independently of heterodimerization and through regulation of receptor expression levels. Thus VEGFR1 and VEGFR2 form heterodimers on co-expressing cells , at least in vitro. VEGFR1–VEGFR2 heterodimers are predicted by computational modelling to comprise 10–50% of the active, signalling, VEGF receptor complexes, and to form at the expense of VEGFR1 homodimers when VEGFR2 populations are larger . VEGFR1–VEGFR2 heterodimers would form as a consequence of VEGFA binding, but not PlGF or VEGFB binding, since these latter ligands bind only to VEGFR1. Still, VEGFR1 can transphosphorylate VEGFR2 in response to PlGF, leading to sensitization of VEGFR2 to subsequent activation by VEGFA . Furthermore, VEGFR1 may modulate VEGF-induced mitogenic signalling via VEGFR2 at least in part through altered VEGFR2 expression levels [51,58]. In haemangioma-derived endothelial cells, low expression of VEGFR1 is associated with constitutive VEGFR2 signalling .
In monocytes, VEGFR1-specific ligands VEGFB and PlGF induce signalling pathways known to operate downstream of most tyrosine kinase receptors such as ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase), PI3K/Akt and the stress kinase p38MAPK. These signal transducers, as well as NFAT1 (nuclear factor of activated T-cells 1), have been implicated in the regulation of monocyte chemotaxis [60,61] (Figure 2).
Vascular smooth muscle cells may respond to PlGF via VEGFR1, in particular under hypoxia, resulting in activation of the ERK1/2 and the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathways . VEGFR1 may also transduce signals for migration and invasion of several cancer cell lines, via the cytoplasmic tyrosine kinase Src and ERK1/2 pathways [63,64].
VEGFR1 biology: in vivo models and disease
Mouse models expressing recombinant variants of VEGFR1 have been instrumental in revealing its complex biology. Flt1−/− (vegfr1−/−) embryos die at E9.0 (embryonic day 9) due to increased proliferation of endothelial cells and a severe disorganization and dysfunction of the vascular system . Deletion of the intracellular domain including the VEGFR1 TK (tyrosine kinase) domain is, however, compatible with vascular development . This suggests that in endothelial cells, VEGFR1 as well as sVEGFR1 may act as a VEGF decoy, serving to spatially control VEGFR2 signalling and formation of angiogenic sprouts . Importantly, mice expressing a TK-deficient VEGFR1 (VEGFR1 TK−/−) fail to mount an inflammatory response in pathologies such as cancer (see below). Membrane insertion of VEGFR1 appears to be important, as 50% of mice expressing only soluble VEGFR1 as a result of deletion of both the transmembrane and the intracellular domains die in utero .
The negative regulatory role of VEGFR1 during vascular development may be exerted by sVEGFR1 (sFlt1), acting by sequestering VEGFA from signalling receptors or by the formation of non-functional heterodimers with VEGFR2 . sFlt1 has important physiological functions such as in vascular maturation  and in the maintenance of corneal avascularity . sFlt1 is expressed in the placenta in a regulated manner during gestation . Overexpression of sFlt1 has been implicated in the aetiology of pregnancy-induced hypertension (pre-eclampsia) . Plasma sFlt1 levels are also elevated in other diseases, such as cancer and ischaemia [74,75].
In addition to its negative regulatory role in vascular development, VEGFR1 is important in mounting an inflammatory response and inflammation-associated angiogenesis (denoted ‘pathological angiogenesis’) through recruitment of bone-marrow-derived myelomonocytic cells followed by deposition of angiogenic growth factors. Consequently, VEGFR1 TK−/− mice show impaired angiogenesis in a wide range of pathological conditions such as tumour growth , metastatic spread of tumours , choroidal neovascularization , rheumatoid arthritis  and cerebral ischaemia .
Regulation of inflammatory cell recruitment via VEGFR1 appears to be exerted mainly through PlGF. Thus PlGF is dispensable for embryonic and adult physiological angiogenesis , but it is crucial for inflammation-associated angiogenesis in a number of diseases (see  for a more detailed review on the properties of PlGF).
VEGFB, another VEGFR1-specific ligand, plays a restricted role in angiogenesis in vivo . Instead, VEGFB serves to promote fatty-acid uptake in endothelial cells, which is important in organs with high metabolic stress, such as the heart . PlGF does not compensate for the loss of VEGFB in fatty-acid transport, neither in vitro  nor in vivo as evidenced by the different phenotypes of vegfb- and plgf-gene-targeted mice [81,85,86]. VEGFB-deficient animals survive development, but display reduced heart size and impaired recovery after cardiac ischaemia [85,86]. The molecular mechanisms underlying these different functions of the two VEGFR1-specific ligands PlGF and VEGFB (i.e. co-receptors, restricted expression, etc.) remain to be identified.
Internalization of VEGFR1
Activated VEGFR1 is most probably internalized through clathrin-mediated endocytosis as it forms a ternary complex with CBL (Cas-Br-M murine ecotropic retroviral transforming sequence homologue) and the adaptor protein CD2AP (CD2-associated protein); the complex in turn associates with clathrin and the early endosome GTPase Rab (Ras-related GTPase) 4 . VEGFR1 does not appear to become recycled, in contrast with VEGFR2 (see below), which is compatible with an important role for VEGFR1 in negative regulation through rapid clearance of VEGFA.
Structure and expression of VEGFR2
VEGFR2 [also known as KDR in the human and Flk1 (fetal liver kinase-1) in the mouse] is a 210–230 kDa glycoprotein that binds VEGFA with a 10-fold lower affinity than VEGFR1 [40,88]. In addition to VEGFA, VEGFR2 binds proteolytically processed VEGFC and VEGFD . Ligand binding involves extracellular Ig-like domains 2 and 3 of VEGFR2 (Figure 1). The parapox virus open-reading-frame-encoded VEGFE is the only known ligand to uniquely bind to VEGFR2.
Alternative splicing results in the generation of sVEGFR2, which is present in various tissues such as the skin, heart, spleen, kidney and ovary, and in plasma. sVEGFR2 binds VEGFC and prevents binding to VEGFR3, consequently inhibiting lymphatic endothelial cell proliferation . Progression of neuroblastoma is accompanied by down-regulation of sVEGFR2 [90–92]. sVEGFR2 may also contribute to vessel maturation by regulating mural cell migration and vessel coverage .
VEGFR2 is expressed most prominently in vascular endothelial cells and their embryonic precursors, with highest expression levels during embryonic vasculogenesis and angiogenesis [93–95]. VEGFR2 is also found in a range of non-endothelial cells such as pancreatic duct cells, retinal progenitor cells, megakaryocytes and haemopoietic cells (see  and references therein). VEGFR2 expression is induced in conjunction with active angiogenesis, such as in the uterus during the reproductive cycle , and in pathological processes associated with neovascularization, such as cancer [98,99]. VEGFR2 expression on tumour cells has been noted for melanoma and haematological malignancies . The detailed expression and functionality of VEGFR2 in different cell types in the adult remains to be determined. Interestingly, Maharaj et al.  detected constitutively phosphorylated VEGFR2 in vivo in adult mouse tissues including the liver, lung, adipose tissue and kidney.
VEGFR2 signal transduction
VEGFR2 is known to transduce the full range of VEGF responses in endothelial cells, i.e. regulating endothelial survival, proliferation, migration and formation of the vascular tube. Major phosphorylation sites in VEGFR2 are Tyr951 in the kinase insert domain, Tyr1054 and Tyr1059 within the kinase domain, and Tyr1175 and Tyr1214 in the C-terminal domain [102,103] (Figure 3). Additional phosphorylation sites in VEGFR2 have been identified at positions 1223, 1305, 1309 and 1319 , but their function(s) are as yet unclear. Moreover, the juxtamembrane tyrosine residue Tyr801 is phosphorylated in isolated intracellular domains ; whether Tyr801 is phosphorylated in the intact VEGFR2 remains to be shown.
VEGFR2 tyrosine phosphorylation sites and signal transduction
Phosphorylated Tyr951 serves as a binding site for the SH2-domain-containing TSAd (T-cell-specific adapter molecule) [102,105], also denoted VRAP (VEGFR-associated protein; however, TSAd is the consensus designation). Tyr951 is phosphorylated during active angiogenesis , but not in quiescent endothelial cells. Mutation of VEGFR2 at Tyr951 as well as TSAd knockdown prevent VEGFA-dependent actin reorganization and migration, but not proliferation, of endothelial cells. TSAd forms a complex with Src in a VEGFA-dependent manner .
Phosphorylation at Tyr1054 and Tyr1059, which are located in the kinase domain activation loop, are critical for kinase activity . Phosphorylation of these residues may be preceded by autophosphorylation at Tyr801 . The positive regulatory tyrosine residues in receptor tyrosine kinases do not as a rule participate in binding of SH2-domain proteins. However, Tyr1059 has been shown to bind Src, which in turn phosphorylates other residues in VEGFR2, such as Tyr1175, as well as downstream signal transducers, such as the actin-binding protein IQGAP1 (IQ-motif-containing GTPase-activating protein 1) . IQGAP1 has been implicated in the regulation of cell–cell contacts, proliferation and migration .
Phosphorylation of Tyr1175 creates a binding site for PLCγ [44,103], but it also binds the adapter proteins SHB (SH2-domain-containing adaptor protein B) and Sck [SHC (Src homology and collagen homology)-related adaptor protein] [109,110]. SHB binds to FAK (focal adhesion kinase), which is tyrosine phosphorylated in VEGF-treated cells  and contributes to endothelial cell attachment and migration . Tyr1175 has also been described to bind SHC or GRB2, which recruit the nucleotide-exchange factor SOS (Son of sevenless) to VEGFR2 [110,113]. This pathway may control Ras activation and mitogenicity in response to VEGF . VEGF-induced Ras activation has been implicated in the generation of pleiotropic lipid mediators, the prostaglandins [e.g. PGI2 (prostaglandin I2)] via cPLA2 (cytoplasmic phospholipase A2) (see  and references therein). However, an important pathway for VEGF-regulated proliferation appears to be through Tyr1175-PLCγ-mediated activation of PKC (protein kinase C) and downstream induction of the ERK pathway leading to cell proliferation . The PKC isoforms PKCβ [116,117], PKCα and PKCζ have been shown to mediate VEGF-induced proliferation . PKCs may also mediate activation of SPK (sphingosine kinase) . Activation of PKCs downstream of VEGF results in serine phosphorylation and activation of PLCβ3, an effector of heterotrimeric G-proteins . PKC activation also results in activation of PKD (protein kinase D) , which promotes nuclear translocation of HDAC (histone deacetylase) 5 and HDAC7 [122,123], followed by phosphorylation of CREB (cAMP-response-element-binding protein) as well as HSP (heat-shock protein) 27 .
Tyr1214 allows recruitment of Nck and the cytoplasmic tyrosine kinase Fyn to VEGFR2. A Nck–Fyn complex mediates phosphorylation of PAK (p21-activated protein kinase) 2 and subsequent activation of Cdc42 (cell division cycle 42) and p38MAPK . HSP27 is also implicated downstream of Tyr1214, in a MAPKAPK2 (MAPK-activated protein kinase 2)-dependent manner .
It has not yet been fully elucidated which phosphorylation sites or domains in VEGFR2 are important for generation of lipid mediators. The docking protein GAB (GRB2-binding protein) 1 contains a binding site for the p85 subunit of PI3K. VEGF-induced recruitment of GAB1 leads to PIP3 (phosphatidylinositol 3,4,5-trisphosphate) generation, which in turn activates the small GTPase Rac, resulting in membrane ruffle formation and cell motility . Flow shear stress has been shown to stimulate VEGFR2 activation and GAB1 tyrosine-residue phosphorylation in a VEGF-independent manner . PI3K in turn promotes activation of PKB (protein kinase B, also called Akt) through PDK (phosphoinositide-dependent kinase) 1 and PDK2 (Figure 3). Akt phosphorylates BAD [Bcl (B-cell lymphoma)-2-associated death promoter] and caspase 9 and thereby inhibits their apoptotic activity [129,130]. VEGFA also induces the expression of the anti-apoptotic proteins Bcl-2 and A1 [131,132], as well as the IAP (inhibitors of apoptosis) family members XIAP (X-chromosome-linked IAP) and survivin, which inhibit the terminal effector caspases 3 and 7 [133,134].
VEGFR2 biology: in vivo models and disease
Vegfr2−/− mice die at E8.5 from impaired development of haemopoietic and endothelial cells , a phenotype similar to that of the vegfa−/− mouse [136,137]. VEGFR2 is therefore believed to be the main transducer of VEGFA effects on endothelial cell differentiation, proliferation, migration and formation of the vascular tube. The Tyr1173 phosphorylation site (corresponding to Tyr1175 in humans) is crucial for endothelial and haemopoietic cell development. Mice with a Y1173F knockin die between E8.5 and E9.5 from lack of endothelial and haemopoietic development , similar to the phenotype of vegfr2−/− mice . The phenotype of PLCγ1-deficient mice mirrors that of Y1173F mice [139,140]. Moreover, PLCγ1 is required for VEGF responsiveness and arterial development in the zebrafish . The loss of binding of the adaptors SHB and Sck may also contribute to the vegfr2 y1173f-like phenotype. SHB is not required for vascular development, but SHB-deficient mice show defects in vessel functionality and impaired tumour growth . In contrast, mice with a VEGFR2 Y1212F substitution (corresponding to position 1214 in the human)  or a Y949F substitution (corresponding to Tyr951 in the human; X. Li and L. Claesson-Welsh, unpublished work) are viable and fertile.
Regulation of vascular permeability may be one of the main in vivo functions of VEGFA, in keeping with its original designation, VPF (vascular permeability factor). Vascular permeability is essential for normal tissue homoeostasis, and dysregulated permeability leading to chronic oedema is an important feature in cancer and in circulatory diseases such as peripheral ischaemia and heart failure. Fenestrae, which can be induced by VEGFA, are found in small numbers in many types of vascular endothelium and are especially numerous in specialized vascular beds in hormone-secreting tissues . VEGFA-induced extravasation of proteins or white blood cells is mediated by VEGFR2 in vivo . The acute increase in vascular permeability induced by VEGFA occurs through venules, not capillaries . Two major mechanisms have been implicated in vascular permeability; creation of transcellular endothelial pores and transient opening of paracellular endothelial junctions .
Transcellular pores such as the VVO (vesiculo-vacuolar organelle) are believed to be assembled by lipid microdomains denoted caveolae that form vesicles/vacuoles spanning the venular endothelium . VEGFR2 is localized in caveolae and occurs in complex with caveolin-1 through its C-terminal tail . Caveolin-1-targeted mice, which lack caveolae, retain VVOs and display increased permeability to plasma albumin and increased tumour growth .
Transient opening of endothelial cell–cell junctions in response to VEGF is well documented in vitro and involves dissolution of tight junctions as well as adherens junctions . VEGF dissolves adherens junction complexes, composed of VE-cadherin (vascular endothelial cadherin), β-catenin, p120-catenin, and α-catenin  through activation of the cytoplasmic tyrosine kinases Src and Yes . Src and Yes phosphorylate VE-cadherin [153,154] and β-catenin [155,156], leading to increased permeability in cell culture models [152,157] and in mice . Enhanced tyrosine phosphorylation of adherens junction proteins may also be a consequence of reduced protein tyrosine phosphatase activity [158,159]. VEGF may furthermore regulate junctional permeability by stimulating endocytosis of VE-cadherin. This pathway is initiated through activation of Src, which via PAK2 phosphorylates VE-cadherin on serine residues .
VEGF-induced permeability also involves eNOS (endothelial nitric oxide synthase)-mediated generation of NO . eNOS is activated either by PLCγ-dependent Ca2+ influx or Akt-mediated phosphorylation at Ser1179 of eNOS [162,163]. Impaired VEGF-induced permeability in eNOS-deficient mice supports a critical role for eNOS-derived NO in the regulation of VEGF-induced vascular permeability . NO in turn regulates microvascular fluid and protein exchange at postcapillary venules by modulating blood flow  or by acting, in a cGMP-PKG (protein kinase G)-dependent manner, on the cytoskeleton to increase the passage of macromolecules . Furthermore, NO-induced S-nitrosylation of β-catenin at Cys619 promotes VEGF-induced endothelial cell permeability .
Internalization of VEGFR2
Activation of VEGFR2 leads to dissociation from caveolin-1 [148,167] and translocation to endosomes . Alternatively, VEGFR2 is translocated to perinuclear caveosomes through caveolar endocytosis [148,169,170]. The degree of internalization of VEGFR2 in vitro is dependent on cell–cell junctions  and may be regulated by other receptor tyrosine kinases such as ephrin-B2 in an as yet unclear manner .
The NRP-binding adaptor synectin and its interaction with myosin VI are required to move VEGFR2-containing endosomes away from the plasma membrane, where PTP1b dephosphorylates VEGFR2 specifically at the Tyr1175 site . Internalized VEGFR2 may be degraded or recycled to the plasma membrane. VEGFR2 is routed for degradation as a consequence of post-translational modifications, i.e. CBL-mediated ubiquitination [171,174,175] or PKC-mediated serine-residue phosphorylation of VEGFR2 at Ser1188 and Ser1191 in the C-terminal tail . Alternatively, activated abluminal VEGFR2 may exit caveolae and associate with ARF (ADP-ribosylation factor) 6 and RAC1 (Ras-related C3 botulinum toxin substrate 1) in focal adhesions . ARF6 is known to promote plasma membrane recycling and co-ordinate actin remodelling .
VEGFR2 signalling is not terminated in conjunction with internalization; instead, the receptor continues to signal from endosomes and during sorting until reaching its final destinations [148,179]. Indeed, internalization of VEGFR2 is required for ERK and Akt activation, whereas p38MAPK is activated only by surface VEGFR2 [7,171,172].
Unlike other receptor tyrosine kinases, VEGFR2 is stored in intracellular Rab4- and Rab11-negative vesicles in resting endothelial cells from where it is delivered to the plasma membrane in a Src-dependent manner, in response to VEGFA .
Structure and expression of VEGFR3
VEGFR3 (also denoted Flt4) is synthesized as a precursor protein of 195 kDa. The precursor is proteolytically cleaved in the fifth Ig-like domain, generating an N-terminal peptide, which remains disulfide-bonded to the mother protein  (Figure 1). VEGFR3 is activated by the binding of VEGFC and VEGFD. Proteolytic processing of VEGFC and VEGFD results in increased affinity for binding to both VEGFR2 and VEGFR3 [181,182]. Processed VEGFC binds with higher affinity to VEGFR3 than to VEGFR2 (for hVEGFC–hVEGFR3, Kd=0.44×10−8 M, and for hVEGFC–hVEGFR2, Kd=2.2×10−8 M, where h is human) . VEGFC binding requires Ig-loops 1 and 2 in VEGFR3 , whereas binding to VEGFR2 involves loops 2 and 3 . hVEGFD shows similar affinity for both VEGFR2 and VEGFR3; in contrast, mouse VEGFD binds only to VEGFR3 . This is the only documented example of species-specific patterns of VEGF–receptor interactions. Furthermore, N-terminal residues in VEGFD are essential for VEGFR3, but not VEGFR2, activation .
Cloning of VEGFR3 identified alternative C-terminal splicing, giving rise to short and long VEGFR3 isoforms [188,189]. The short VEGFR3 isoform lacks the 65 C-terminal amino acid residues due to a retroviral insertion and occurs only in the human genome. The long isoform is predominantly expressed, but, interestingly, in thyroid diseases, the short variant is abundantly expressed. This may suggest a different function of the two isoforms . Notably, the short isoform lacks the two most C-terminal phosphorylation sites, which are phosphorylated by VEGFR3 in receptor homodimers, but not by VEGFR2 in VEGFR2–VEGFR3 heterodimers .
VEGFR3 expression starts in the primary vascular plexus at E8.5 during mouse development . At a later stage of embryogenesis, VEGFR3 is expressed in venous endothelial cells in the cardinal vein, which subsequently gives rise to VEGFR3-expressing lymphatics . VEGFR3 has an essential role in lymphatic endothelial cells, but its expression is induced in endothelial cells in conjunction with active angiogenesis , such as in the tumour vasculature or in endothelial tip cells of angiogenic sprouts in the developing retina . Moreover, zebrafish VEGFR3 is expressed in the tip cells of developing intersegmental arterial vessels .
VEGFR3 signal transduction
Binding of VEGFC/VEGFD to VEGFR3 leads to kinase activation and phosphorylation of at least five C-terminal tyrosine residues in VEGFR3 (Tyr1230, Tyr1231, Tyr1265, Tyr1337 and Tyr1363)  (Figure 4). Mutational analyses show that residues Tyr1063 and Tyr1068, located in the VEGFR3 kinase domain activation loop, are fundamental for kinase activity .
VEGFR3 tyrosine phosphorylation sites and signal transduction
The roles of the different phosphorylation sites have been studied in vitro. As mentioned above, tyrosine residues in the kinase activation loop are not considered as accessible for SH2-protein binding. Still, phosphorylated Tyr1063 has been shown to interact with the adaptor protein CRK I/II (C10 regulator of kinase; originally identified in a C10 retrovirus), which activates the JNK (c-Jun N-terminal kinase) pathway leading to cell survival . On the other hand, mutation of Tyr1337 leads to reduced binding of SHC–GRB2 and reduced mitogenic signalling . Phosphorylation of Tyr1230 and Tyr1231 creates a docking site for SHC–GBR2, which promotes signalling in the ERK1/2 and PI3K/Akt pathways . The PI3K pathway is critical in the development of lymphatics by regulating lymphendothelial migration . Migration of lymphendothelial cells in vitro is induced by both VEGFC and a mutant VEGFC156S that binds specifically to VEGFR3 and not to VEGFR2.
Integrins can induce SRC-dependent phosphorylation of VEGFR3 at tyrosine residues 830, 833, 853, 1063, 1333 and 1337. This phosphorylation is independent of VEGFC/VEGFD and activation of the VEGFR3 kinase. Integrin-mediated VEGFR3 phosphorylation leads to recruitment of the adaptor proteins CRKI/II and SHC to VEGFR3, thereby inducing activation of JNK . These findings suggest that blocking receptor ligands is not sufficient to suppress VEGFR3 signalling.
There are several studies describing heterodimers between VEGFR2 and VEGFR3 [16,191,204] (Figure 1) and, interestingly, the heterodimers appear to be functionally different from homodimers of each receptor. Indeed, it has been stated that VEGFR3 needs to be associated with VEGFR2 to induce at least certain VEGFC and VEGFD-dependent cellular responses . On the other hand, VEGFR3 homodimers have been implicated in three-dimensional organization of endothelial cells and lumen formation . VEGFR2–VEGFR3 heterodimers appear to be biologically active in angiogenesis, on the basis of treatment with receptor-neutralizing antibodies . That VEGFR2–VEGFR3 heterodimers are functionally distinct is indicated by the fact that heterodimeric VEGFR3 is phosphorylated only at three of its five potential C-terminal tyrosine phosphorylation sites; the two most C-terminal tyrosine residues appear not to be accessible for the VEGFR2 kinase .
VEGFR3 biology: in vivo models and disease
VEGFC and VEGFR3 are critical regulators of lymphendothelial function. Patients with certain variants of hereditary lymphoedema carry mutations in VEGFR3, resulting in an inactive tyrosine kinase [206–208]. In addition, the Chy mouse mutant, whose phenotype is characterized by accumulation of chylous ascites, carries a point mutation in the VEGFR3 tyrosine kinase domain, which renders the kinase enzymatically inactive . The phenotype of naturally occurring VEGFR3 mutants strongly indicates that, physiologically, VEGFR3 signal transduction is critical in the regulation of lymphatic vessel function.
During development, however, VEGFR3 plays an important role in blood vascular development. Vegfr3−/− mice die at E10.5, before the formation of lymphatics, due to cardiovascular failure and impaired hierarchical organization of the vasculature . Gene inactivation to eliminate expression of VEGFC alone, or both VEGFC and VEGFD, unexpectedly results in defects mainly in lymphatic vessels, whereas blood vessels are unaffected .
Studies on recombinant mice expressing mutated or truncated forms of VEGFR3 have shown that VEGFR3 has to be expressed and functional to allow lymphatic development. In contrast, blood vessel development is maintained in mice expressing a kinase-deficient VEGFR3 retaining the extracellular ligand-binding region . These results support the concept that VEGFR3 expression in blood vascular endothelial cells regulates VEGFR2 signalling by heterodimerization. VEGFR3 may also regulate VEGFR2 signalling by trapping VEGFC.
The importance of the VEGFR2–VEGFR3 cross-talk is furthermore underscored by the effects of VEGFR3 neutralizing antibodies, which inhibit both VEGFR3 homodimerization and heterodimerization with VEGFR2, resulting in suppression both of vascular and lymphatic endothelial cell migration and vessel sprouting .
In vivo, VEGFC-induced migration and sprouting of lymphendothelial precursor cells from restricted regions of the cardinal vein is required for development of the lymphatic system  and, as discussed above, PI3K may be critical for lymphendothelial migration . An important role for the PI3K pathway downstream of VEGFR3 is supported by in vivo results. The gene Pik3r1 encodes three proteins (p85α, p55α and p50α) that serve as regulatory subunits of class IA PI3Ks. Gene inactivation leading to loss of all protein products of this gene results in chylous ascites, indicative of impaired lymphatic function . Furthermore, VEGFC-mediated Akt activation is required for embryonic and adult lymphangiogenesis . Finally, Ras proteins regulate VEGFR3 expression and activity in lymphatic signalling .
Internalization of VEGFR3
Similar to VEGFR2, VEGFR3 internalization is crucial for pro-angiogenic downstream signalling. VEGFR3 internalization and signalling involving RAC1, Akt and ERK is regulated by ephrin B2 .
VEGF co-receptors are broadly defined in the present review as VEGF-binding cell-surface-expressed molecules that are devoid of intrinsic catalytic activity, but which modulate the signal-transduction output downstream of VEGFRs. However, we do not categorically exclude that signals may be transmitted downstream of co-receptors also independently of VEGFRs.
HS and the more abundantly sulfated, mast-cell-derived, H (heparin) modulate VEGF biology in several ways by binding not only VEGF, but also receptors and co-receptors such as NRP1 (Figure 5A). All VEGFA isoforms except VEGFA121 and the VEGFAxxxb forms bind HS/H . Moreover, the fourth Ig-loop of VEGFR1 and the sixth and seventh Ig-loops of VEGFR2 have been shown to directly interact with HS/H [220,221]. NRP1, but not NRP2 (see below), binds HS/H . Simultaneous HS/H-binding to several or all components in the signalling complex is expected to increase its stability . Moreover, presentation of VEGFA165 to VEGFR2 in trans, by HSPGs (HS proteoglycans) expressed on adjacent cells such as pericytes, further increases the signalling amplitude and duration  (Figure 5B), most probably by blocking internalization of the receptor.
Schematic outline of the interactions of VEGFR2 with its co-receptors HS/H, NRP1 and integrins
HS serves as a reservoir for growth factors, and controlled release allows formation of growth-factor gradients . Thus tip cells of sprouting blood vessels migrate in response to VEGFA164 gradients, and these gradients are shaped by interactions with HSPG . The role of HS in regulating retinal vascular development has been studied in mice that selectively express single VEGF isoforms. Interestingly, VEGFA164/164 mice (expressing only VEGFA164 and none of the other VEGFA isoforms) show normal vascular development, whereas VEGFA120/120 mice (expressing only VEGFA120 and none of the other VEGFA isoforms) exhibit severe defects in vascular outgrowth and patterning .
There are two NRP homologues, NRP1 and NRP2, which are 130 kDa transmembrane proteins with a small cytoplasmic tail lacking intrinsic catalytic function . NRP1 may homomultimerize and can also form heteromultimers with NRP2 . There are several alternatively spliced NRP1 and NRP2 isoforms . NRPs were first identified as receptors for class 3 semaphorins, a family of soluble molecules with neuronal guidance functions, implicated in modulating the development of the nervous and vascular systems [231–233]. NRP1 was later shown to bind exon 8-encoded regions of VEGFA isoforms such as VEGFA165. Heparin-mediated VEGFR2–NRP1 complex formation depends on heparin-binding regions both in VEGFA isoforms (encoded by exons 6 and 7) and in NRP1 (b1b2) . Therefore VEGFA121 (lacking exon 7) also binds directly to NRP1, but without promoting complex formation between NRP1 and VEGFR2 . NRP2 on the other hand associates with VEGFR3 in a VEGFC- or VEGFD-dependent manner . The NRPs are organized in extracellular subdomains (Figure 5): the a1-a2 domain is homologous with components of the complement system and binds semaphorins; the b1-b2 domain shares homology with coagulation factors V and VIII, binds VEGFA and HS/H, and supports semaphorin-binding to the a1-a2 domain; and the c domain is homologous with the MAM (protease maprin, A5-protein, PTPμ) domain . Both NRP homologues bind VEGFA165, but with different affinities . In addition, NPR1 binds VEGFB, PlGF and VEGFE, whereas NRP2 binds VEGFA145 and VEGFC . NRP1 is modified, in a cell-type-specific manner, by heparan and chondroitin sulfation, which may allow indirect binding of growth factors other than VEGFA to the glycosaminoglycan side chains .
NRP1 modulates VEGFR signalling, leading to enhanced migration  and survival [239,240] of endothelial cells in vitro. Furthermore, NRP1 has been implicated in VEGFR2-mediated permeability  and in VEGFA-induced three-dimensional biology, such as vessel sprouting and branching , NRPs have also been strongly implicated in intracellular trafficking of VEGFR2 by association of the NRP1 C-terminal SEA (Ser-Glu-Ala, the last three residues in the NRP1 C-terminal tail) motif to the PDZ domain of the adaptor synectin (also called GIPC) [240,243] (Figure 5A). The exact molecular mechanism whereby NRP1 modulates VEGF biology is unclear, but altered VEGFR2 signalling in the presence of NRP1 of both of p38MAPK  and p130CAS (Crk-associated substrate)  have been reported. It remains to be shown whether NRP1 transduces VEGF signals independently of VEGFR2 through its C-terminal PDZ-binding domain or whether it modulates VEGFR2 signalling, e.g. by influencing the tertiary structure of the receptor or the stability of the signalling complex.
During development, NRP1 is preferentially expressed in arteries, and NRP2 in veins and lymphatics [245–247]. Both overexpression and disruption of NRP1 in mice lead to embryonic lethality at E12.5–13.5 due to either an excess of vessel formation or a range of vascular abnormalities respectively . NRP2-knockout mice show normal development of arteries and veins, but exhibit marked deviations in the development of capillaries and small lymphatics . In agreement, NRP2 mediates VEGFC-induced lymphatic sprouting in a VEGFR3-dependent manner . NRP1/NRP2-double-knockout mice display a more severe phenotype and die in utero at E8.5 with a completely avascular yolk sac . Treatment with a neutralizing anti-NRP1 antibody reduces angiogenesis and vascular remodelling in vivo and has an additive effect with anti-VEGFA antibody therapy in reducing tumour growth and tumour vessel organization . Treatment with a neutralizing anti-NRP2 antibody reduces lymphangiogenesis and functional lymphatics in tumours, while leaving established lymphatics unaffected. The reduced lymphatic function is accompanied by reduced metastatic spread .
Integrins are transmembrane heterodimers that mediate cell-matrix adhesion by specific binding to extracellular matrix components, such as collagen, fibronectin, vitronectin and laminin. In cells plated on vitronectin, VEGF induces complex formation between VEGFR2 and the αVβ3 integrin (Figure 5C). The β3 integrin subunit binds VEGFR2 through its extracellular domain and the complex is stabilized by the αV subunit [254,255]. The VEGFR2–αVβ3-integrin association is important for full VEGFR2 activity, for activation of p38MAPK and FAK, and for recruitment of actin-binding vinculin to initiate endothelial cell migration [256,257]. VEGFA-dependent binding of integrin αVβ3 to NRP1 may prevent interaction with VEGFR2 . In vivo, the VEGFR2–αVβ3 interaction is required for tumour-induced and wound angiogenesis as well as in the recruitment of bone-marrow-derived cells to angiogenic sites [259,260]. Complex formation between VEGFR2 and β1 integrin induced by matrix-bound VEGF leads to prolonged phosphorylation of Tyr1214 of VEGFR2 and association of β1 integrin with focal adhesions .
Complex formation between VEGFR3 and αVβ1 integrins in response to VEGFC treatment of cells plated on fibronectin has also been reported .
VEGFR signalling affects a number of vital processes during development, in adult physiology and pathology. Therefore studies on VEGFR signalling have received ample interest from a broad community of academia, the pharmaceutical industry and clinical medicine. However, the recent challenges in the application of VEGF-targeted cancer therapy, i.e. resistance to VEGF neutralization  and increased invasion and metastasis [263,264], have very clearly shown that we need to learn more about how VEGF affects blood vessels in intact tissues under different metabolic conditions (see  for a review on VEGFR-targeted therapy in cancer). Such information is urgently needed to control the impact of vascular targeting in diseases.
VEGFR signalling has until now mostly been studied in vitro. These studies have provided important knowledge on the impact of VEGF on endothelial cell function, which in turn has promoted the development of anti-angiogenic drugs for treatment of diseases characterized by excess angiogenesis. However, a number of parameters of in vivo vascular function cannot be accurately modelled in monolayer cultures of immortalized, transformed or even primary endothelial cells. Such parameters include (i) that cell–cell junctions may not be properly organized, (ii) that an appropriate vascular basement membrane and supporting cells are missing, (iii) that the three-dimensional context may not be represented, and (iv) that co-receptors may not be adequately expressed. Therefore progress in research on VEGF receptor signalling now requires not only tools for sensitive and accurate biochemical analyses, but also ambitious in vivo models.
Bcl-2-associated death promoter
Cas-Br-M murine ecotropic retroviral transforming sequence homologue
cytoplasmic phospholipase A2
C10 regulator of kinase
endothelial nitric oxide synthase
focal adhesion kinase
Fms-like tyrosine kinase
inhibitor(s) of apoptosis
IQ-motif-containing GTPase-activating protein 1
c-Jun N-terminal kinase
kinase insert domain receptor
mitogen-activated protein kinase
non-catalytic region of tyrosine kinase adaptor protein
p21-activated protein kinase
protein kinase C
placental growth factor
protein tyrosine phosphatase
Src homology 2
SH2-domain-containing adaptor protein B
Src homology and collagen homology
SHC-related adaptor protein
T-cell-specific adapter molecule
vascular endothelial cadherin
vascular endothelial growth factor
VEGF receptor tyrosine kinase
This work was supported by Uppsala University, the Swedish Cancer Foundation [contract number 10 0543], the Swedish Science Council [contract number K2011-67X-12552-14-5], the Knut and Alice Wallenberg Foundation, a postdoctoral grant from the Spanish Ministry of Science and Education (MEC) (to S.T.), a postdoctoral grant from the Spanish Association of Liver Diseases (AEEH) (to S.T.), and the Gustaf Adolf Johansson Foundation at Uppsala University (to S.K.).