VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling - PubMed (original) (raw)

. 2011 Sep 11;13(10):1202-13.

doi: 10.1038/ncb2331.

Georgia Zarkada, Harri Nurmi, Lars Jakobsson, Krista Heinolainen, Denis Tvorogov, Wei Zheng, Claudio A Franco, Aino Murtomäki, Evelyn Aranda, Naoyuki Miura, Seppo Ylä-Herttuala, Marcus Fruttiger, Taija Mäkinen, Anne Eichmann, Jeffrey W Pollard, Holger Gerhardt, Kari Alitalo

Affiliations

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Tuomas Tammela et al. Nat Cell Biol. 2011.

Abstract

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

K.A. is the chairman of the Scientific Advisory Board of Circadian.

Figures

Figure 1

Figure 1

Blood vascular hyperplasia and excessive filopodia projection in mice with a targeted deletion of Vegfr3 in the endothelium. (a,b) Visualization of blood vessels by isolectin B4 (iB4) staining of Vegfr3iΔEC and wild-type littermate retinas at P5. Yellow dots indicate filopodia at the vascular front in b. Scale bars, 100 μm (a) and 50 μm (b). (c–f) Quantitative analysis of the retinas shown in a and b. (c) iB4-positive surface area normalized to total area. (d) Number of vessel branching points. (e) Number of filopodia per length of vascular front. (f) BrdU-positive cells per iB4 area (see Supplementary Fig. S2). In all cases, Cre activity was induced for 48 h before the mice were killed. c–e show data from one litter containing 5 Vegfr3iΔEC and 3 wild-type mice. (f) Data from one litter containing 3 Vegfr3iΔEC and 4 wild-type mice. (g,h) Endomucin staining of E11.5 mouse hindbrains after Cre induction for 24 h before the mice were killed. Yellow asterisks indicate the hindbrain midline in g, and yellow dots indicate filopodia in h. Scale bars, 100 μm (g) and 20 μm (h). (i–k) Quantitative analysis of the Vegfr3iΔEC and wild-type hindbrains; n =3 Vegfr3iΔEC and 5 wild-type embryos. (i) Endomucin-positive surface area normalized to total area. (j) Number of vessel branching points in the subventricular side. (k) Number of vessel sprouts in the pial side (see Supplementary Fig. S3). (l) PECAM-1 staining of LLC tumour xenografts 11 days after implantation into Vegfr3iΔEC or wild-type littermate mice. Scale bar, 50 μm. (m) Quantification of PECAM-1-positive area in the tumours shown in l; n = 5 Vegfr3iΔEC and 5 wild-type mice. (n) Fold increase in vascular area 4 days after transduction with adenoviral vectors encoding VEGF (AdVEGF), normalized to AdVEGF-B in Vegfr3iΔEC versus wild-type mice (see Supplementary Fig. S4); n =3 ears per group. **P <0.005, *P <0.05. Error bars, s.e.m.

Figure 2

Figure 2

Role of VEGFR-3 tyrosine kinase activity in angiogenesis. (a) Intra-embryonic injection of FITC–dextran (green) into the cardiac outflow tract at E11.5 showing homogeneous perfusion of the embryo. Scale bar, 200 μm. (b,c) Immunoprecipitation (IP) of VEGFR-3 (b) or VEGFR-2 (c) of embryos stimulated with VEGF, VEGF-C or BSA followed by western blotting (WB) for phosphotyrosine (pY), VEGFR-3 (R3) or VEGFR-2 (R2). N = 9 (b) and 8 (c) embryos per lane. (d) Immunoprecipitation of VEGFR-2 from hBECs transduced with pMX–VEGFR3–StreptagII retrovirus. Adherent cells were stimulated with VEGF-C, whereas detached cells were replated on collagen I or poly-L-lysine, and subjected to the indicated inhibitors. Uncropped images of blots are shown in Supplementary Fig. S9a. (e) Schematic illustration showing the expected VEGFR-3 activity following the indicated genetic perturbations of Vegfr3. (f) iB4 staining of mouse retinas at P5 48 h after 4-OHT administration. A, artery; V, vein. Scale bar, 100 μm. (g–i) Quantitative analysis of the retinas shown in f. (g) Isolectin B4 (iB4)-positive surface area normalized to total area. (h) Number of vessel branching points. (i) Number of filopodia per length of vascular front. Data pooled from 4 litters containing altogether 8 i_Δ_EC/i_Δ_EC, 4 i_Δ_EC/KD, 6 +/i_Δ_EC, 5 KD/+ and 7 wild-type pups. *P<0.05. Error bars, s.e.m.

Figure 3

Figure 3

An increased level of VEGFR-2 signalling contributes to vascular hyperplasia in Vegfr3iΔEC retinas. (a) Isolectin B4 staining (in green) of Vegfr3iΔEC retinas after treatment with VEGFR-3- or VEGFR-2-blocking antibodies during P3–P5. Non-specific rat IgG was used as a control. Arrowheads indicate abnormally thick vessels. Scale bar, 100 μm. (b) Statistical analysis showing the percentage vessel area increase in Vegfr3iΔEC versus wild-type littermate mice in every treatment group (individual experiments; n = 4, 5 and 4 Vegfr3iΔEC pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively; and 6, 3 and 5 wild-type pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively). (c) qRT-PCR analysis of Vegfr1 gene (also known as Flt1) expression; n = 4 Vegfr3iΔEC and 3 wild-type pups. In all analyses of the retina, Cre activity was induced for 48h before the mice were killed. *P < 0.05, ***P < 0.001. Error bars, s.e.m. (d) Cultured HUVECs subjected to siRNA-mediated silencing of VEGFR3 expression (VEGFR3 siRNA) and stimulation with VEGF for the indicated times. VEGFR-2 was immunoprecipitated (IP) followed by immunoblotting (IB) for phosphotyrosine (pY) and VEGFR-2. Numbers below the blots indicate relative intensities of pY to VEGFR-2, normalized to control siRNA at the same time point. Note the increased pVEGFR-2 signal at 30 min and 60min (red). Immunoprecipitation and western blot analysis for VEGFR-3 from the same lysates is shown below. Uncropped images of blots are shown in Supplementary Fig. S9b.

Figure 4

Figure 4

A decreased level of Notch signalling underlies excessive angiogenesis in Vegfr3iΔEC retinas. (a) Fold changes in Hey1, Hey2, Nrarp and Dll4 mRNA levels in the retinas of Vegfr3iΔEC and wild-type littermate pups at P5. mRNA levels were normalized to Cadh5 to compensate for the increased endothelial cell numbers in Vegfr3iΔEC retinas. *P < 0.05; n = 4 Vegfr3iΔEC and 3 wild-type pups. Error bars, s.e.m. (b,c) Vessel area quantification (b) and isolectin B4 (iB4) staining (c) of Vegfr3iΔEC and wild-type littermate retinas at P5 following administration of Jagged1 peptide mimetics (Jag1) or scrambled peptides (SC-Jag1) and 4-OHT for 48h. Scale bar, 100 μm. ***P < 0.001; n = 3 Vegfr3iΔEC and 4 wild-type pups treated with SC-Jag1 and 4 Vegfr3iΔEC and 4 wild-type pups treated with Jag1. Data pooled from 2 individual experiments. Error bars, s.e.m. (d) A 10 day chimaeric embryoid body derived from wild-type DsRed-expressing embryonic stem cells (red), mixed in a 1:1 ratio with embryonic stem cells having one functional Vegfr3 allele (Vegfr3+/LacZ) and stained for iB4 (green). Red arrowheads indicate tip cells of wild-type origin; green arrowheads point to Vegfr3 heterozygous cells. Scale bar, 200 μm. (e) High-magnification image of a sprout showing a mosaic distribution of the cells. DNA in blue. Scale bar, 20 μm. (f,g) Quantification of the tip cell genotype in all sprouts (f; 65.89%±2.5% s.e.m.; n = 621 sprouts), in sprouts that exhibited a 1:1 contribution of wild-type and Vegfr3+/LacZ cells (g; 61.8%± 1.8% s.e.m.; n = 360 sprouts) and in sprouts with a 1:1 contribution of wild-type and Vegfr3+/LacZ cells following treatment with DAPT (h; 53.7%±2.7% s.e.m.; n = 325 sprouts). **P < 0.01, **P < 0.05. Error bars, s.e.m. (i) Mosaic retina of a P5.5 pup derived from a wild-type blastocyst injected with Vegfr3+/LacZ embryonic stem cells and stained for iB4. β-galactosidase activity (in black, arrow) indicates a Vegfr3+/LacZ cell. Scale bar, 50 μm.

Figure 5

Figure 5

Vegfc haploinsufficiency leads to instability of sprout fusion points and inefficient angiogenesis. (a) Isolectin B4 (iB4) staining (green) of retinas from Vegfc+/− mice and their wild-type littermates at P5. (b–f) Quantitative analysis of the retinas shown in a; data pooled from two litters containing altogether 6 Vegfc+/− and 9 wild-type pups. (b) iB4-positive surface area normalized to total area. (c) Extent of vascular plexus migration from the optic stalk (OS). (d) Number of vessel branching points. (e) Number of sprouts. (f) Filopodia per length of vascular front. (g) Fold changes in Hey1, Hey2 and Nrarp mRNA levels analysed by qRT-PCR in the retinas of Vegfc+/− and wild-type pups at P5 (data pooled from two litters containing altogether 7 Vegfc+/− and 6 wild-type pups). (h) Number of failed fusions per vascular loop in the retinas of Vegfc+/− and Vegfc+/+ pups at P5 (n = 6 Vegfc+/− and 9 wild-type pups, data pooled from 2 litters). (i) iB4 (green) and collagen IV (red) staining of Vegfc+/− or wild-type littermate retinas at P5. Arrowheads indicate empty basement membrane sleeves. (j) iB4 (white), VEGF-C (red) and Tie2 (green) immunostaining in wild-type mouse retinas at P5. Arrows indicate VEGF-C- and Tie2-positive macrophages at the angiogenic front. (k) iB4 staining (green) of P5 retinas of op/op pups and op/+ littermate controls. (l–o) Quantitative analysis of the retinas shown in k; n = 5 op/op and 4 op/+ pups. Dashed line in a and k indicates a similar distance from the optic stalk (OS). (l) iB4-positive surface area normalized to total area. (m) Extent of vascular plexus migration from the optic stalk. (n) Number of vessel branching points. (o) Number of sprouts. (p) Fold changes in Hey1, Hey2 and Nrarp mRNA levels analysed by qRT-PCR in the retinas of op/op pups and op/+ pups at P5 (n = 5 op/op and 3 op/+ pups). Scale bars, 100 μm (a,k) and 50 μm (i,j). * P < 0.05, ** P < 0.01, ***P < 0.001. Error bars, s.e.m.

Figure 6

Figure 6

VEGF-C promotes Notch signalling in endothelial cells through VEGFR-3 and PI(3)K. (a–d) Fold changes in Notch target gene and DLL4 levels in hBECs stimulated with 200 ng ml−1 VEGF-C, and treated with Dll4-Fc conditioned medium (a), transfected with VEGFR3 siRNA or control siRNA (b), in conditions where 50% of hBECs express membrane-bound Dll4 (Dll4-TM; c) or treated with the PI(3)K inhibitor LY294002 (d). Cells were stimulated for 1 h before lysis. Expression of GAPDH was used as the normalization control. Note the successful transduction of hBECs with retroviruses encoding Dll4-TM in c, as evaluated by qRT-PCR. (e) Fold increase in PI(3)K activity in VEGFR3 versus control silenced hBECs after stimulation with VEGF-C (100 ng ml−1) for 15 min. Data pooled from 2 individual experiments, each containing 3 replicates. * denotes P values versus control group (*P <0.05, **P <0.01, ***P <0.001) and # denotes P values between groups (# P <0.05, ## P <0.01). Error bars, s.e.m.

Figure 7

Figure 7

VEGFR-3 interacts with the transcription factor FoxC2 to control angiogenesis. (a) Fold change in the level of FOXC2 mRNA expression following stimulation of hBECs with 200 ng ml−1 VEGF-C (n = 3 plates per group). (b) Immunostaining for FoxC2 (red) and isolectin B4 (iB4; green) in Vegfr3iΔEC and wild-type littermate pups at P5. Arrowheads indicate FoxC2-negative tip cells. (c) Quantification of FoxC2-positive nuclei from the retinas shown in b. Nuclei in the area of iB4-positive endothelial cells were quantified at the angiogenic front (n = 3 pups per group). (d,e) Fold change in the level of Foxc2 mRNA expression in Vegfr3iΔEC and wild-type littermate retinas (d), and in Vegfc+/− or wild-type littermate retinas (e) at P5 (n = 3 pups per group). (f) iB4 staining (white) in Foxc2+/−; Vegfr3+/−, Foxc2+/−, Vegfr3+/− or wild-type littermate retinas at P5. Yellow dots in the lower panels indicate filopodia. (g–i) Quantitative analysis of the retinas shown in f. (g) iB4-positive surface area normalized to total area. (h) Number of vessel branching points. (i) Filopodia per length of vascular front. Data pooled from 2 litters; n = 3 Foxc2+/−; Vegfr3+/−, 4 Foxc2+/−, 4 Vegfr3+/− and 4 wild-type pups. Scale bars, 50 μm. *P<0.05, **P<0.01, ***P< 0.001. Error bars, s.e.m. (j) Schematic of VEGF-C-expressing macrophages in vessel anastomosis and branch maintenance during developmental angiogenesis. Initially, 2 tip cells that lead vascular sprouts are chaperoned to fuse by a macrophage (green). VEGF-C expression (purple) ensues in the macrophage, activating VEGFR-3 in the tip cells, which leads to the expression of Notch target genes and decreased sensitivity to the VEGF gradient in the cells. Vegfr3 loss-of-function (LOF) leads to decreased Notch signalling. A simplified summary of the ‘active’ (green) and ‘passive’ (red) signalling pathways originating from VEGFR-3 is shown in the upper left corner. Only the ‘active’ pathway is targetable by inhibitors.

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