We used neutralizing antibodies to deduce the effects of the different VEGFR dimeric complexes in the EB model. VEGFR3-neutralizing antibodies, which bind to the ligand- binding site of VEGFR3 (Pytowski et al, 2005), blocked VEGFA-induced heterodimers although VEGFA fails to directly bind to VEGFR3. This indicates a scenario where dimerization is initiated by binding of ligand to one receptor molecule, rather than simultaneous bridging between two receptor molecules. Binding of ligand is likely to induce changes in the conformation of the extracellular domain, which in turn may trigger dimerization with the second receptor molecule. Binding of the neutralizing antibody to the second receptor molecule, in this case VEGFR3, may block dimerization either by steric hindrance or by locking the conformation of VEGFR3 in a state incompatible with dimerization. Interestingly, electron microscopical analysis of VEGFR2 homodimers show points of contacts between the receptor molecules involving Ig-like loop 7, which supports that receptor extracellular domain folding is modulated during the dimerization process (Ruch et al, 2007). As it is likely that VEGFR3 neutralization of the VEGFA- induced sprouts was a result of blocking of heterodimers, we propose that both VEGFR2 homodimers and VEGFR2/-3 heterodimers contribute to angiogenic sprouting. As judged from the more modest response to VEGFC, compared with VEGFA, in angiogenic sprouting in the 3D EBs, VEGFR3 homodimers on the other hand may not contribute to sprout- ing. The extent of VEGFC-induced sprouting may very well be context dependent. We observed relatively efﬁcient VEGFC- induced angiogenic sprouting compared with VEGFA, in adeno-associated virus-transduced muscle (Supplementary Figure S2). Overexpression of VEGFC in the mouse cornea, chorioallantoic membrane or mouse skin has previously been shown to induce blood vascular angiogenesis (Oh et al, 1997; Cao et al, 1998; Saaristo et al, 2002). Interestingly, VEGFC- induced blood vascular angiogenesis is negatively regulated by ongoing lymphangiogenesis and increases with increasing distance from the lymphangiogenic site (Benest et al, 2008). The extent of VEGFC-induced sprouting may be dependent on the concomitant production of VEGFA to drive essential aspects of vascular endothelial cell biology that VEGFC may complement but not replace. In vivo, simultaneous exposure of cells to VEGFA and VEGFC, in addition to many other growth modulatory factors, would be the expected scenario. In this context, it is interesting that EBs treated with a combination of VEGFA and VEGFC showed increased hetero- dimerization as detected by PLA, accompanied by a marked increase in vessel branching (Supplementary Figure S3). Ligand-dependent increase in commitment to the endothelial cells lineage and increased expression of receptors may contribute to this relative increase in receptor heterodimer- ization. Our data agree excellently with those of Tammela et al (2008) who showed that VEGFR3 is upregulated during active angiogenesis and expressed on tip cells of angiogenic sprouts. Furthermore, there are previous indications in the literature that heterodimerization of VEGFRs may be of biological relevance. Goldman et al (2007) used receptor neutralizing antibodies to show cooperative signalling of VEGFR2 and VEGFR3 in lymphatic migration and proliferation. VEGFR2 has been suggested to be required for VEGFC-induced differ- entiation of vascular progenitor cells (Suzuki et al, 2005). & 2010 European Molecular Biology Organization VEGF receptor heterodimers I Nilsson et al VEGFC, but not the VEGFR3-speciﬁc ligand VEGFC156S (Kirkin et al, 2001), induced differentiation of VEGFR3-ex- pressing progenitors. On the other hand, Matsumura et al (2003) reported that VEGFR3 negatively regulates VEGFR2 in the maintenance of vascular integrity. We have previously shown that VEGFR3 heterodimerized to VEGFR2 does not become phosphorylated on two of its most C-terminal tyr- osine phosphorylations sites (Dixelius et al, 2003). A similar scenario, with loss of speciﬁc tyrosine phosphorylation, may be true for VEGFR2 when dimerized with VEGFR3. However, as the VEGFC-induced VEGFR2 tyrosine phosphorylation is relatively weak (see Figure 1), it has not been technically feasible to determine VEGFR2 tyrosine phosphorylation pattern in response to VEGFC. Taken together, it seems very likely that the molecular communication between VEGFR2 and VEGFR3 exerted via receptor heterodimerization is biologically relevant in angio- genesis and serves to modulate angiogenic sprouting. It is important to remember that receptor dimerization is guided not only by the growth factors but also by the availability of receptors whose expression levels may be highly plastic dependent on the physiological or pathological context. This is implied by our results using the 3D EB model (see Figure 5), where VEGFA-induced sprouts showed VEGFR3 expression throughout the length of the sprout. In contrast, VEGFC-induced sprouts showed higher expression of VEGFR3 and higher extent of VEGFR2/-3 heterodimerization in the tip. Therefore, the extent of heterodimerization is likely to vary with receptor expression level and be different during the various stages of the angiogenic process, and provide an additional avenue for cells to ﬁne tune their responses to different VEGFs. Detailed studies on how homodimer versus heterodimer ratios impact proliferation, survival, migration and differentiation of endothelial cells will reveal the span of information that can be transmitted by VEGFA and VEGFC. It is for example an interesting possibility that signalling by VEGFR2/-3 heterodimers contribute to the early develop- mental function of VEGFR3 in vascular pruning (Dumont et al, 1998). Materials and methods Endothelial and ES cell culture Primary human saphenous vein endothelial cells (HSaVECs) and human umbilical vein endothelial cells (HUVECs) were purchased from Promocell (Heidelberg, Germany) and cultured on gelatin- coated dishes in endothelial cell basal medium supplemented with 5% fetal bovine serum (FBS) and bullet kit (Promocell). Cells were used between passage 2 and 8. PAE cells overexpressing human VEGFR3 (PAE/VEGFR3) and wild-type controls were cultured on gelatin-coated dishes in F12 medium supplemented with 10% FBS (Invitrogen, San Diego, CA). HSaVECs were treated with different growth factors as indicated. Typically, cells were seeded in eight-well chamber slides (5 104 per well), kept overnight in starvation medium and thereafter subjected to treatment for 8 min at 371C, with the following human ligands: VEGFA121 (R&D), VEGFA165 (PeproTech), VEGFA145, VEGFA189 (both from Relia Tech GmbH, Braunschweig, Germany) all at 50 ng/ ml. This concentration found to be close to saturating on a linear scale as determined by dose-response analyses (not shown). Effects were compared with those of VEGFC (in-house production), VEGFD (Vegenics Limited, Toorak, Australia), human platelet-derived growth factor (PDGFB; PeproTech), all given at 100 ng/ml. In some cases, pre-incubation on ice for 15 min with neutralizing antibodies against human VEGFR2 (IMC-1121b; 15 mg/ml; Zhu et al, 2003) or VEGFR3 (hF4-3C5; 15 mg/ml; Persaud et al, 2004) was performed. The EMBO Journal VOL 29 | NO 8 | 2010 1385