Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 die of vascular overgrowth, and we are interested in how flt-1 normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular overgrowth, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-1 to produce blood vessels. Flt-1−/− embryonic stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. Flt-1 mutant embryos also had an increased endothelial cell mitotic index, indicating that aberrant endothelial cell division occurs in vivo in the absence of flt-1. Theflt-1 mutant vasculature of the cultures was partially rescued by mitomycin C treatment, consistent with a cell division defect in the mutant background. Analysis of cultures at earlier time points showed no significant differences until day 5, whenflt-1 mutant cultures had increased β-galactosidase+ cells, indicating that the expansion of flt-1 responsive cells occurs after day 4. Mitomycin C treatment blocked this early expansion, suggesting that aberrant division of angioblasts and/or endothelial cells is a hallmark of theflt-1 mutant phenotype throughout vascular development. Consistent with this model is the finding that expansion of platelet and endothelial cell adhesion molecule+ and VE-cadherin+ vascular cells in theflt-1 mutant background first occurs between day 5 and day 6. Taken together, these data show that flt-1 normally modulates vascular growth by controlling the rate of endothelial cell division both in vitro and in vivo.

Introduction

Blood vessels form by coordinating several cellular processes, including cell division and morphogenesis (reviewed in Folkman & D'Amore,1 Weinstein,2 and Conway et al3). Some of the mitogenic signals that promote division of endothelial cells and their precursors are known, but how these signals are modulated to initiate cell divisions only when and where they are needed is not known in detail. After blood vessels initially form, maturation and remodeling steps involve the recruitment of ancillary cells, such as smooth muscle and pericytes. These cells and the extracellular matrix that is also produced can negatively modulate endothelial cell division.4-8 However, modulators of endothelial cell mitogenesis at the earliest stages of blood vessel formation have not been identified.

The vascular endothelial growth factor (VEGF) signaling pathway is clearly critical to both early endothelial cell division and morphogenesis, and its regulation is complex (reviewed in Ferrara & Davis-Smyth9 and Neufeld et al10). Mouse embryos lacking even one copy of the VEGF gene die in utero with severe vascular defects, and vascular development in differentiating embryonic stem (ES) cells is compromised in VEGF-A+/− andVEGF-A−/− ES cells in a dose-dependent manner.11-13 Moreover, even modestly elevated levels of VEGF lead to vascular abnormalities,14 and large doses of VEGF invariably severely compromise both vascular development and neovascularization in adult organisms.15-17 These findings suggest that VEGF signaling must be precisely controlled during vascularization to result in proper vessels. The location and duration of VEGF expression provide the first level of control,18-21 but other components of the pathway are likely to be involved in fine-tuning the signal.

Two high-affinity receptors, flk-1 and flt-1, participate in VEGF signal transduction and are candidates to be involved in fine-tuning mechanisms. Both receptors are membrane-spanning receptor tyrosine kinases that bind VEGF with high affinity,22-26 but their effects on VEGF signaling are very different. Mice or ES cells lacking flk-1 have little or no blood vessel formation, suggesting that many downstream effects of VEGF on endothelial cells are mediated through flk-1.27,28 Specifically, numerous studies show that VEGF signaling through flk-1 produces a strong mitogenic signal for endothelial cells.29-32 

In contrast, VEGF binding to flt-1 does not produce a strong mitogenic signal, and flt-1−/− mice die at mid-gestation with vascular overgrowth and disorganization.23,29,33 This phenotype was reported to result from increased numbers of cells called hemangioblasts that can give rise to both hematopoietic and endothelial cells.34 However, invoking control of an early cell fate switch as the exclusive cellular mechanism of flt-1 action is inconsistent with evidence that flt-1 is expressed in mature endothelial cells, including tumor vasculature.35-37 It is also inconsistent with a molecular model of flt-1 action, suggesting that flt-1 can sequester VEGF ligand and, thus, modulate signaling through flk-1, because flk-1 signaling affects multiple endothelial processes, including cell division.38,39 Moreover, VEGF addition to flt-1–expressing trophoblast cells inhibits cell division, and 2 recent studies using chimeric receptors suggested that flt-1 signaling may counteract the positive mitogenic signal from flk.40-42 

Thus, we asked if flt-1 could negatively modulate endothelial mitogenesis developmentally, and to address this question we analyzed the cellular mechanism responsible for theflt-1−/− phenotype in both ES cell cultures and embryos. The flt-1 mutant ES cell cultures and embryos had vascular overgrowth that was caused primarily by aberrant endothelial cell division, and this deregulated mitogenesis in the vascular lineage was seen throughout the stages of vascular development. Thus, flt-1 acts early in vascular development to modulate vessel formation by affecting the rate of cell division in embryonic endothelial cells and their precursors.

Materials and methods

Cell culture and in vitro differentiation

Wild type (WT, +/+), hemizygous mutant (flt-1+/−), and homozygous mutant for the targeted flt-1 mutation (flt-1−/−)33 ES cells were maintained and differentiated in vitro as attached cultures as described previously.43 

For mitomycin C treatment, ES cell cultures were differentiated to day 6, then incubated with mitomycin C (Sigma) at 30 μg/mL diluted in differentiation media for 2 hours at 37°C. After incubation in fresh differentiation medium for 48 hours (to day 8), cultures were fixed and stained with the appropriate antibodies. For earlier times, cultures were incubated with mitomycin C as described earlier on day 4 or day 5, then incubated in fresh medium for 24 hours (to day 5 or day 6) before fixation and staining.

Antibody staining and image analysis

ES cell cultures were rinsed in phosphate-buffered saline (PBS) and fixed for 5 minutes in ice-cold methanol:acetone (50:50) or fresh 4% paraformaldehyde (for VE-cadherin staining). Fixed cultures were reacted with antibodies as described previously.13,43 In double-labeling experiments, cultures were first incubated with rabbit anti–β-galactosidase or rabbit antiphosphohistone H3 antibodies and the appropriate secondary, then blocked in staining media (3% fetal bovine serum [FBS], 0.1% NaN3 in PBS) with 5% donkey serum before the addition of rat antimouse platelet and endothelial cell adhesion molecule (PECAM). In triple-labeling experiments, rabbit polyclonal antiphosphohistone H3 incubation was followed by incubation with rat antimouse PECAM and, subsequently, staining with the DNA dye topro-3 (Molecular Probes) at 1:1000 for 5 minutes at room temperature. All cultures were rinsed in PBS and viewed with an Olympus IX-50 inverted microscope by using epifluorescence or a Zeiss LSM 410 confocal microscope.

Primary antibodies and dilutions used were rat antimouse PECAM at 1:1000 (MEC 13.3; Pharmingen); rat antimouse intercellular adhesion molecule 2 (ICAM-2) at 1:500 (3C4; Pharmingen), rabbit polyclonal anti–β-galactosidase at 1:300 (Cappel Labs), rabbit polyclonal antiphosphohistone H3 at 1:500 (Upstate Biotechnology), and rat antimouse VE-cadherin at 1:100 (11D4.1; Pharmingen). Secondary antibodies and dilutions used were donkey antirabbit immunoglobulin G (IgG; H + L) TRITC cross-absorbed at 1:100 (Jackson Immunoresearch) for antiphosphohistone H3 and β-galactosidase, donkey antirat IgG (H + L) B-phycoerythrin cross-absorbed at 1:300 (Jackson Immunoresearch) for PECAM and ICAM-2, donkey antirat IgG (H + L) fluorescein isothiocyanate (FITC) cross-absorbed at 1:100 (Jackson Immunoresearch) for PECAM, and goat antirat IgG (H + L) Alexa 488 cross-absorbed at 1:100 (Molecular Probes) for PECAM and VE-cadherin.

Quantitative image analysis of day 8 ES cell cultures reacted with the appropriate antibodies was performed as previously described.13 Sequential nonoverlapping areas completely covered with cells were photographed at ×10 magnification, so that the total area photographed per well was more than 60% of the well area. For earlier time points, β-galactosidase–stained wells were photographed, and only areas covered with cells were used for analysis. Digital images were generated and analyzed by using Adobe Photoshop (version 5.0, Adobe Systems). Quantitation of the stained area for each image was performed by using an Image Processing Tool Kit (Rev. 2.1; Reindeer Games, Asheville, NC). Stained area averages for each well were calculated, and the average of 3 to 4 wells for each condition was used to determine SD values.

β-Galactosidase detection

β-Galactosidase detection was performed by using a modified protocol.44 Cultures were rinsed twice in 0.1 M phosphate buffer (pH 7.3) and fixed with glutaraldehyde fix solution (0.2% glutaraldehyde, 5 mM EGTA [pH 7.3], 2 mM MgCl2 in 0.1 M phosphate buffer [pH 7.3]) for 5 minutes. After washing 3 times for 5 minutes with phosphate buffer, cultures were incubated for 3 hours (day 8 ES cultures) or 5 hours (early time course experiments) at 37°C in X-gal staining solution (0.625 mg/mL X-gal; Sigma), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, in wash buffer (2 mM MgCl2, 0.02% Nonidet-P40 in 0.1 M sodium phosphate buffer [pH 7.3]), then rinsed and stored in wash buffer at 4°C.

RNA analysis

Total RNA was isolated from day 7 ES cell cultures by centrifugation through a CsCl gradient.45 RNase protection assays for PECAM were performed by using a modified protocol.13,46 In vitro transcription of PECAM-dCPa (nt 1425-1904) was used to generate a 32P-labeled antisense RNA probe. Overnight hybridization at 45°C with the PECAM probe and a β-actin internal control probe was followed by digestion with RNase A and RNase T1. Protected fragments were then electrophoresed through a 5% acrylamide urea (8 mM gel) and quantified by using a PhosphorImager (Molecular Dynamics).

Fluorescent-activated cell sorter analysis

Day 8 ES cell cultures were rinsed twice with PBS and dissociated with 0.2% collagenase (Sigma; 0.15% type II, 0.05% type XI in PBS) for approximately 2 hours with repeated passage through a 20-gauge needle. The cells were rinsed in FBS/PBS (1:1), resuspended in cold staining media (3% FBS + 0.01% sodium azide in PBS), and incubated on ice for 20 minutes. Cells were then incubated with 100 μg/mL biotin-coupled ICAM-2 antibody in staining medium for 45 minutes at 4°C. After 3 washes with cold staining medium, cells were resuspended in staining medium with 25 μg/mL streptavidin-phycoerythrin (Southern Biologicals) and incubated for 45 minutes at 4°C. After 3 washes with cold PBS, the cells were fixed and stored at 4°C in 1% paraformaldehyde. Flow cytometry data were collected with a Becton Dickinson FACSCAN.

Mitotic index calculations

WT and flt-1−/− ES cell cultures were differentiated in chamber slide wells (Nunc) to day 6 or 7, fixed, and triple-labeled with rabbit antiphosphohistone H3, rat antimouse PECAM, and the DNA binding dye topro-3. Slides were mounted in AquaPolymount (LifeSciences). Confocal images were analyzed by using Adobe Photoshop (version 5.0, Adobe Systems) software. Triple-labeled images were counted in the following 4 ways: (1) the total number of cells per field, (2) the total number of phosphohistone H3+ cells per field, (3) the number of PECAM+ cells with endothelial morphology per field, and (4) the number of PECAM+/phosphohistone H3+ cells with endothelial morphology per field. Endothelial mitotic indices were calculated on a per field basis by dividing the number of PECAM+, phosphohistone H3+ cells by the total number of PECAM+ cells. Nonendothelial mitotic indices were also calculated on a per field basis by dividing the number of PECAM, phosphohistone H3+ cells by the total number of PECAM cells. Data were collected from multiple fields of multiple wells and averaged for each day.

Embryo immunohistochemistry

Flt-1+/− mice maintained on the CD-1 background were intercrossed to obtain embryos. Embryos were dissected from the maternal decidua at day 8.5 (the morning of the plug is day 0.5), heads were removed and saved at −20°C for genotyping by using a modification of a published protocol,33 and the rest of the embryo was fixed in Serra fixative47 or cold 4% paraformaldehyde at 4°C overnight. The embryos were dehydrated through a methanol series and stored at −20°C in 100% methanol. Embryos were embedded in paraffin, sectioned at 10 μm on a Zeiss Microm, dewaxed in Histoclear, and rehydrated. Sections fixed in paraformaldehyde were incubated in 0.02% Protease XXIV (Sigma) in PBS for 4 minutes, then washed 3 times in PBS. After blocking in 0.25% H2O2 in PBS for 15 minutes, primary antibody (1:250 dilution in 5% goat serum/PBS) was added, and sections were incubated overnight at 4°C in humidified chambers. After 3 washes in PBS, secondary antibody (1:300 dilution of goat antirabbit or antirat IgG–horseradish peroxidase [Accurate] in 5% goat serum/PBS) was added, and incubation was overnight as before. After 3 washes in PBS, sections were incubated in 3′-diaminobenzidine tetrahydrochloride substrate to which 3 mg/mL NiSO4 was sometimes added (for blue color) for 15 minutes. Slides were rinsed in PBS, incubated in a 1:10 000 dilution of DAPI (1 mg/mL stock) in H2O for 10 minutes, mounted using Glycergel (Dako), and visualized with a Nikon Eclipse E800 microscope outfitted with DIC optics and epifluorescence. To count mitotic endothelial nuclei, alternate sections were stained with PECAM and phosphohistone H3. The DAPI-stained nuclei were used to overlay digital images.

Results

Flt-1−/−ES cell cultures have increased vascularization

ES cells undergo a differentiation program in vitro that mimics early murine yolk sac development, including primitive hematopoietic development and blood vessel formation.43,48-51Hematoendothelial development begins when a mesodermally derived hemangioblast population arises at days 2 to 3 of differentiation,52 and angioblasts can also differentiate directly from mesoderm. The endothelial cells of primitive blood vessels are differentiated from angioblasts by coexpression of PECAM and ICAM-2, both adhesion receptors of the immunoglobulin superfamily.13,53,54 We  initially investigated the cellular mechanism of flt-1 in day 8 cultures, when the PECAM+/ICAM-2+ vasculature is well established.

Flt-1+/− and flt-1−/−ES cells were engineered so that Escherichia coli lacZ is expressed under flt-1 regulatory control in the targeted gene.33 These ES cells and WT (+/+) controls were stained for β-galactosidase activity or for PECAM expression at day 8 (Figure 1). Theflt-1−/− cultures had a dramatically expanded β-galactosidase expression domain compared with theflt-1+/− cultures (Figure 1A). The β-galactosidase+ cells in theflt-1−/− cultures were found in large circular sheets, with areas of normal-looking vasculature at the edge of the sheets (Figure 1A,B, arrow). Immunofluorescent antibody staining for PECAM, ICAM-2, or VE-cadherin showed a similar pattern in theflt-1−/− cultures (Figure 1B and data not shown), suggesting that most of the β-galactosidase–expressing cells were endothelial cells. The β-galactosidase– and antibody-stained cells were elongated and interconnected, indicating that they were endothelial cells. This criterion is important, because subsets of hematopoietic cells can also react with the antibodies to PECAM or ICAM-2. Only a ring of intensely β-galactosidase+ cells (Figure 1A, arrowheads) did not appear to stain for PECAM or ICAM-2 by double-label immunofluorescent antibody staining (data not shown). These cells were found in both WT and mutant cultures, and they reacted with a flt-1 antisense RNA probe in the WT background (data not shown), indicating that they are nonvascular flt-1–expressing cells.

Fig. 1.

Flt-1−/−ES cell cultures have increased vascularization.

Day 8 differentiated flt-1−/− (A-C),flt-1+/− (D-F), and WT (G-I) cultures were processed for β-galactosidase detection (A,D,G) or reacted with an antibody to PECAM (B,E,H). A and B show one quadrant of the relatively large β-galactosidase+ (A) or PECAM+ (B) sheet of cells that characterizes the flt-1−/−phenotype. In contrast, an extensive vascular plexus is found in bothflt-1+/− (E) and WT (H) ES cell cultures. Arrowheads (A) outline an intensely stained β-galactosidase+ ring of cells that surrounds most of the β-galactosidase+ cells. Arrows (A,B) point toflt-1−/− vasculature that looks WT. (C,F,I) Phase contrast images of PECAM-labeled fields in B,E,H. Magnification is ×10.

Fig. 1.

Flt-1−/−ES cell cultures have increased vascularization.

Day 8 differentiated flt-1−/− (A-C),flt-1+/− (D-F), and WT (G-I) cultures were processed for β-galactosidase detection (A,D,G) or reacted with an antibody to PECAM (B,E,H). A and B show one quadrant of the relatively large β-galactosidase+ (A) or PECAM+ (B) sheet of cells that characterizes the flt-1−/−phenotype. In contrast, an extensive vascular plexus is found in bothflt-1+/− (E) and WT (H) ES cell cultures. Arrowheads (A) outline an intensely stained β-galactosidase+ ring of cells that surrounds most of the β-galactosidase+ cells. Arrows (A,B) point toflt-1−/− vasculature that looks WT. (C,F,I) Phase contrast images of PECAM-labeled fields in B,E,H. Magnification is ×10.

The increase in endothelial cells observed in mutant cultures was quantitated in several ways (Figure 2). RNase protection analysis of day 8 cultures with a PECAM antisense RNA probe revealed that PECAM RNA levels were 2.5- to 3.3-fold higher inflt-1−/− cultures compared with WT cultures (Figure 2A). Quantitative image analysis on day 8 ICAM-2–labeled cultures used digital images of vascular immunofluorescence to determine the percentage area stained, which approximates the amount of vasculature (see “Materials and methods” section for detailed protocols). Flt-1−/− cultures exhibited nearly a 2-fold increase in ICAM-2 staining area over WT, whereasflt-1+/− cultures had essentially WT levels (Figure 2B). ICAM-2 antibody-stained cultures were also processed for fluorescent-activated cell sorting (FACS; Figure 2C).Flt-1−/−–attached cultures contained a population of ICAM-2+ cells that was significantly increased over WT levels (compare 39% with 25%, respectively), whereas flt-1+/− cultures had WT numbers of ICAM-2+ cells. Similar FACS results were obtained with antibodies to PECAM (data not shown). Taken together, these data show that the lack of flt-1 results in increased numbers of vascular endothelial cells.

Fig. 2.

Flt-1−/− ES cell cultures have increased numbers of endothelial cells.

(A) RNase protection assay using an antisense PECAM RNA probe on day 8 WT, flt-1+/−, andflt-1−/− attached cultures. Protected fragments were separated on a polyacrylamide-urea gel and quantified by using a PhosphorImager. Protected PECAM signal was normalized to a β-actin signal, and the normalized PECAM band densities forflt-1+/− and flt-1−/−samples were compared with WT (+/+) samples. Sample 1 and sample 2 are RNAs from separate differentiations. Each bar is the average of 3 experiments performed on a particular sample. (B) Quantitative image analysis of the ICAM-2+ area on day 8 WT (+/+), flt-1+/−, andflt-1−/− attached cultures. Each bar represents the average area stained with ICAM-2 antibody from 3 wells. This experiment was repeated (data not shown), and similar quantitative trends were obtained. (C) Fluorescent cell sorting of ICAM-2–labeled day 8 WT (+/+), flt-1+/−, andflt-1−/− ES cell cultures. The plots in dotted lines are controls without primary antibody.

Fig. 2.

Flt-1−/− ES cell cultures have increased numbers of endothelial cells.

(A) RNase protection assay using an antisense PECAM RNA probe on day 8 WT, flt-1+/−, andflt-1−/− attached cultures. Protected fragments were separated on a polyacrylamide-urea gel and quantified by using a PhosphorImager. Protected PECAM signal was normalized to a β-actin signal, and the normalized PECAM band densities forflt-1+/− and flt-1−/−samples were compared with WT (+/+) samples. Sample 1 and sample 2 are RNAs from separate differentiations. Each bar is the average of 3 experiments performed on a particular sample. (B) Quantitative image analysis of the ICAM-2+ area on day 8 WT (+/+), flt-1+/−, andflt-1−/− attached cultures. Each bar represents the average area stained with ICAM-2 antibody from 3 wells. This experiment was repeated (data not shown), and similar quantitative trends were obtained. (C) Fluorescent cell sorting of ICAM-2–labeled day 8 WT (+/+), flt-1+/−, andflt-1−/− ES cell cultures. The plots in dotted lines are controls without primary antibody.

Lack of flt-1 leads to increased endothelial cell division

To investigate the cellular mechanism(s) responsible for the increased vascularization seen in the absence of flt-1, the hypothesis that flt-1−/− endothelial cells have a higher rate of cell division than WT endothelial cells was tested. Day 6 and day 7 ES cell cultures were labeled with antibodies to the vascular marker PECAM and to the mitotic marker phosphohistone H3,55 then stained with a DNA-binding dye (topro-3; Figure3). Visual observation suggested that day 6 and day 7 flt-1−/− ES cell cultures had more PECAM+ cells that colabeled with the antiphosphohistone H3 antibody than WT controls (compare Figure 3A,C with B,D and E with F).

Fig. 3.

Flt-1−/− ES cell cultures have mitotic endothelial cells.

Day 7 (A-D) or day 6 (E,F) WT (A,C,E), andflt-1−/− (B,D,F) attached cultures were labeled with antibodies to PECAM (green) and phosphohistone H3 (red), then stained with the nuclear marker topro-3 (blue). The arrowhead (B) shows a phosphohistone H3+ nonendothelial cell (PECAM), whereas the arrow (B) points to a phosphohistone H3+ endothelial cell (PECAM+). Notice the increase in phosphohistone H3+/PECAM+ cells inflt-1−/− cultures relative to WT cultures. All panels are confocal images at ×40 magnification.

Fig. 3.

Flt-1−/− ES cell cultures have mitotic endothelial cells.

Day 7 (A-D) or day 6 (E,F) WT (A,C,E), andflt-1−/− (B,D,F) attached cultures were labeled with antibodies to PECAM (green) and phosphohistone H3 (red), then stained with the nuclear marker topro-3 (blue). The arrowhead (B) shows a phosphohistone H3+ nonendothelial cell (PECAM), whereas the arrow (B) points to a phosphohistone H3+ endothelial cell (PECAM+). Notice the increase in phosphohistone H3+/PECAM+ cells inflt-1−/− cultures relative to WT cultures. All panels are confocal images at ×40 magnification.

To quantitate the apparent increase in mitotic PECAM+ cells in flt-1−/− cultures, confocal images from day 6 or day 7 fixed cultures processed as in Figure 3 were used to calculate cell counts and mitotic indices for both endothelial and nonendothelial cell populations (Table 1and Figure 4; see “Materials and methods” section for details). In all cases endothelial cells of theflt-1−/− cultures had a higher mitotic index than WT endothelial cells. To control for differential growth rates, a nonendothelial cell mitotic index was obtained for each experiment (Table 1). There was little difference between WT andflt-1−/− nonendothelial cell mitotic indices within a given experiment, in contrast to increases in theflt-1−/− endothelial cell mitotic index. Each endothelial cell mitotic index was normalized to its companion nonendothelial cell mitotic index (Figure 4; Table 1, far right column). Day 6 flt-1−/− cultures had normalized endothelial cell mitotic indices that were 3- to 4-fold higher than normal, and similar but less dramatic trends were observed in day 7 cultures (Figure 4, compare black bars with gray bars). These results indicate that the increased vascularization seen in day 8flt-1−/− ES cell cultures is caused, at least in part, by an increased endothelial cell division rate in the absence of flt-1.

Table 1.

Comparison of endothelial and nonendothelial mitotic indices in wild type and flt-1−/− embryonic stem cell cultures

Cell no.*Mitotic indexEI/NEI × 1001-155
EndothelialNonendothelial1-153EndothelialNonendothelial
Day 6      
 WT Exp 1 1 543 4 006 2.31 1.61 143.5 
−/−Exp 1 2 719 2 052 4.02 1.22 329.5  
 WT Exp 2 1 050 3 060 2.66 1.99 133.7 
−/−Exp 2 1 072 2 374 6.14 1.32 465.2  
Day 7      
 WT Exp 1 3 554 11 303 1.88 1.78 105.6 
−/−Exp 1 4 797 7 820 2.83 2.10 134.8  
 WT Exp 2 1 777 6 498 1.18 1.24 95.2 
−/−Exp 2 3 216 4 703 3.25 1.02 318.6 
Cell no.*Mitotic indexEI/NEI × 1001-155
EndothelialNonendothelial1-153EndothelialNonendothelial
Day 6      
 WT Exp 1 1 543 4 006 2.31 1.61 143.5 
−/−Exp 1 2 719 2 052 4.02 1.22 329.5  
 WT Exp 2 1 050 3 060 2.66 1.99 133.7 
−/−Exp 2 1 072 2 374 6.14 1.32 465.2  
Day 7      
 WT Exp 1 3 554 11 303 1.88 1.78 105.6 
−/−Exp 1 4 797 7 820 2.83 2.10 134.8  
 WT Exp 2 1 777 6 498 1.18 1.24 95.2 
−/−Exp 2 3 216 4 703 3.25 1.02 318.6 

EI, endothelial index; NEI, nonendothelial index; WT, wild type; Exp, experiment.

*

Total number of nuclei counted.

Percentage of replicating cells as determined by labeling with the mitotic marker phosphohistone H3.

Number of nuclei with PECAM-labeling along the cell border.

F1-153

Nuclei of cells that did not exhibit PECAM-labeling.

F1-155

Formula used to express each endothelial index as a percentage of its companion nonendothelial index.

Fig. 4.

Flt-1−/− ES cell cultures have an elevated endothelial cell mitotic index.

Days 6 and 7 WT (+/+) and flt-1−/−triple-labeled images were used to calculate nonendothelial cell and endothelial cell mitotic indices for 2 separate differentiation experiments (Table 1). Endothelial cell mitotic indices were expressed as a percentage of the nonendothelial cell mitotic index calculated for each experimental condition. The dotted black line represents the nonendothelial mitotic index for each experiment converted to 100%, and it was used as a baseline for comparison of endothelial mitotic indices.

Fig. 4.

Flt-1−/− ES cell cultures have an elevated endothelial cell mitotic index.

Days 6 and 7 WT (+/+) and flt-1−/−triple-labeled images were used to calculate nonendothelial cell and endothelial cell mitotic indices for 2 separate differentiation experiments (Table 1). Endothelial cell mitotic indices were expressed as a percentage of the nonendothelial cell mitotic index calculated for each experimental condition. The dotted black line represents the nonendothelial mitotic index for each experiment converted to 100%, and it was used as a baseline for comparison of endothelial mitotic indices.

If aberrant endothelial cell division contributes to theflt-1 mutant phenotype, then blocking cell division during ES cell differentiation may affect the phenotype. Thus, day 6 ES cell cultures were treated with the replication inhibitor mitomycin C before incubation for an additional 2 days (Figure5). Untreatedflt-1−/− cultures fixed on day 6 had slightly increased numbers of PECAM+ cells compared with day 6 WT cultures (compare Figure 5A with B). Treated day 8flt-1−/− had half as much vasculature as untreated genotype-matched controls, accompanied by a dramatic decrease in the labeling of nuclei with antiphosphohistone H3 (compare Figure 5D with F,H). In some cases, mitomycin C-treatedflt-1−/− vasculature at day 8 was branched and appeared WT in morphology (Figure 5G), suggesting that blocking cell division during days 6 to 8 of differentiation can partially compensate for the lack of flt-1 in vascular development. Mitomycin C treatment also affected vascular growth in WT cultures, which is predicted because blood vessel formation requires endothelial cell division. The treated WT cultures had 2- to 3-fold less vasculature and less branching than untreated controls (compare Figure 5C with E,H). Thus, treatment with mitomycin C, an inhibitor of replication, partially rescues the flt-1 mutant vascular phenotype.

Fig. 5.

Mitomycin C treatment partially rescues theflt-1−/− vascular phenotype.

Day 6 ES cell cultures were fixed (A,B), left untreated (C,D), or treated with mitomycin C (E-G). Some cultures (C-G) were differentiated for an additional 48 hours. Cultures were labeled with an antibody to PECAM (green), and some cultures (C-F) were also labeled with the mitotic marker antiphosphohistone H3 (red). Notice the abundance of phosphohistone H3–labeled figures in untreated (C-D) cultures compared with treated (E-F) cultures. (G) Example of a treatedflt-1−/− culture that morphologically resembled WT vasculature. (H) Quantitative image analysis of the PECAM+ area of day 8 WT (+/+) andflt-1−/− (−/−) cultures treated with mitomycin C (red) or left untreated (green). Each bar represents the average stained area from at least 3 wells stained with PECAM antibody. Magnification was ×10 except C (×20) and G (×4).

Fig. 5.

Mitomycin C treatment partially rescues theflt-1−/− vascular phenotype.

Day 6 ES cell cultures were fixed (A,B), left untreated (C,D), or treated with mitomycin C (E-G). Some cultures (C-G) were differentiated for an additional 48 hours. Cultures were labeled with an antibody to PECAM (green), and some cultures (C-F) were also labeled with the mitotic marker antiphosphohistone H3 (red). Notice the abundance of phosphohistone H3–labeled figures in untreated (C-D) cultures compared with treated (E-F) cultures. (G) Example of a treatedflt-1−/− culture that morphologically resembled WT vasculature. (H) Quantitative image analysis of the PECAM+ area of day 8 WT (+/+) andflt-1−/− (−/−) cultures treated with mitomycin C (red) or left untreated (green). Each bar represents the average stained area from at least 3 wells stained with PECAM antibody. Magnification was ×10 except C (×20) and G (×4).

Flt-1 mutation affects division of vascular precursor cells

To determine when the flt-1 mutation first affects vascular development, we investigated earlier time points of ES cell differentiation. To establish when cells expressing lacZ under control of the flt-1 promoter were first affected by the lack of flt-1 protein, we analyzed an early time course of ES cell differentiation. We plated cells directly after dispase treatment, then processed wells of each genotype for lacZ expression on days 2 to 6 of differentiation (Figure 6). The percentage of lacZ-expressing cells was equivalent betweenflt-1+/− and flt-1−/−cultures on days 2 to 4, and only on day 5 was there a significant increase in the percentage of lacZ-expressing cells in the flt-1 mutant background (Figure 6A). To determine if this expansion was the result of aberrant cell division, wells were treated with mitomycin C on day 4 or day 5, then compared with control untreated wells 24 hours later. Day 5 flt-1−/− mutant cultures treated with mitomycin C 24 hours earlier had fewer lacZ-expressing cells than paired untreated controls (compare Figure 6C with D). The day 5 mitomycin C-treated wells were, in fact, similar to untreated wells fixed at day 4 (compare Figure 6B with C). Day 6flt-1−/− mutant cultures treated with mitomycin C 24 hours earlier also had fewer lacZ-expressing cells than paired untreated controls (compare Figure 6E with F). These results show that the earliest expansion of lacZ-expressing cells in theflt-1−/− mutant cultures can be inhibited by mitomycin C, suggesting that the expansion results from aberrant cell division.

Fig. 6.

Mitomycin C–sensitive expansion of β-galactosidase–expressing cells in flt-1−/− ES cell cultures at earlier times.

(A) Quantitative image analysis of the β-galactosidase+areas of flt-1+/− (light blue bars) andflt-1−/− (dark blue bars) ES cell cultures on days 2 to 5 of in vitro differentiation. For days 2 and 3, the bars represent the average β-galactosidase+ area for 9 individual attached ES cell clumps. For days 4 and 5, the bars represent the average β-galactosidase+ area for 2 culture wells. The asterisk (*) indicates significance atP < .001. (B-F) Days 4 to 6flt-1−/− ES cell cultures untreated (B,D,F) or treated with mitomycin C (C,E) and stained for β-galactosidase activity. (B) Day 4 flt-1+/− culture. (C) Day 5flt-1−/− culture treated on day 4 with mitomycin C. Note decrease in stained area relative to (D) untreated day 5 flt-1−/− culture. (E) Day 6flt-1−/− culture treated on day 5 with mitomycin C. Note decrease in stained area relative to (F) untreated day 6 flt-1−/− culture. Original magnification, ×20.

Fig. 6.

Mitomycin C–sensitive expansion of β-galactosidase–expressing cells in flt-1−/− ES cell cultures at earlier times.

(A) Quantitative image analysis of the β-galactosidase+areas of flt-1+/− (light blue bars) andflt-1−/− (dark blue bars) ES cell cultures on days 2 to 5 of in vitro differentiation. For days 2 and 3, the bars represent the average β-galactosidase+ area for 9 individual attached ES cell clumps. For days 4 and 5, the bars represent the average β-galactosidase+ area for 2 culture wells. The asterisk (*) indicates significance atP < .001. (B-F) Days 4 to 6flt-1−/− ES cell cultures untreated (B,D,F) or treated with mitomycin C (C,E) and stained for β-galactosidase activity. (B) Day 4 flt-1+/− culture. (C) Day 5flt-1−/− culture treated on day 4 with mitomycin C. Note decrease in stained area relative to (D) untreated day 5 flt-1−/− culture. (E) Day 6flt-1−/− culture treated on day 5 with mitomycin C. Note decrease in stained area relative to (F) untreated day 6 flt-1−/− culture. Original magnification, ×20.

Because both endothelial cells and a nonendothelial cell population express flt-1 promoter-driven β-galactosidase, we investigated the expression of several vascular markers in the ES cell cultures. Cultures were stained with PECAM or VE-cadherin from days 2 to 6 of differentiation (Figure 7), because both markers are expressed early in vascular development. PECAM was expressed throughout the time course, but before day 5 only clumps of PECAM+ cells were seen, and no significant differences were seen among the different genotypes (data not shown). By day 5 both WT and flt-1+/− cultures had some areas of PECAM+ vasculature, but surprisingly theflt-1−/− mutant day 5 cultures had few PECAM+ cells and most were still in clumps (Figure 7A-C). By day 6 all cultures had PECAM+ vasculature, and theflt-1−/− mutant cultures had as much or more PECAM+ vessels compared with WT orflt-1+/− cultures (Figure 7G-I). Treatment offlt-1−/− cultures from days 5 to 6 with mitomycin C reduced the number of PECAM+ cells (data not shown). VE-cadherin+ cells were not seen in any cultures until day 5 (data not shown). Similar to the PECAM pattern, on day 5 WT and flt-1+/− cultures had VE-cadherin+ vasculature, whereas theflt-1−/− mutant cultures had only a few VE-cadherin+ cells that were not organized into vessels (Figure 7D-F). By day 6 cultures of all genotypes had VE-cadherin+ vessels (Figure 7J-L). These results show thatflt-1−/− mutant cultures did not have expansion of either PECAM+ or VE-cadherin+vascular cells until between days 5 and 6 of differentiation, when expansion of both β-galactosidase–expressing cells and PECAM-expressing cells was sensitive to mitomycin C.

Fig. 7.

Expression of vascular markers in differentiating ES cell cultures.

Wt (+/+) (A,D,G,J), flt-1+/−(B,E,H,K), and flt-1−/− (C,F,I,L) ES cell cultures were fixed on day 5 (A-F) or day 6 (G-L) and labeled with antibodies to PECAM (A-C,G-I) or VE-cadherin (D-F,J-L), and the appropriate fluorescent-labeled secondary antibody. Arrows (C,F) point to sparse PECAM+ and VE-cadherin+ cells in day 5 flt-1−/− cultures. Original magnifications ×20, except D-F at ×40.

Fig. 7.

Expression of vascular markers in differentiating ES cell cultures.

Wt (+/+) (A,D,G,J), flt-1+/−(B,E,H,K), and flt-1−/− (C,F,I,L) ES cell cultures were fixed on day 5 (A-F) or day 6 (G-L) and labeled with antibodies to PECAM (A-C,G-I) or VE-cadherin (D-F,J-L), and the appropriate fluorescent-labeled secondary antibody. Arrows (C,F) point to sparse PECAM+ and VE-cadherin+ cells in day 5 flt-1−/− cultures. Original magnifications ×20, except D-F at ×40.

Flt-1−/−embryos have increased mitoses

To determine if the aberrant endothelial cell division seen in the absence of flt-1 during ES cell differentiation also occurred in vivo, day 8.5 embryos were stained with the antiphosphohistone H3 antibody (Figure 8). Theflt-1−/− mutant embryos had numerous mitotic nuclei in several vascular areas, including the lining of yolk sac blood islands (Figure 8B,C,E,F) and the allantois (Figure 8F). In contrast, nonmutant embryos had far fewer mitotic nuclei in those areas (Figure 8A,D). The increase in mitotic nuclei was specific to vascular areas in vivo, because embryonic structures such as the neural tube and somites had roughly equivalent numbers of mitotic nuclei regardless of the genetic background (data not shown). Digital overlays of alternate sections stained with PECAM and phosphohistone H3 (Figure 8D-F) were used to calculate the endothelial mitotic indices in vivo. The endothelial mitotic index of flt-1−/− embryos was 2.8% (n = 1270), double that of WT+/+ embryos whose endothelial mitotic index was 1.4% (n = 425). Thus, the aberrant endothelial cell division documented during ES cell differentiation in the absence of flt-1 is also a hallmark of the mutant phenotype in vivo.

Fig. 8.

Flt-1 −/− embryos have increased mitoses in endothelial cells.

Transverse sections of day 8.5 embryos were processed for immunohistochemistry by using antiphosphohistone H3 to detect mitotic nuclei (A-C), and overlays of adjacent sections were processed individually (see “Materials and methods” section) for immunohistochemistry with antiphospohistone H3 (red), anti-PECAM (green), and DAPI (blue) (D-F). Visualization of yolk sacs offlt-1+/− (A) or WT (+/+) (D) embryos that were phenotypically normal showed few mitotic nuclei in vascular areas (arrow in D). In contrast,flt-1−/− embryos (B,C,E,F) exhibited vascular overgrowth and numerous mitotic nuclei (red; E,F) in PECAM+regions (green; E,F) of the yolk sac and allantois (F, left part of panel). (A-C) Asterisks denote the lumina of blood islands in the yolk sac, and arrows point to mitotic nuclei abutting the endoderm with the long axis perpendicular to the long axis of the endoderm cells, a characteristic of dividing endothelial cells. In contrast, the arrowhead in C points to a mitotic nucleus in the endoderm with the long axis parallel to the long axis of the endoderm cells, a characteristic of dividing endoderm. The arrowhead in B points to a mitotic nucleus of unknown cell type. (D-F) Arrows point to mitotic nuclei of PECAM+ cells. En, visceral endoderm of the yolk sac.

Fig. 8.

Flt-1 −/− embryos have increased mitoses in endothelial cells.

Transverse sections of day 8.5 embryos were processed for immunohistochemistry by using antiphosphohistone H3 to detect mitotic nuclei (A-C), and overlays of adjacent sections were processed individually (see “Materials and methods” section) for immunohistochemistry with antiphospohistone H3 (red), anti-PECAM (green), and DAPI (blue) (D-F). Visualization of yolk sacs offlt-1+/− (A) or WT (+/+) (D) embryos that were phenotypically normal showed few mitotic nuclei in vascular areas (arrow in D). In contrast,flt-1−/− embryos (B,C,E,F) exhibited vascular overgrowth and numerous mitotic nuclei (red; E,F) in PECAM+regions (green; E,F) of the yolk sac and allantois (F, left part of panel). (A-C) Asterisks denote the lumina of blood islands in the yolk sac, and arrows point to mitotic nuclei abutting the endoderm with the long axis perpendicular to the long axis of the endoderm cells, a characteristic of dividing endothelial cells. In contrast, the arrowhead in C points to a mitotic nucleus in the endoderm with the long axis parallel to the long axis of the endoderm cells, a characteristic of dividing endoderm. The arrowhead in B points to a mitotic nucleus of unknown cell type. (D-F) Arrows point to mitotic nuclei of PECAM+ cells. En, visceral endoderm of the yolk sac.

Discussion

Our data support a model whereby flt-1 normally affects early vascular development by negatively modulating cell division in the vascular lineage. The identification of this cellular mechanism of flt-1 action suggests that flt-1 is critical for the fine tuning of VEGF-mediated vessel growth that is required to form proper blood vessels. It also strongly suggests that flt-1 may affect blood vessel formation in similar ways in both the embryo and the adult. Embryos and differentiated ES cells lacking flt-1 have increased vascularization and numbers of endothelial cells accompanied by an increased endothelial cell mitotic index. In contrast, the nonendothelial cell mitotic index is similar in both genetic backgrounds, indicating that the increased mitotic rate in the flt-1−/−background is endothelial cell specific.

The ability of mitomycin C to partially rescue theflt-1−/− vascular phenotype further supports the conclusion that deregulated endothelial cell division is responsible for the flt-1 mutant phenotype. The WT cultures were also affected, which was expected because endothelial cell division is a critical component of normal blood vessel formation.56 A caveat is that mitomycin C inhibits division in all cells, so lack of division in nonendothelial cells could indirectly affect the endothelial cell phenotype. This scenario cannot be ruled out, but the increased endothelial mitotic index in theflt-1 mutant background and its diminution with mitomycin C suggest that a substantial part of the rescue is likely to result from direct effects on endothelial cell division. This model can be more precisely tested by expressing genes that modulate cell division under the control of endothelial-specific regulatory sequences in the mutant ES cells.

Flt-1 modulates cell division in the vascular lineage at the earliest stages of vascular development. The first documented difference in ES cell cultures was at day 5, when flt-1−/−mutant cultures had more cells expressing β-galactosidase under control of the flt-1 promoter than flt-1+/−cultures. The exact identity of these cells is unclear because we have identified a nonvascular, flt-1–expressing cell population in ES cell cultures, and several cell types such as trophoblasts and monocyte/macrophages express flt-1 in vivo.40,57-59However, because endothelial cells also express flt-1, it is likely that at least a subpopulation of these cells are vascular precursor cells. In any case, the expansion of β-galactosidase–expressing cells in the flt-1−/− mutant background could be blocked by mitomycin C from days 4 to 5 onward, indicating that the expansion of this cell population resulted from aberrant cell division. Interestingly, vascular cells expressing PECAM and/or VE-cadherin were much less prevalent in the flt-1−/− mutant cultures on day 5, suggesting that different subpopulations of vascular precursor cells may be affected by the flt-1 mutation at different times. The expansion of PECAM+ and/or VE-cadherin+ vascular cells was not evident until day 6 in the flt-1−/− mutant background, and this expansion was also blocked by mitomycin C. Thus, flt-1 has a major role in modulating cell division in the vascular lineage starting at days 4 to 5 of ES cell differentiation, just before formation of the first primitive blood vessels.

Other processes can also affect the number of endothelial cells, including cell fate decisions and programmed cell death. Appreciable endothelial cell death is not observed during days 5 to 8 of normal ES cell differentiation (V.L.B., unpublished observation), so inhibition of apoptosis is unlikely to make a major contribution to the flt-1 mutant phenotype. Our results do not formally exclude that, in addition to an effect on vascular cell division, flt-1 may alter cell fate by affecting hemangioblast formation,34 but our results are not consistent with this model. We see no significant differences between normal and mutant cultures until day 5, well beyond the peak of hemangioblast formation at days 2.5 to 3.0.52In the hemangioblast study, increased PECAM and β-galactosidase staining during differentiation of flt-1−/−EBs was interpreted as increased hemangioblast numbers, but the lack of a definitive hemangioblast marker makes it impossible to distinguish between hemangioblasts, angioblasts, and differentiated endothelial cells using these criteria. Moreover, in our hands the expansion of the vascular lineage was blocked by mitomycin C at its earliest detection on days 4 to 5, suggesting that the major effect of the flt-1 mutation on vascular growth results from aberrant cell division.

The identification of flt-1 as an early modulator of cell division in vascular development is consistent with several elegant studies showing that flt-1 affects endothelial cell mitogenesis in cultured endothelial cells.31,41,42 Extending this model of flt-1 action to the earliest stages of development has several implications. First, it suggests that deregulation of proliferation can be sufficient to disrupt developmental processes. Other recent investigations of the role of the cell cycle in development support this hypothesis.60 Second, the data suggest that flt-1 can modulate the endothelial cell cycle developmentally by affecting one or more molecular signaling pathways, although which pathways are affected is not entirely clear. Deletion of the flt-1 tyrosine kinase domain does not disrupt vascular development,58 suggesting that signaling through this domain is not necessary for flt-1 to affect the endothelial cell cycle developmentally. Signaling through flk-1 does produce a strong endothelial mitogenic signal, and flk-1 selective inhibitors partially rescue the flt-1−/−phenotype in ES cell cultures (D. Roberts and V.L.B., unpublished results). This finding suggests that flt-1 affects vascular development at least in part by modulating VEGF-mediated flk-1 signaling, and this modulation could occur in several ways.

A soluble form of flt-1, sflt-1, is expressed during development61 and ES cell differentiation (J.B.K. and V.L.B., unpublished results), and it can inhibit VEGF-dependent endothelial cell division.38,39 Thus, sflt-1 can bind VEGF and prevent ligand-induced dimerization of the flk-1 receptor. The full-length receptor can also theoretically form an inactive heterodimer with flk-1, as suggested by a recent study using chimeric receptors.41 In addition, ligand engagement of flt-1 may modulate flk-1 signaling at points downstream in the signal transduction pathway. This model is supported by the inhibitor sensitivity of chimeric receptors and a study implicating nitric oxide as a mediator of flt-1 effects on the flk-1 mitogenic pathway.42,62 Importantly, these models of flt-1 action are not mutually exclusive, and it is likely that flt-1 uses some combination of these actions to modulate endothelial cell division developmentally. The identification of the cellular mechanism of flt-1 action suggests ways to test these molecular models.

Flt-1−/− mutant embryos had increased mitoses in areas rich in endothelial cells and an increased mitotic index as well, indicating that aberrant endothelial cell division contributes to the mutant phenotype in vivo. To our knowledge this is the first demonstration that flt-1 affects endothelial cell division in vivo. The ability of flt-1 to negatively modulate endothelial cell division in vivo indicates that it is an endogenous negative regulator of blood vessel formation. Our data show that flt-1 regulates vascular growth from the earliest stages of vascular development, and it is likely to modulate angiogenesis in the adult organism by a similar cellular mechanism. Several molecules, such as angiostatin and endostatin, negatively regulate pathologic blood vessel formation when administered exogenously, and some of these regulators are likely to control pathologic vascularization by endogenous production.63-65However, these angiogenesis inhibitors have surprisingly little effect on normal blood vessel formation. Clearly, our knowledge of how blood vessel formation is negatively regulated is sparse compared with what is known of positive regulation.

VEGF expression is up-regulated in many pathologies with vascular components, such as cancer and chronic inflammation.66-70Thus, flt-1 could potentially negatively modulate pathologic vascularization, as described here for vascular development, and therapeutics that specifically block flt-1 action may help rather than hinder pathologic vascularization. Conversely, VEGF treatment can in some cases promote vascularization of ischemic limbs,71,72but our lack of understanding about how VEGF signaling is normally exquisitely fine-tuned has hampered our ability to produce functional vessels therapeutically. Flt-1 clearly participates in the modulation of VEGF-mediated vascular growth, and understanding the role of flt-1 in controlling this process should help in designing better therapies. In any case, defining the cellular mechanism of flt-1 action in endothelial cells developmentally suggests alternative ways to modulate blood vessel formation in vivo.

We thank Guo-Hua Fong for supplying the flt-1 mutant ES cell lines and mice and the pflt probe. We thank Bob Duronio, Anthony LaMantia, and Cam Patterson for critical reading of the manuscript; Susan Whitfield for artwork; and fellow lab members for fruitful discussion.

Supported by grants from the National Institutes of Health (HL43174) and Glaxo-Wellcome to V.L.B. V.L.B. was supported by a National Institutes of Health Career Development Award (HL02908), and J.B.K. was supported by a predoctoral fellowship from the Department of Defense (DAMD 17-00-1-0379).

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Folkman
J
D'Amore
PA
Blood vessel formation: what is its molecular basis?
Cell.
87
1996
1153
1155
2
Weinstein
BM
What guides early embryonic blood vessel formation?
Dev Dyn.
215
1999
2
11
3
Conway
EM
Collen
D
Carmeliet
P
Molecular mechanisms of blood vessel growth.
Cardiovasc Res.
49
2001
507
521
4
Orlidge
A
D'Amore
PA
Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells.
J Cell Biol.
105
1987
1455
1462
5
Madri
JA
Pratt
BM
Yannariello-Brown
J
Matrix-driven cell size change modulates aortic endothelial cell proliferation and sheet migration.
Am J Pathol.
132
1988
18
27
6
Ziats
NP
Anderson
JM
Human vascular endothelial cell attachment and growth inhibition by type V collagen.
J Vasc Surg.
17
1993
710
718
7
Podesta
F
Roth
T
Ferrara
F
Cagliero
E
Lorenzi
M
Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication.
Diabetologia.
40
1997
879
886
8
Underwood
PA
Bean
PA
Whitelock
JM
Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.
Atherosclerosis.
141
1998
141
152
9
Ferrara
N
Davis-Smyth
T
The biology of vascular endothelial growth factor.
Endocr Rev.
18
1997
4
25
10
Neufeld
G
Cohen
T
Gengrinovitch
S
Poltorak
Z
Vascular endothelial growth factor (VEGF) and its receptors.
FASEB J.
13
1999
9
22
11
Ferrara
N
Carver-Moore
K
Chen
H
et al
Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.
Nature.
380
1996
439
442
12
Carmeliet
P
Ferreira
V
Breier
G
et al
Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature.
380
1996
435
439
13
Bautch
VL
Redick
SD
Scalia
A
Harmaty
M
Carmeliet
P
Rapoport
R
Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A.
Blood.
95
2000
1979
1987
14
Miquerol
L
Langille
BL
Nagy
A
Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression.
Development.
127
2000
3941
3946
15
Drake
CJ
Little
CD
Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.
Proc Natl Acad Sci U S A.
92
1995
7657
7661
16
Lee
RJ
Springer
ML
Blanco-Bose
WE
Shaw
R
Ursell
PC
Blau
HM
VEGF gene delivery to myocardium: deleterious effects of unregulated expression.
Circulation.
102
2000
898
901
17
Springer
ML
Hortelano
G
Bouley
DM
Wong
J
Kraft
PE
Blau
HM
Induction of angiogenesis by implantation of encapsulated primary myoblasts expressing vascular endothelial growth factor.
J Gene Med.
2
2000
279
288
18
Breier
G
Albrecht
U
Sterrer
S
Risau
W
Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation.
Development.
114
1992
521
532
19
Monacci
WT
Merrill
MJ
Oldfield
EH
Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues.
Am J Physiol.
264
1993
C995
1002
20
Dumont
DJ
Fong
GH
Puri
MC
Gradwohl
G
Alitalo
K
Breitman
ML
Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development.
Dev Dyn.
203
1995
80
92
21
Miquerol
L
Gertsenstein
M
Harpal
K
Rossant
J
Nagy
A
Multiple developmental roles of VEGF suggested by a LacZ-tagged allele.
Dev Biol.
212
1999
307
322
22
Shibuya
M
Yamaguchi
S
Yamane
A
et al
Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family.
Oncogene.
5
1990
519
524
23
de Vries
C
Escobedo
JA
Ueno
H
Houck
K
Ferrara
N
Williams
LT
The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.
Science.
255
1992
989
991
24
Terman
BI
Dougher-Vermazen
M
Carrion
ME
et al
Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.
Biochem Biophys Res Commun.
187
1992
1579
1586
25
Quinn
TP
Peters
KG
De Vries
C
Ferrara
N
Williams
LT
Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium.
Proc Natl Acad Sci U S A.
90
1993
7533
7537
26
Shibuya
M
Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1).
Int J Biochem Cell Biol.
33
2001
409
420
27
Shalaby
F
Rossant
J
Yamaguchi
TP
et al
Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.
Nature.
376
1995
62
66
28
Shalaby
F
Ho
J
Stanford
WL
et al
A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.
Cell.
89
1997
981
990
29
Waltenberger
J
Claesson-Welsh
L
Siegbahn
A
Shibuya
M
Heldin
CH
Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.
J Biol Chem.
269
1994
26988
26995
30
Bernatchez
PN
Soker
S
Sirois
MG
Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent.
J Biol Chem.
274
1999
31047
31054
31
Kanno
S
Oda
N
Abe
M
et al
Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.
Oncogene.
19
2000
2138
2146
32
Gille
H
Kowalski
J
Li
B
et al
Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.
J Biol Chem.
276
2001
3222
3230
33
Fong
GH
Rossant
J
Gertsenstein
M
Breitman
ML
Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.
Nature.
376
1995
66
70
34
Fong
GH
Zhang
L
Bryce
DM
Peng
J
Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice.
Development.
126
1999
3015
3025
35
Plate
KH
Breier
G
Weich
HA
Risau
W
Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.
Nature.
359
1992
845
848
36
Brown
LF
Berse
B
Jackman
RW
et al
Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract.
Cancer Res.
53
1993
4727
4735
37
Barleon
B
Hauser
S
Schollmann
C
et al
Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells.
J Cell Biochem.
54
1994
56
66
38
Kendall
RL
Thomas
KA
Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.
Proc Natl Acad Sci U S A.
90
1993
10705
10709
39
Kendall
RL
Wang
G
Thomas
KA
Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR.
Biochem Biophys Res Commun.
226
1996
324
328
40
Ahmed
A
Dunk
C
Kniss
D
Wilkes
M
Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells.
Lab Invest.
76
1997
779
791
41
Rahimi
N
Dayanir
V
Lashkari
K
Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells.
J Biol Chem.
275
2000
16986
16992
42
Zeng
H
Dvorak
HF
Mukhopadhyay
D
Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) receptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways.
J Biol Chem.
276
2001
26969
26979
43
Bautch
VL
Stanford
WL
Rapoport
R
Russell
S
Byrum
RS
Futch
TA
Blood island formation in attached cultures of murine embryonic stem cells.
Dev Dyn.
205
1996
1
12
44
Hogan
B
Beddington
R
Constantini
F
Lacy
E
Manipulating the Mouse Embryo: A Laboratory Manual.
2nd edition.
1994
Cold Spring Harbor Laboratory Press
Plainview, NY
45
Chirgwin
JM
Przybyla
AE
MacDonald
RJ
Rutter
WJ
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry.
18
1979
5294
5299
46
Melton
DA
Krieg
PA
Rebagliati
MR
Maniatis
T
Zinn
K
Green
MR
Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.
Nucl Acids Res.
12
1984
7035
7056
47
Serra
JA
Histochemical tests for protein and amino acids: the characterization of basic proteins.
Stain Technol.
21
1946
5
18
48
Risau
W
Sariola
H
Zerwes
HG
et al
Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies.
Development.
102
1988
471
478
49
Wang
R
Clark
R
Bautch
VL
Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development.
Development.
114
1992
303
316
50
Vittet
D
Prandini
MH
Berthier
R
et al
Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.
Blood.
88
1996
3424
3431
51
Wiles
MV
Keller
G
Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture.
Development.
111
1991
259
267
52
Choi
K
Kennedy
M
Kazarov
A
Papadimitriou
JC
Keller
G
A common precursor for hematopoietic and endothelial cells.
Development.
125
1998
725
732
53
Newman
PJ
Berndt
MC
Gorski
J
et al
PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.
Science.
247
1990
1219
1222
54
Xu
H
Bickford
JK
Luther
E
Carpenito
C
Takei
F
Springer
TA
Characterization of murine intercellular adhesion molecule-2.
J Immunol.
156
1996
4909
4914
55
Hendzel
MJ
Wei
Y
Mancini
MA
et al
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
Chromosoma.
106
1997
348
360
56
Clark
ER
Clark
EL
Microscopic observations on the growth of blood capillaries in the living mammal.
Am J Anat.
64
1939
251
299
57
Ahmed
A
Li
XF
Dunk
C
Whittle
MJ
Rushton
DI
Rollason
T
Colocalisation of vascular endothelial growth factor and its Flt-1 receptor in human placenta.
Growth Factors.
12
1995
235
243
58
Hiratsuka
S
Minowa
O
Kuno
J
Noda
T
Shibuya
M
Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice.
Proc Natl Acad Sci U S A.
95
1998
9349
9354
59
Sawano
A
Iwai
S
Sakurai
Y
et al
Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans.
Blood.
97
2001
785
791
60
Myster
DL
Duronio
RJ
To differentiate or not to differentiate?
Curr Biol.
10
2000
R302
304
61
Carmeliet
P
Moons
L
Luttun
A
et al
Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.
Nat Med.
7
2001
575
583
62
Bussolati
B
Dunk
C
Grohman
M
Kontos
CD
Mason
J
Ahmed
A
Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.
Am J Pathol.
159
2001
993
1008
63
O'Reilly
MS
Holmgren
L
Shing
Y
et al
Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.
Cell.
79
1994
315
328
64
O'Reilly
MS
Boehm
T
Shing
Y
et al
Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.
Cell.
88
1997
277
285
65
Cao
Y
Endogenous angiogenesis inhibitors: angiostatin, endostatin, and other proteolytic fragments.
Prog Mol Subcell Biol.
20
1998
161
176
66
Ito
A
Hirota
S
Mizuno
H
et al
Expression of vascular permeability factor (VPF/VEGF) messenger RNA by plasma cells: possible involvement in the development of edema in chronic inflammation.
Pathol Int.
45
1995
715
720
67
Dvorak
HF
Detmar
M
Claffey
KP
Nagy
JA
van de Water
L
Senger
DR
Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation.
Int Arch Allergy Immunol.
107
1995
233
235
68
Proescholdt
MA
Heiss
JD
Walbridge
S
et al
Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain.
J Neuropathol Exp Neurol.
58
1999
613
627
69
Brown
LF
Berse
B
Jackman
RW
et al
Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.
Hum Pathol.
26
1995
86
91
70
Viglietto
G
Maglione
D
Rambaldi
M
et al
Upregulation of vascular endothelial growth factor (VEGF) and downregulation of placenta growth factor (PlGF) associated with malignancy in human thyroid tumors and cell lines.
Oncogene.
11
1995
1569
1579
71
Takeshita
S
Zheng
LP
Brogi
E
et al
Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.
J Clin Invest.
93
1994
662
670
72
Isner
JM
Pieczek
A
Schainfeld
R
et al
Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb.
Lancet.
348
1996
370
374

Author notes

Victoria L. Bautch, CB# 3280, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; e-mail:bautch@med.unc.edu.