VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts
2010; Springer Nature; Volume: 29; Issue: 8 Linguagem: Inglês
10.1038/emboj.2010.30
ISSN1460-2075
AutoresIngrid Nilsson, Fuad Bahram, Xiujuan Li, Laura Gualandi, Sina Koch, Malin Jarvius, Ola Söderberg, Andrey Anisimov, Ivana Kholová, Bronislaw Pytowski, Megan E. Baldwin, Seppo Ylä‐Herttuala, Kari Alitalo, Johan Kreuger, Lena Claesson‐Welsh,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle11 March 2010Open Access VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts Ingrid Nilsson Ingrid Nilsson Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SwedenPresent address: Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Fuad Bahram Fuad Bahram Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Xiujuan Li Xiujuan Li Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Laura Gualandi Laura Gualandi Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Sina Koch Sina Koch Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Malin Jarvius Malin Jarvius Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Ola Söderberg Ola Söderberg Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Andrey Anisimov Andrey Anisimov Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Ivana Kholová Ivana Kholová Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, FinlandPresent address: Pathology, Laboratory Centre, Tampere University Hospital, Finland Search for more papers by this author Bronislaw Pytowski Bronislaw Pytowski Imclone Systems Corporation, New York, NY, USA Search for more papers by this author Megan Baldwin Megan Baldwin Vegenics Limited, Toorak, Victoria, Australia Search for more papers by this author Seppo Ylä-Herttuala Seppo Ylä-Herttuala Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Search for more papers by this author Kari Alitalo Kari Alitalo Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Johan Kreuger Johan Kreuger Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden Search for more papers by this author Lena Claesson-Welsh Corresponding Author Lena Claesson-Welsh Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Ingrid Nilsson Ingrid Nilsson Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SwedenPresent address: Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Fuad Bahram Fuad Bahram Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Xiujuan Li Xiujuan Li Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Laura Gualandi Laura Gualandi Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Sina Koch Sina Koch Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Malin Jarvius Malin Jarvius Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Ola Söderberg Ola Söderberg Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Andrey Anisimov Andrey Anisimov Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Ivana Kholová Ivana Kholová Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, FinlandPresent address: Pathology, Laboratory Centre, Tampere University Hospital, Finland Search for more papers by this author Bronislaw Pytowski Bronislaw Pytowski Imclone Systems Corporation, New York, NY, USA Search for more papers by this author Megan Baldwin Megan Baldwin Vegenics Limited, Toorak, Victoria, Australia Search for more papers by this author Seppo Ylä-Herttuala Seppo Ylä-Herttuala Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Search for more papers by this author Kari Alitalo Kari Alitalo Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Johan Kreuger Johan Kreuger Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden Search for more papers by this author Lena Claesson-Welsh Corresponding Author Lena Claesson-Welsh Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Ingrid Nilsson1,‡, Fuad Bahram1,‡, Xiujuan Li1, Laura Gualandi1, Sina Koch1, Malin Jarvius1, Ola Söderberg1, Andrey Anisimov2, Ivana Kholová3, Bronislaw Pytowski4, Megan Baldwin5, Seppo Ylä-Herttuala3, Kari Alitalo2, Johan Kreuger6 and Lena Claesson-Welsh 1 1Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden 2Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland 4Imclone Systems Corporation, New York, NY, USA 5Vegenics Limited, Toorak, Victoria, Australia 6Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden ‡These authors contributed equally to this work *Corresponding author. Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjoldsv. 20, Uppsala 751 85, Sweden. Tel.: +46 18 471 4363; Fax: +46 18 55 89 31; E-mail: [email protected] The EMBO Journal (2010)29:1377-1388https://doi.org/10.1038/emboj.2010.30 Present address: Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting. Introduction The mammalian vascular endothelial growth factor (VEGF) family includes VEGFA, VEGFB, VEGFC, VEGFD and placenta growth factor (PlGF). These ligands bind in an overlapping pattern to three different receptor tyrosine kinases denoted VEGF receptor 1 (VEGFR1), VEGFR2 and VEGFR3 (Olsson et al, 2006). Although the VEGFRs are not exclusively expressed on vascular cells, there is prominent expression and important function of VEGFR1 on hematopoietic and endothelial cells, of VEGFR2 on vascular endothelial cells and of VEGFR3 on vascular and lymphatic endothelial cells (LECs). The active VEGF/VEGFR signalling complexes also include co-receptors, such as heparan sulphate proteoglycans (Esko and Selleck, 2002) and neuropilins (Geretti et al, 2008). The VEGFRs transduce their effects according to the consensus scheme for receptor tyrosine kinases. Binding of ligand leads to dimerization of receptors. This close apposition confers structural reorganization of the receptor intracellular domain and exposure of the kinase active site (Hubbard, 1999). Kinase activity catalyses the transfer of phosphate groups to tyrosine residues, which serve as substrates for the kinase. Such acceptor sites are found both on the partner in the dimer as well as on cytoplasmic signalling molecules. Tyrosine phosphorylation initiates signal transduction cascades, which ultimately become established as cellular responses such as survival, proliferation and motility. Certain VEGF ligands bind to more than one VEGFR, potentially allowing receptors to form heterodimers in addition to homodimers. We have shown earlier that VEGFR2/-3 heterodimers are formed in cultured LECs in response to VEGFC (Dixelius et al, 2003). In accordance, processing of VEGFC during synthesis allows binding to both VEGFR2 and -3 (Joukov et al, 1997; Alitalo et al, 2005). Heterodimerization was accompanied by a loss of phosphorylation of C-terminal tyrosine residues in VEGFR3 (Dixelius et al, 2003). The underlying mechanism may involve different substrate specificities of the VEGFR2 and VEGFR3 kinases. Alternatively, VEGFR3 may undergo a conformational change when engaged in a heterodimer with VEGFR2, and thereby, certain acceptor sites become hidden or otherwise inaccessible. Thus, it is important to determine the composition of receptor complexes formed under different conditions, as this will be of consequence for the biological response. Various sophisticated recombinant animal models have greatly aided our understanding of the biology of the VEGFRs. All three receptors are required for proper embryonic development. Gene inactivation of vegfr1 is lethal at E8.5–9 due to increased endothelial proliferation leading to obstruction of vessels (Fong et al, 1995, 1999). Inactivation of vegfr2 leads to arrest in endothelial differentiation, causing embryonic lethality at E8.5 (Shalaby et al, 1995). Inactivation of vegfr3 is lethal slightly later, at E10.5, due to the lack of remodelling of the vascular network (Dumont et al, 1998). In subsequent development, VEGFR3 is critical for lymphatic vessel function. Thus, during a restricted phase in early development, both VEGFR2 and VEGFR3 serve critical functions in the formation of the vascular tree. A number of cell types co-express VEGFR2 and VEGFR3, potentially allowing formation of heterodimers. Expression of VEGFR3 is induced during angiogenic sprouting in the adult, both in physiological and pathological conditions (Tammela et al, 2008). In differentiating stem cell cultures, VEGFR3 is expressed by subpopulations of endothelial cells in growing vessels, some of which may transdifferentiate to become early lymphatic structures (Kreuger et al, 2006; Adams and Alitalo, 2007). In addition, VEGFR2 may be variably expressed by LECs (Petrova et al, 2002), and on collecting lymphatic vessels that otherwise are characterized by high expression levels of VEGFR3 (Saaristo et al, 2002). Here, we investigate the function of VEGFR2/-3 heterodimerization in intact endothelial cells and angiogenic sprouts, using a newly developed in situ proximity ligation assay (in situ PLA) (Soderberg et al, 2006; Jarvius et al, 2007). This method is based on the use of oligonucleotide-conjugated antibodies, which when brought in close proximity by binding to epitopes on, for example, dimerized VEGFRs, allow a rolling-circle amplification detected by a fluorescently labelled probe. We show that endogenous unmanipulated VEGFRs form heterodimers in response to VEGFA or VEGFC. We present evidence that heterodimers are present on tip cell filopodia, and that neutralization of VEGFR3 to prevent heterodimer formation induced by VEGFA leads to a marked decrease in sprouting activity. It is concluded that VEGFR2/-3 heterodimers have a significant function in the positive regulation of angiogenic sprouting. Results Detection of VEGFC-induced VEGFR2/-3 heterodimers in endothelial cells by immunoprecipitation To study the induction of VEGFR2/-3 heterodimers, we examined receptor expression in different endothelial cell cultures. On reducing gels, VEGFR3 migrates as three molecular weight species, 195, 175 and 125 kDa (Pajusola et al, 1994). The mature, cell surface expressed VEGFR2 appears as a 250 kDa band on immunoblots. Using porcine aortic endothelial (PAE) cells transfected to express VEGFR3 as control, we compared expression of receptors in primary human saphenous and umbilical vein-derived endothelial cells (HSaVECs and HUVECs). Both cell types co-expressed VEGFR2 and VEGFR3 (Figure 1A); we chose HSaVECs for further experimentation. Figure 1.Induction of VEGFR2/-3 heterodimers in co-expressing endothelial cells. (A) Total cell lysates derived from HUVECs, HSaVECS or PAE cells; either untransfected or expressing VEGFR3 were immunoblotted to detect VEGFR3 (left panel). After reduction of disulphide bridges, VEGFR3 migrates as three species of apparent mw 195, 175 and 125 kDa. PAE, HUVEC and HSaVEC lysates were also immunoblotted to detect VEGFR2, which migrates as two species around the 250 kDa marker (right panel). (B) HSaVEC lysates from cells treated or not with VEGFA or VEGFC for 8 min were used for immunoprecipitation (ip) of VEGFR2 followed by immunoblotting for VEGFR3 (upper panel) to detect receptor heterodimerization. This was followed by immunoblotting to detect phosphorylated VEGFR2 (middle panel) using the 4G10 mAb, and immunoblotting to show equal loading of VEGFR2 (lower panel). (C) Immunoprecipitation of VEGFR3 from HSaVECs treated as in (B) followed by immunoblotting to detect co-immunoprecipitation of VEGFR2 (upper panel), phosphorylation of VEGFR3 (middle panel) and VEGFR3 loading (lower panel). Download figure Download PowerPoint Treatment of HSaVECs with processed VEGFC, followed by immunoprecipitation of VEGFR2 and immunoblotting for VEGFR3, showed complex formation between the receptors. Figure 1B displays the prominent 125 kDa band and the weaker 195–175 kDa VEGFR3 bands in the VEGFR2 immunoprecipitate. Heterodimers were not detected in response to VEGFA in this assay (Figure 1B, upper panel). The effect of VEGFA on VEGFR2 tyrosine phosphorylation was more prominent than that of VEGFC (Figure 1B, middle panel), in agreement with the data from earlier biochemical analyses, showing higher affinity of VEGFA than VEGFC, for binding to VEGFR2 (Joukov et al, 1997). Similarly, treatment with VEGFC but not with VEGFA, allowed detection of the 250 kDa VEGFR2 band in immunoblots of VEGFR3 immunoprecipitates from HSaVECs (Figure 1C, upper panel). These data show that VEGFR2 and VEGFR3 form heterodimeric signalling complexes in primary cells in response to VEGFC. in situ PLA reveals VEGFR2/-3 heterodimerization in response to VEGFA or VEGFC To demonstrate the formation of heterodimers in intact cells, we used in situ PLA (see schematic outline in Figure 2A). We used an experimental design where cells were incubated with VEGFA, VEGFC or vehicle for 8 min, fixed and then probed with primary antibodies raised in different species and directed towards the intracellular domains of VEGFR2 or VEGFR3. Antibodies against the intracellular domain were preferred, as they would not be disturbed by the ligand–receptor interaction. This was followed by incubation with two sets of secondary antibodies conjugated with oligonucleotide, unique for each type of secondary antibody. Ligation of the oligonucleotides by a bridging probe in a proximity-dependent manner, allows a rolling-circle amplification. Finally, this product is detected by complementary fluorescent probes. As shown in Figure 2B, treatment of HSaVECs with VEGFC induced formation of heterodimers in situ, distributed on the plasma membrane as well as in the cytoplasm. The number of heterodimers/cell increased 100-fold from basal, in response to VEGFC treatment (see quantification in Figure 2C). Figure 2.In situ PLA detection of VEGFR2/-3 heterodimers in intact HSaVECs. (A) Schematic outline of the in situ PLA strategy showing: (i) dimerized receptors (VEGFR2 in blue and VEGFR3 in grey) reacting with primary antibodies; (ii) close proximity of oligonucleotide-ligated secondary antibodies allows a rolling-circle amplification (RCA); (iii) detection of the RCA product by a fluorescently labelled probe. (B) Detection of heterodimers (in red) in HSaVECs treated with vehicle (–), VEGFA or VEGFC for 8 min on cells labelled with FITC-conjugated phalloidin (green). Inset in the VEGFC panel shows high magnification to clearly visualize the PLA spots representing heterodimers. Scale bar=10 μm. (C) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with vehicle (–), VEGFA (A) or VEGFC (C) in cells preincubated or not with neutralizing antibodies blocking ligand binding to VEGFR2 or VEGFR3. n=6. (D) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with different human VEGF isoforms (VEGFA121, 145, 165 or 189) or VEGFC for 8 min. n=6. (E) Quantification of VEGFR2/-3 heterodimers in response to VEGFA, VEGFC, VEGFD or PDGFB. Growth factors are indicated as A (VEGFA), C (VEGFC), D (VEGFD) and P (PDGFB). n=6. Note that a different batch of PLA probes was used in this analysis compared with other panels in the figure (see Materials and methods). (F) Turnover of VEGFR2/-3 heterodimers in HSaVECs. Cells were treated with VEGFC for different time periods from 10 min to 24 h and samples were processed for detection of in situ PLA signals. n=6. Asterisks in panels C–F indicate the degree of significance (**P<0.01, ***P<0.001). Download figure Download PowerPoint Of note, VEGFR2/-3 heterodimerization also increased significantly (25-fold) when cells were treated with VEGFA for 8 min (see Figure 2B and C). Therefore, despite the fact that VEGFA fails to induce receptor heterodimerization as detected by co-immunoprecipitation, VEGFR2/-3 heterodimers were induced by VEGFA in the in situ PLA. To show the specificity of the reactions, we preincubated cells with antibodies previously shown to specifically neutralize either human VEGFR2; IMC-1121b (Zhu et al, 2003) or human VEGFR3; hF4-3C5 (Persaud et al, 2004), by binding to the ligand-binding part of the extracellular domains. As shown in Figure 2C, blocking with either of the neutralizing antibodies quenched the appearance of heterodimers to the level of basal heterodimerization recorded in unstimulated cells. Different VEGFA isoforms (VEGFA121, 145, 165 and 189) were analysed for their ability to induce VEGFR2/-3 heterodimers. Isoforms with a higher propensity for binding to heparan sulphate and extracellular matrix, that is VEGFA145 and VEGFA189 (Kawamura et al, 2008), induced heterodimers more efficiently than the relatively more soluble isoforms VEGFA121 and VEGFA165 (Figure 2D). In repeated experiments, however, VEGFC induced heterodimer formation more efficiently than any of the VEGFA isoforms. Further, VEGFD also induced VEGFR2/-3 heterodimerization, in agreement with that processed human VEGFD, but not mouse VEGFD, binds to VEGFR2 (Baldwin et al, 2001). The effect was less efficient than that of VEGFC but more prominent than that of VEGFA. To control for the specificity, we analysed the effect of PDGFB, which does not bind to either of the VEGF receptors. As shown in Figure 2E, VEGFR2/-3 heterodimers were not induced by PDGFB. Binding of ligand induces internalization and degradation of receptor tyrosine kinases. Figure 2F shows HSaVECs treated with VEGFC for different time periods, followed by in situ PLA. Formation of heterodimers peaked at 10 min and the ligand-induced heterodimers remained detectable for up to 2 h. With prolonged incubations, the in situ PLA signals were lost, in agreement with the clearance of receptors. Combined, these data provide evidence that heterodimers are formed in a ligand-dependent and specific manner in intact cells. Detection of homodimerization of VEGFRs by in situ PLA We next examined the pattern of VEGFR2 and VEGFR3 homodimerization induced by VEGFA and VEGFC. For this purpose, monoclonal antibodies against either VEGFR2 or VEGFR3 were divided in pools, which were ligated with either a 'plus' oligonucleotide or a 'minus' oligonucleotide. The PLA reaction indicating for example VEGFR2 homodimers, would occur only as a result of close proximity of a plus-ligated antibody with a minus-ligated antibody, whereas pairs consisting of plus–plus or minus–minus ligated antibodies would not give rise to PLA signals (see Figure 3A for a schematic outline). Consequently, we could score only 50% of the actual homodimerization events, namely when antibodies combined in plus–minus and minus–plus constellations. Moreover, we could not directly compare the relative extent of receptor homo- and heterodimerization, as different combinations of antibodies had to be used to detect the different receptor complexes. Figure 3.VEGFR2/-3 homo- and heterodimers induced by VEGFA or VEGFC. (A) Schematic outline of primary antibody ligation with oligonucleotide plus and minus strands to detect VEGFR homodimers. Only pairing of antibodies with plus and minus strands allow initiation of the rolling-circle amplification. (B) VEGFR2/-3 heterodimerization. HSaVECs were treated for 8 min with either VEGFA or VEGFC. Heterodimerization was 3–4-fold more efficiently induced by VEGFC. n=6. (C) VEGFR2 homodimers. Equal mixtures of VEGFR2 monoclonal antibodies ligated with plus and minus strands of oligonucleotides (as outlined in A) were used to detect VEGFR2 homodimers on HSaVECs treated for 8 min with VEGFA or VEGFC as above. n=6. (D) VEGFR3 homodimers. Equal mixtures of VEGFR3 monoclonal antibodies ligated with plus and minus strands of oligonucleotides were used to detect VEGFR3 homodimers on HSaVECs treated for 8 min with VEGFA or VEGFC. n=6. (E) Schematic outline of VEGFR homo- and heterodimerization induced by VEGFA or VEGFC. Note that the relative distribution of homodimers versus heterodimers cannot be accurately determined due to the inherent difference in affinity of different antibodies. Asterisks in panels B–D indicate the degree of significance (***P<0.001). Download figure Download PowerPoint As shown in Figure 3B, VEGFA and VEGFC consistently induced heterodimers and VEGFC was about 3–4-fold more potent in this regard. VEGFR2 homodimers were induced only by VEGFA and not by VEGFC (Figure 3C). VEGFR3 homodimers were induced by VEGFC (Figure 3D). There was a slight tendency for VEGFA-induced VEGFR3 homodimerization, but still the effect was negligible compared with that of VEGFC. Figure 3E concludes on these results; VEGFA induces VEGFR2 homodimers and VEGFR2/-3 heterodimers. VEGFC induces VEGFR3 homodimers and VEGFR2/-3 heterodimers. Although no firm conclusions can be made as reasoned above, homodimers of VEGFR3 seemed to be roughly two-fold more frequent than heterodimers in VEGFC-treated cells. Moreover, homodimers of VEGFR2 were eight-fold more frequent than heterodimers in VEGFA-treated cells. VEGFR2/-3 heterodimers are formed in blood and lymphatic endothelium in differentiating EBs To identify the biological significance of VEGF receptor heterodimerization, we exploited the embryoid body (EB) model of angiogenesis (Jakobsson et al, 2007). In this model system, differentiating mouse embryonic stem (ES) cells efficiently form capillary structures in response to angiogenic growth factors. In the first set of experiments, EBs were cultured in the so-called two-dimensional (2D) setup, seeded onto tissue culture slides. The advantages of the 2D setup are that these cultures readily can be scaled up to allow for biochemical analyses, such as immunoprecipitation, and that the 2D setup also makes it feasible to analyse the relatively rare events whereby immature capillaries in EB cultures begin to transdifferentiate into early lymphatic structures (Kreuger et al, 2006). Figure 4A shows co-expression of VEGFR2 and VEGFR3 in endothelial cells in vascular structures formed in 2D EBs differentiated for 12 days, when endothelial cells constitute about 5% of the total cellular pool (Magnusson et al, 2004). The vascular identity of the cells co-expressing VEGFR2 and VEGFR3 was validated by positive immunostaining for VE-cadherin (lower panel in Figure 4A). As shown in Figure 4B, left panel, immunoprecipitation/immunoblotting analyses demonstrated VEGFC-induced VEGFR2/-3 heterodimerization in the EB model. Parallel VEGFR2 immunoprecipitation from VEGFA or VEGFC-treated EBs followed by immunoblotting for VEGFR3 (Figure 4B, right panel) showed VEGFC-, but also to some extent, VEGFA-induced receptor heterodimerization. Figure 4.Formation of vessel structures in differentiating 2D EBs involves both VEGFR2 and VEGFR3. (A) Expression of VEGFRs in 2D EB vascular structures. EBs differentiating in 2D cultures for 12 days in the presence of VEGFA shows vessel-like structures co-expressing VEGFR2 and VEGFR3 (upper panels; merged immunostainings to the right), or VE-cadherin and VEGFR3 (lower panels; merged immunostainings to the right). Scale bar=50 μm (upper), 100 μm (lower). (B) Complex formation between VEGFRs in 2D EBs. Left: Immunoprecipitation (ip) of VEGFR3 from day 12 EBs treated with vehicle (–) VEGFA (A) or VEGFC (C) for 15 min followed by immunoblotting for VEGFR2 (upper left panel). Immunoblotting for VEGFR3 (lower left panel) shows equal loading of VEGFR3. Right: Parallel aliquots of cell lysate were analysed by immunoprecipitation of VEGFR2 followed by immunoblotting for VEGFR3 (upper right panel). VEGFR2 immunoblotting (lower right panel) showed equal loading. (C) Heterodimers in CD31-positive cells. Formation of VEGFR2/-3 heterodimers as detected by in situ PLA (red spots) on 2D EBs immunostained for CD31 (green), in response to vehicle (–), VEGFA or VEGFC. Scale bar=10 μm. (D) Quantification of PLA spots in CD31-positive cells as in C. n=8. (E) Identification of LYVE1-positive cells. EBs in 2D cultures were treated with VEGFA or VEGFC until day 12, and immunostained to detect expression of CD31 (green) and LYVE1 (red). Panels to the right show merged immunostainings. Lymphatic vascular structures expressing LYVE1 but not CD31 are indicated by arrows in the VEGFC-treated cultures. Single, rather than vessel-organized LYVE1-positive cells, are indicated by asterisk. Scale bar=100 μm. (F) Heterodimers in LYVE1-positive cells. EBs in 2D culture were treated as indicated above and processed for immunostaining to detect CD31 (blue) and LYVE1 (white), followed by in situ PLA to detect VEGFR2/-3 heterodimers (red). Scale bar=10 μm. (G) Quantification of PLA spots in LYVE1-positive cells as in (F). n=8. Asterisks in panels D and G indicate the degree of significance (**P<0.01, ***P<0.001). Download figure Download PowerPoint We next used in situ PLA to examine VEGFR2/-3 heterodimer formation in intact 2D EBs. As shown in Figure 4C (quantification in Figure 4D), VEGFA and VEGFC induced heterodimers to an extent similar to that detected in the HSaVEC cultures (see Figure 2 for comparison). Occasional PLA signals were detected also in cells expressing low or non-detectable levels of CD31, prompting us to ask whether some of these cells could be LECs or progenitors thereof. We have shown earlier that differentiation of ES cells allows formation of lymphatic endothelial precursors that express lower levels of CD31, which can be identified by virtue of expression of lymphatic markers such as LYVE1 (Kreuger et al, 2006). As shown in Figure 4E, VEGFA-treated EB cultures contained LYVE1-positive cells that also expressed CD31. A certain fraction of the LYVE1-expressing cells lacked detectable CD31 expression (arrow in Figure 4E). LYVE1-positive cells with distinct morphology, possibly corresponding to monocytic CD45-positive cells, were also detected (asterisk in Figure 4E; see Kreuger et al, 2006). LYVE1-positive cells contained VEGFR2/-3 heterodimers, particularly in response to VEGFC, as shown using in situ PLA (Figure 4F; quantification in Figure 4G). The number of PLA signals/cell was lower in this analysis; this is in part likely due to that co-staining for LYVE1/CD31 together with PLA required additional washing steps and therefore, loss in sensitivity. However, the relationship between the different conditions (control, VEGFA, VEGFC) remained the same. Tip cell filopodia assemble VEGFR2/-3 heterodimers in response to VEGFA or VEGFC Next, the function of VEGFR2/-3 heterodimers in angiogenic sprouting was addressed. For this purpose, EBs were placed in a three-dimensional (3D) collagen I matrix that is permissive for the formation of high-quality angiogenic sprouts, shown to develop a complete basement membrane, to be luminized and covered by
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