(a) Immunoblotting of lysates with antibodies against phospho-VEGFR2 Tyr1175 (P-R2) and VEGFR2 (R2). After overnight serum-starvation, HDMEC and hemEC were pretreated with 2 μg ml⁻¹ VEGF neutralizing antibody (VEGF Ab; + lanes) or isotype control mouse IgG (− lanes) for 48 hours.
(b) Normalized results of quantitative multiplex ELISA for several targets of VEGFR2 signaling. Data were normalized by dividing experimental values by α-tubulin values. HDMEC and three different hemEC were treated with either control siRNA (black, light gray and medium gray bars) or VEGFR2-specific siRNA (R2 siRNA) (dark gray and white bars). Also, cells were treated with vehicle (PBS + 0.1% BSA, black bars) or 25 ng ml⁻¹ VEGF for 15 min (light gray bars, to measure protein phosphorylation) or 12 h (light gray bars, to measure protein expression). Finally, the effect of adding 50 ng ml⁻¹ recombinant sVEGFR1 (sR1) was tested (medium gray bars). Control experiments showed that levels of VEGFR2 transcripts were reduced by > 80% in cells treated with VEGFR2 siRNA (not shown). Error bars represent SD +1 (n = 3).
(c) Increased cell migration of hemEC compared to HDMEC. Cells were treated as described in Fig. 1b. Data expressed as relative fluorescence units (RFU). Error bars represent SD of +1 (n = 3).
(d) Increased proliferation of hemEC without exogenous VEGF by FACS analyses of BrdU incorporation. Cells were treated as described in Fig. 1b. Numbers indicate the percentage of BrdU positive cells. Error bars represent SD of +1 (n = 3).

(a) In vivo interaction of NFATc2 with VEGFR1 promoter assayed by chromatin immunoprecipitation.
Top: HDMEC were stimulated with (+) or without (−) VEGF (25 ng ml⁻¹), β1 integrin stimulating antibody (ITGB1 Ab, 10 μg ml⁻¹) or control IgG for 30 min and the amount of VEGFR1 promoter fragments in the precipitate was quantitated using real-time PCR as described in Supplementary Methods. * P < 0.05 as compared with the value in unstimulated cells; error bar represents SD+1 (n = 3).
Bottom: In HDMEC and hemEC2, the presence of VEGFR1 promoter fragments in the precipitates was detected using PCR followed by agarose gel electrophoresis. Similar results were obtained with hemEC4 and hemEC21A.
(b) Real-time quantitative PCR was used to measure VEGFR1 (black bars) and COX-2 (light gray bars) transcripts in lysates of HDMEC and hemEC incubated in the absence (−) or presence (+) of 10 μg ml⁻¹ β1 integrin antibody (ITGB1 Ab; + lanes) for 6 h. Transcript levels were normalized to GAPDH. Levels in HDMEC without the antibody were set at 1. *P < 0.05 by Mann-Whitney test; error bars represent SD of +1 (n = 3).
(c) Immunocytochemical detection of active (HUTS-21) and total β1 integrin (total) on cell surface of HDMEC (left) transfected with vector, mutant VEGFR2 (Mut VEGFR2) or mutant TEM8 (Mut TEM8), compared with hemEC2^(VEGFR2) (right) transfected with vector, wild-type VEGFR2 (WT VRGFR2) or wild-type TEM8 (WT TEM8). Similar results were obtained with hemEC4^(TEM8) and hemEC21A. White scale bars are 50 μm.

(a) Top: Immunoblotting of cell lysates from control cells (HUVEC, HDMEC) and all nine hemEC subjected to immunoprecipitation (IP) with anti-VEGFR2 antibodies or control IgG. The blot was incubated with antibodies to VEGFR2 (R2), β1 integrin (ITGB1) and TEM8.
Bottom: The expression levels of VEGFR1, VEGFR2, β1 integrin and TEM8 in HDMEC and hemEC. Immunoblotting was performed with whole cell lysates using antibodies to VEGFR1, VEGFR2, β1 integrin or TEM8. The levels of actin are shown as loading controls.
(b) Top: Immunoblotting of lysates from 293-EBNA cells subjected to immunoprecipitation (IP) with antibody to VEGFR2 or control IgG. Cells were stably transfected with combinations of constructs encoding wild-type (WT) and mutant (Mut) VEGFR2 (His-tagged and puromycin-resistant) and TEM8 (HA-tagged and hygromycin B-resistant). Transfected cells were selected for both puromycin (10 μg ml⁻¹) and hygromycin B (300 μg ml⁻¹) resistance. Gel electrophoresis followed by immunoblotting with antibody to β1 integrin (ITGB1), His-tag (R2-His) and HA-tag (TEM8-HA).
Bottom: The expression levels of β1 integrin, TEM8 and VEGFR2 in 293 cells transfected with constructs encoding VEGFR2 and TEM8 as described in Fig. 5b Top. Immunoblotting was performed with whole cell lysates using antibodies to VEGFR2, β1 integrin or TEM8. The levels of actin are shown as loading controls.
(c) Stimulation of VEGFR1 transcript levels by VEGF or β1 integrin stimulating antibodies (ITGB1 Ab) in HDMECs requires TEM8, NFATc2 and VEGFR2. Cells were treated with specific siRNAs or control siRNA and cultured without (black bars) or with VEGF (light gray bars) or β1 integrin antibody (dark gray bars) for 6 h. Control experiments showed transcript levels for each siRNA target gene to be reduced by > 80% (not shown). Relative amounts of VEGFR1 transcripts (normalized with GAPDH) determined in total RNA with quantitative PCR. Error bars represent SD of +1 (n = 3). * P < 0.05 as compared with the values in control cells.
(d) In vivo interaction of NFATc2 with VEGFR1 promoter assayed by chromatin immunoprecipitation. HDMEC and hemEC4^(TEM8) were transfected with retroviral vector or constructs encoding mutant TEM8 (Mut TEM8) , wild-type TEM8 (WT TEM8) or wild-type VEGFR2 (WT R2). The presence of VEGFR1 promoter fragments in the precipitates was detected using PCR followed by agarose gel electrophoresis. Transfection efficiency was determined by GFP expression (> 95%). Similar results were obtained with hemEC2^(VEGFR2) and hemEC21A.
(e) Increased proliferation and migration of HDMEC following stable transfection with construct encoding mutant TEM8. HDMEC were stably transfected with retroviral vector or constructs encoding wild-type (WT) TEM8 or mutant (Mut) TEM8. HemEC4^(TEM8) cells with empty vector were included for comparison. For proliferation analysis, cells were labeled with BrdU and analyzed by flow cytometry. Bar heights represent proportions of BrdU-positive cells. For migration analysis, cells were assayed as described in Fig. 1d. *P < 0.05; **P < 0.01 comparing proliferation or migration to vector control. Error bars represent SD+1 (n = 3).
(f) Diagram summarizing pathways that are affected in hemangioma. In control endothelial cells, such as HDMEC (left), activation of NFAT and VEGFR1 (R1) expression is controlled by a functional interaction between integrin, the VEGFR2 extracellular domain and TEM8. This results in high levels of VEGFR1 (and COX-2, DSCR-1) and low levels of VEGF-dependent VEGFR2 signaling. In hemEC (right), repressed NFAT activation causes low expression of VEGFR1 (and COX-2, DSCR-1). This results in high level VEGF-mediated signaling through activation of VEGFR2-tyrosine kinase. Consequently, proliferation and migration are upregulated. The effects of mutations in TEM8 and VEGFR2 on VEGFR2 phosphorylation and downstream pathways are indirect and linked to downregulated activation of integrin-NFAT and low VEGFR1 expression. In hemEC4^(TEM8), mutant TEM8 has a dominant negative effect on the integrin-NFAT-VEGFR1 pathway. In patient 4 this provides a sufficient genetic explanation for the hemangioma signaling phenotype. The C482R mutation in hemEC2^(VEGFR2)/17B^(VEGFR2) represents a loss-of-function mutation in the context of VEGFR1 expression, but it does not appear to be sufficient to induce a hemEC-like phenotype when overexpressed in HDMEC. There are two possible reasons; one possibility is that viral expression of mutant VEGFR2 does not reach the required level (in hemEC2 and 17B the ratio between wild-type and mutant protein is 1:1, but this ratio may need to be even more in favor of the mutant in a cell that already expresses two wild-type alleles); a second possibility is that the VEGFR2 mutation is associated with an additional genetic or physiological change to generate the hemangioma cellular phenotype of repressed integrin-NFAT activity and low VEGFR1 expression. In hemEC from patients where we have not yet found mutations, we speculate that their increased β1 integrin/VEGFR2/TEM8 complex formation and reduced integrin/NFAT/VEGFR1 activity is associated with mutations in other, as yet unidentified, cell surface (stippled bars) or cytoplasmic (grey areas with arrows) components of the pathway.

(a) Mean concentrations of VEGFR1 protein by ELISA in culture media (for measuring sVEGFR1) and cell lysates (for measuring total VEGFR1) of control cells (cbEPC, HUVEC, HFSEC, HDMEC) and nine different hemEC. Cells were incubated in serum-free medium for 24 h in the absence (−) or presence (+) of 25 ng ml⁻¹ VEGF. For all hemEC, the levels of VEGFR1 protein were significantly lower (P < 0.05) than the value for unstimulated HDMEC. Error bars represent SD of +1 (n = 3).
(b) Effects of retroviral overexpression of full-length membrane-bound form of VEGFR1 (mR1; + lanes) or Y1213F mutated VEGFR1 (Mut mR1) on levels of VEGFR2 (R2) and ERK and their phosphorylated forms (P-R2 and P-ERK) in HDMEC and hemEC. Control cells were transfected with empty vector. VEGFR1 expression was detected using antibody to VEGFR1.
(c) NFAT is a transcriptional activator of the VEGFR1 promoter.
Top: The difference in relative VEGFR1 promoter activity in HDMEC when transiently transfected with wild-type (WT) or mutated (Mut) VEGFR1 promoter-luciferase construct, and when incubated without (−) or with (+) VEGF for 3 h. Luciferase activity in HDMEC with wild-type VEGFR1 and without VEGF set at 1. Data are expressed as means of three independent experiments. *P < 0.05 as compared with the value for cells transfected with wild-type VEGFR1 promoter construct; error bars represent SD of +1 (n = 3).
Bottom: DNA binding activity of NFAT to biotin-labeled VEGFR1- or control COX-2 promoter oligonucleotides. An oligonucleotide representing the promoter of COX-2, a known NFAT target gene, used as a positive control. Cell lysates from HDMEC incubated without (−) or with (+) VEGF for 15 min were used.
(d) Real-time quantitative PCR used to measure DSCR1, MCP-1, COX-2, NFATc1 and NFATc2 transcripts in lysates of HDMEC and hemEC incubated in the absence (−) or presence (+) of 25 ng ml⁻¹ of VEGF for 6 h. Transcript levels were normalized to GAPDH. Levels in HDMEC without VEGF were set at 1. For hemEC, the levels of DSCR1, MCP-1 and COX-2 mRNA were significantly lower (P < 0.05) than the value for HDMEC. Error bars represent SD of +1 (n = 3).
(e) Relative transcript levels (to VEGFR2) of the NFAT target genes, VEGFR1 and COX-2, in control tissues (placenta, foreskin), hemangioma tissues, control cells (cbEPC, HUVEC, HFSEC, HDMEC) and hemEC using real-time quantitative PCR. Hemangioma tissues consisted of 3 proliferating phase hemangiomas (hem75, hem76, and hem82) and 3 involuting hemangiomas (I-39, I-47 and I-52) (see also48). Cultured cells were incubated without (−) or with (+) VEGF (25 ng ml⁻¹) for 6 hours. To allow comparisons to be made between the tissues and the cultured endothelial cells, we chose to compare the VEGFR1 and COX-2 transcript levels relative to VEGFR2 (assuming the two VEGF receptors are endothelial cell-specific48). The VEGFR1/VEGFR2 or COX-2/VEGFR2 transcript ratios for placenta and HDMEC without VEGF were set at 1. No significant differences between VEGFR2 transcript levels (relative to GAPDH) in HDMEC and hemEC were found (not shown).
(f) Treatment with cell-permeable selective NFAT inhibitor downregulates VEGFR1 expression and induces phosphorylation of VEGFR2 and the downstream target ERK.
Top: Relative amounts of VEGFR1 (R1) transcripts determined by real-time quantitative PCR in lysates of cells not treated (−; black bars) or treated (+; light gray bars) with the cell-permeable NFAT inhibitor III (INCA-6; Calbiochem). HDMEC were treated with 10−30 μM inhibitor for 12 h. Value for lysates of untreated cells in the experiment with 10 μM INCA-6 was set at 1. * P < 0.05 as compared with the value in cells without inhibitor; error bars represent SD of +1 (n = 3). Bottom: Immunoblotting performed as described in Fig. 1a of cell lysates incubated with indicated concentrations of INCA-6.

(a) Left: Portions of nucleotide sequence tracings of genomic DNA from HDMEC (control) and genomic DNA and cDNA from cells of hemangioma patient 4 show heterozygosity for G and A (arrows) in TEM8 in hemEC4.
Right: Diagrams illustrating the domain structure of wild-type TEM8 protein variant 1 (top), mutant TEM8-A326T and TEM8 variant 3. Selected exons (1, 12, 13 and 18) within the TEM8 gene are shown (bottom). Variant 1 transcript is generated by splicing exon 12 to exon 13; variant 3 transcript is generated by splicing exon 12 to a cryptic exon within intron 12 and terminates upstream of exon 13.
(b) Left: The effects of overexpressing constructs coding for TEM8 in HDMEC. Lysates from cells transfected with vector or constructs encoding HA-tagged wild-type TEM8, HA-tagged A326T mutant TEM8 or variant 3 were used for immunoblotting with antibodies against VEGFR1 (R1), phospho-VEGFR2 Tyr1175 (P-R2), VEGFR2 (R2), HA-tag (HA), actin, phospho-ERK (P-ERK) and ERK. Transfection efficiency was determined by GFP expression (> 95%).
Right: Quantitation of VEGFR1 and TEM8 expression. To determine VEGFR1 promoter activity, VEGFR1 promoter-luciferase reporter was transfected into HDMEC transfected with retroviral constructs described in Fig. 4b left (black bars). TEM8 expression was measured by real-time PCR (light gray bars). The bar graph shows fold stimulation, with the value in HDMEC transfected with retroviral vector arbitrarily set at 1. * P < 0.05 as compared with the value in cells transfected with retroviral vector. Error bars represent SD +1 (n = 3). The changes in VEGFR1 promoter activity correlate with changes in VEGFR1 protein expression.
(c) Left: Immunoblotting of lysates prepared from hemEC4^(TEM8), hemEC2^(VEGFR2) and hemEC21A transfected with retroviral constructs as described in Fig. 4b.
Right: Quantitative assay of VEGFR1 and TEM8 expression as described in Fig. 4b. For this assay, a mixture of equal amounts hemEC2^(VEGFR2) , hemEC4^(TEM8) and hemEC21A lysates was used. The bar graph shows levels of VEGFR1 promoter activity (black bars) and TEM8 transcript levels (light gray bars), relative to the value in HDMEC transfected with retroviral vector arbitrarily set at 1 in Fig. 4b. *P < 0.05 as compared with the value in cells transfected with retroviral vector. Error bars represent SD+1 (n = 3). The changes in VEGFR1 promoter activity correlate with changes in VEGFR1 protein expression.
(d) Portions of nucleotide sequence tracings of genomic DNA from cells of control individuals (HDMEC) and cells (hemEC) from hemangioma patients 2 and 17 demonstrate heterozygosity for T and C (arrows) in VEGFR2 in hemEC. Sequencing of blood-derived DNA demonstrated heterozygosity for T and C alleles in both patients. The T-to-C transition results in a substitution of Cys482 by arginine in the Ig-like domain V (arrow) in the extracellular domain of VEGFR2 (at right).
(e) Left: The effects of overexpressing constructs coding for VEGFR2 in HDMEC. Following retroviral transfer of expression constructs into HDMEC, lysates from cells transfected with vector or constructs encoding His-tagged wild-type VEGFR2 or His-tagged mutant VEGFR2 were subjected to immunoblotting with antibodies to VEGFR1 (R1), phospho-VEGFR2 Tyr1175 (P-R2), His-tag (His), actin, phospho-ERK (P-ERK) and ERK. Transfection efficiency was determined by GFP expression (> 95%).
Right: VEGFR1 (black bars) and VEGFR2 (light gray bars) protein concentrations determined by ELISA in lysates of HDMEC transfected with retroviral vector, or constructs encoding wild-type or mutant VEGFR2. *P < 0.05, **P < 0.01 as compared with the value for cells transfected with vector. Error bars represent SD of +1 (n = 3).
(f) Left: Immunoblotting of lysates prepared from hemEC4^(TEM8), hemEC2^(VEGFR2), hemEC17B^(VEGFR2) and hemEC21A transfected with retroviral constructs.
Right: Quantitative ELISA of VEGFR1 (black bars) and VEGFR2 (light gray bars). Protein levels were determined in a mixture of equal amounts of lysates from hemEC2^(VEGFR2) , hemEC4^(TEM8) and hemEC17B^(VEGFR2) and hemEC21A, stably transfected with retroviral constructs as described in Fig. 4e. *P < 0.05 as compared with the value for cells transfected with retroviral vector. Error bars represent SD+1 (n = 3).
(g) Immunoblots of lysates of hemEC2^(VEGFR2) overexpressing wild-type or mutant forms of VEGFR2 probed with antibodies against VEGFR1 (R1), VEGFR2 (R2) and actin. Constructs, stably transfected into cells using retrovirus, included empty vector and constructs encoding wild-type VEGFR2, the T1444C/C482R mutation, T1444A/C482S, A1416T/Q472H, VEGFR2 lacking the cytoplasmic domain, A2852T/Y951F, A3176T/Y1057F, and A3524T/Y1175F. A Q472H substitution, reported as a potential hemangioma mutation4, did not affect VEGFR2 stimulation of VEGFR1 expression. This is consistent with the finding that the Q472H VEGFR2 allele is common (about 50%) among “control” chromosomes. Similar results were obtained with hemEC4^(TEM8) and hemEC21A.