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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Program in Genetics and Molecular Biology,
Department of Biology, University of North Carolina at Chapel Hill,
Chapel Hill.
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 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+/ 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 Thus, we asked if flt-1 could negatively modulate endothelial
mitogenesis developmentally, and to address this question we analyzed
the cellular mechanism responsible for the
flt-1 Cell culture and in vitro differentiation
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
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- 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,
 -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-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.
Flt-1 Flt-1+/
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 in
flt-1
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; Figure
3). 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).
To quantitate the apparent increase in mitotic PECAM+ cells
in flt-1
If aberrant endothelial cell division contributes to the
flt-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 (Figure
5). Untreated flt-1
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 between flt-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 6 flt-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 the
flt-1/ mutant cultures can be inhibited by
mitomycin C, suggesting that the expansion results from aberrant
cell division.
Because both endothelial cells and a nonendothelial cell population
express flt-1 promoter-driven
Flt-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.
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 The ability of mitomycin C to partially rescue the
flt-1 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 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 3.0.52
In the hemangioblast study, increased PECAM and 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 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 VEGF expression is up-regulated in many pathologies with vascular components, such as cancer and chronic inflammation.66-70 Thus, 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,71,72 but 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.
Submitted May 18, 2001; accepted November 8, 2001.
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.
Reprints: Victoria L. Bautch, CB# 3280, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; e-mail: bautch{at}med.unc.edu.
1. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell. 1996;87:1153-1155[CrossRef][Medline] [Order article via Infotrieve]. 2. Weinstein BM. What guides early embryonic blood vessel formation? Dev Dyn. 1999;215:2-11[CrossRef][Medline] [Order article via Infotrieve].
3.
Conway EM, Collen D, Carmeliet P.
Molecular mechanisms of blood vessel growth.
Cardiovasc Res.
2001;49:507-521
4.
Orlidge A, D'Amore PA.
Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells.
J Cell Biol.
1987;105:1455-1462 5. Madri JA, Pratt BM, Yannariello-Brown J. Matrix-driven cell size change modulates aortic endothelial cell proliferation and sheet migration. Am J Pathol. 1988;132:18-27[Abstract]. 6. Ziats NP, Anderson JM. Human vascular endothelial cell attachment and growth inhibition by type V collagen. J Vasc Surg. 1993;17:710-718[CrossRef][Medline] [Order article via Infotrieve]. 7. Podesta F, Roth T, Ferrara F, Cagliero E, Lorenzi M. Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication. Diabetologia. 1997;40:879-886[CrossRef][Medline] [Order article via Infotrieve]. 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. 1998;141:141-152[CrossRef][Medline] [Order article via Infotrieve].
9.
Ferrara N, Davis-Smyth T.
The biology of vascular endothelial growth factor.
Endocr Rev.
1997;18:4-25
10.
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z.
Vascular endothelial growth factor (VEGF) and its receptors.
FASEB J.
1999;13:9-22 11. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439-442[CrossRef][Medline] [Order article via Infotrieve]. 12. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435-439[CrossRef][Medline] [Order article via Infotrieve].
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.
2000;95:1979-1987 14. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development. 2000;127:3941-3946[Abstract].
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.
1995;92:7657-7661
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.
2000;102:898-901 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. 2000;2:279-288[CrossRef][Medline] [Order article via Infotrieve]. 18. Breier G, Albrecht U, Sterrer S, Risau W. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development. 1992;114:521-532[Abstract].
19.
Monacci WT, Merrill MJ, Oldfield EH.
Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues.
Am J Physiol.
1993;264: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. 1995;203:80-92[Medline] [Order article via Infotrieve]. 21. Miquerol L, Gertsenstein M, Harpal K, Rossant J, Nagy A. Multiple developmental roles of VEGF suggested by a LacZ-tagged allele. Dev Biol. 1999;212:307-322[CrossRef][Medline] [Order article via Infotrieve]. 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. 1990;5:519-524[Medline] [Order article via Infotrieve].
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.
1992;255:989-991 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. 1992;187:1579-1586[CrossRef][Medline] [Order article via Infotrieve].
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.
1993;90:7533-7537 26. Shibuya M. Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol. 2001;33:409-420[CrossRef][Medline] [Order article via Infotrieve]. 27. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995;376:62-66[CrossRef][Medline] [Order article via Infotrieve]. 28. Shalaby F, Ho J, Stanford WL, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997;89:981-990[CrossRef][Medline] [Order article via Infotrieve].
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.
1994;269:26988-26995
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.
1999;274:31047-31054 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. 2000;19:2138-2146[CrossRef][Medline] [Order article via Infotrieve].
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.
2001;276:3222-3230 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. 1995;376:66-70[CrossRef][Medline] [Order article via Infotrieve]. 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. 1999;126:3015-3025[Abstract]. 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. 1992;359:845-848[CrossRef][Medline] [Order article via Infotrieve].
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.
1993;53:4727-4735 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. 1994;54:56-66[CrossRef][Medline] [Order article via Infotrieve].
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.
1993;90:10705-10709 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. 1996;226:324-328[CrossRef][Medline] [Order article via Infotrieve]. 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. 1997;76:779-791[Medline] [Order article via Infotrieve].
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.
2000;275:16986-16992
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.
2001;276:26969-26979 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. 1996;205:1-12[CrossRef][Medline] [Order article via Infotrieve]. 44. Hogan B, Beddington R, Constantini F, Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd edition. Plainview, NY: Cold Spring Harbor Laboratory Press; 1994. 45. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299[CrossRef][Medline] [Order article via Infotrieve].
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.
1984;12:7035-7056 47. Serra JA. Histochemical tests for protein and amino acids: the characterization of basic proteins. Stain Technol. 1946;21:5-18. 48. Risau W, Sariola H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development. 1988;102:471-478[Abstract]. 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. 1992;114:303-316[Abstract].
50.
Vittet D, Prandini MH, Berthier R, et al.
Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.
Blood.
1996;88:3424-3431 51. Wiles MV, Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 1991;111:259-267[Abstract]. 52. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998;125:725-732[Abstract].
53.
Newman PJ, Berndt MC, Gorski J, et al.
PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.
Science.
1990;247:1219-1222 54. Xu H, Bickford JK, Luther E, Carpenito C, Takei F, Springer TA. Characterization of murine intercellular adhesion molecule-2. J Immunol. 1996;156:4909-4914[Abstract]. 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. 1997;106:348-360[CrossRef][Medline] [Order article via Infotrieve]. 56. Clark ER, Clark EL. Microscopic observations on the growth of blood capillaries in the living mammal. Am J Anat. 1939;64:251-299[CrossRef]. 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. 1995;12:235-243[Medline] [Order article via Infotrieve].
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.
1998;95:9349-9354
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.
2001;97:785-791 60. Myster DL, Duronio RJ. To differentiate or not to differentiate? Curr Biol. 2000;10:R302-304[CrossRef][Medline] [Order article via Infotrieve]. 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. 2001;7:575-583[CrossRef][Medline] [Order article via Infotrieve].
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.
2001;159:993-1008 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. 1994;79:315-328[CrossRef][Medline] [Order article via Infotrieve]. 64. O'Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997;88:277-285[CrossRef][Medline] [Order article via Infotrieve]. 65. Cao Y. Endogenous angiogenesis inhibitors: angiostatin, endostatin, and other proteolytic fragments. Prog Mol Subcell Biol. 1998;20:161-176[Medline] [Order article via Infotrieve]. 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. 1995;45:715-720[Medline] [Order article via Infotrieve]. 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. 1995;107:233-235[CrossRef][Medline] [Order article via Infotrieve]. 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. 1999;58:613-627[Medline] [Order article via Infotrieve]. 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. 1995;26:86-91[CrossRef][Medline] [Order article via Infotrieve]. 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. 1995;11:1569-1579[Medline] [Order article via Infotrieve]. 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. 1994;93:662-670. 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. 1996;348:370-374[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  S.-W. Jin and C. Patterson The Opening Act: Vasculogenesis and the Origins of Circulation Arterioscler. Thromb. Vasc. Biol., May 1, 2009; 29(5): 623 - 629. [Abstract] [Full Text] [PDF]
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 C. Ruiz de Almodovar, D. Lambrechts, M. Mazzone, and P. Carmeliet Role and Therapeutic Potential of VEGF in the Nervous System Physiol Rev, April 1, 2009; 89(2): 607 - 648. [Abstract] [Full Text] [PDF]
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 E. Kurtagic, M. P. Jedrychowski, and M. A. Nugent Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L534 - L546. [Abstract] [Full Text] [PDF]
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 N. C. Kappas, G. Zeng, J. C. Chappell, J. B. Kearney, S. Hazarika, K. G. Kallianos, C. Patterson, B. H. Annex, and V. L. Bautch The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching J. Cell Biol., October 20, 2008; 181(5): 847 - 858. [Abstract] [Full Text] [PDF]
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 W. Zheng, L. P. Christensen, and R. J. Tomanek Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H794 - H800. [Abstract] [Full Text] [PDF]
|
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 J.-i. Nishi, T. Minamino, H. Miyauchi, A. Nojima, K. Tateno, S. Okada, M. Orimo, J. Moriya, G.-H. Fong, K. Sunagawa, et al. Vascular Endothelial Growth Factor Receptor-1 Regulates Postnatal Angiogenesis Through Inhibition of the Excessive Activation of Akt Circ. Res., August 1, 2008; 103(3): 261 - 268. [Abstract] [Full Text] [PDF]
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M. T. Holderfield and C. C.W. Hughes Crosstalk Between Vascular Endothelial Growth Factor, Notch, and Transforming Growth Factor-{beta} in Vascular Morphogenesis Circ. Res., March 28, 2008; 102(6): 637 - 652. [Abstract] [Full Text] [PDF]
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 H. T. Yuan, S. Venkatesha, B. Chan, U. Deutsch, T. Mammoto, V. P. Sukhatme, A. S. Woolf, and S. A. Karumanchi Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival FASEB J, October 1, 2007; 21(12): 3171 - 3183. [Abstract] [Full Text] [PDF]
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 D. Menendez, A. Inga, J. Snipe, O. Krysiak, G. Schonfelder, and M. A. Resnick A Single-Nucleotide Polymorphism in a Half-Binding Site Creates p53 and Estrogen Receptor Control of Vascular Endothelial Growth Factor Receptor 1 Mol. Cell. Biol., April 1, 2007; 27(7): 2590 - 2600. [Abstract] [Full Text] [PDF]
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 G. Zeng, S. M. Taylor, J. R. McColm, N. C. Kappas, J. B. Kearney, L. H. Williams, M. E. Hartnett, and V. L. Bautch Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation Blood, February 15, 2007; 109(4): 1345 - 1352. [Abstract] [Full Text] [PDF]
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H. Wang, P. C. Charles, Y. Wu, R. Ren, X. Pi, M. Moser, M. Barshishat-Kupper, J. S. Rubin, C. Perou, V. Bautch, et al. Gene Expression Profile Signatures Indicate a Role for Wnt Signaling in Endothelial Commitment From Embryonic Stem Cells Circ. Res., May 26, 2006; 98(10): 1331 - 1339. [Abstract] [Full Text] [PDF]
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 L. D. Covassin, J. A. Villefranc, M. C. Kacergis, B. M. Weinstein, and N. D. Lawson Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish PNAS, April 25, 2006; 103(17): 6554 - 6559. [Abstract] [Full Text] [PDF]
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N. F. Voelkel, R. W. Vandivier, and R. M. Tuder Vascular endothelial growth factor in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L209 - L221. [Abstract] [Full Text] [PDF]
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J.E. Ferguson III, R. W. Kelley, and C. Patterson Mechanisms of Endothelial Differentiation in Embryonic Vasculogenesis Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2246 - 2254. [Abstract] [Full Text] [PDF]
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 R. Summer, D. N. Kotton, S. Liang, K. Fitzsimmons, X. Sun, and A. Fine Embryonic Lung Side Population Cells Are Hematopoietic and Vascular Precursors Am. J. Respir. Cell Mol. Biol., July 1, 2005; 33(1): 32 - 40. [Abstract] [Full Text] [PDF]
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 G. Keller Embryonic stem cell differentiation: emergence of a new era in biology and medicine Genes & Dev., May 15, 2005; 19(10): 1129 - 1155. [Abstract] [Full Text] [PDF]
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S. A. McGrath-Morrow, C. Cho, C. Cho, L. Zhen, D. J. Hicklin, and R. M. Tuder Vascular Endothelial Growth Factor Receptor 2 Blockade Disrupts Postnatal Lung Development Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 420 - 427. [Abstract] [Full Text] [PDF]
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D. M. Roberts, A. L. Anderson, M. Hidaka, R. L. Swetenburg, C. Patterson, W. L. Stanford, and V. L. Bautch A Vascular Gene Trap Screen Defines RasGRP3 as an Angiogenesis-Regulated Gene Required for the Endothelial Response to Phorbol Esters Mol. Cell. Biol., December 15, 2004; 24(24): 10515 - 10528. [Abstract] [Full Text] [PDF]
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 A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers, A. T. Van Oosterom, and E. A. De Bruijn Vascular Endothelial Growth Factor and Angiogenesis Pharmacol. Rev., December 1, 2004; 56(4): 549 - 580. [Abstract] [Full Text] [PDF]
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I. NILSSON, C. ROLNY, Y. WU, B. PYTOWSKI, D. HICKLIN, K. ALITALO, L. CLAESSON-WELSH, and S. WENNSTROM Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development FASEB J, October 1, 2004; 18(13): 1507 - 1515. [Abstract] [Full Text] [PDF]
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J. B. Kearney, N. C. Kappas, C. Ellerstrom, F. W. DiPaola, and V. L. Bautch The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis Blood, June 15, 2004; 103(12): 4527 - 4535. [Abstract] [Full Text] [PDF]
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 D. M. Roberts, J. B. Kearney, J. H. Johnson, M. P. Rosenberg, R. Kumar, and V. L. Bautch The Vascular Endothelial Growth Factor (VEGF) Receptor Flt-1 (VEGFR-1) Modulates Flk-1 (VEGFR-2) Signaling During Blood Vessel Formation Am. J. Pathol., May 1, 2004; 164(5): 1531 - 1535. [Abstract] [Full Text] [PDF]
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 L. Lai, B. L. Bohnsack, K. Niederreither, and K. K. Hirschi Retinoic acid regulates endothelial cell proliferation during vasculogenesis Development, December 29, 2003; 130(26): 6465 - 6474. [Abstract] [Full Text] [PDF]
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Y. Wu, M. Moser, V. L. Bautch, and C. Patterson HoxB5 Is an Upstream Transcriptional Switch for Differentiation of the Vascular Endothelium from Precursor Cells Mol. Cell. Biol., August 15, 2003; 23(16): 5680 - 5691. [Abstract] [Full Text] [PDF]
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E. Mata-Greenwood, B. Meyrick, S. J. Soifer, J. R. Fineman, and S. M. Black Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L222 - L231. [Abstract] [Full Text] [PDF]
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 S. A. Feeney, D. A. C. Simpson, T. A. Gardiner, C. Boyle, P. Jamison, and A. W. Stitt Role of Vascular Endothelial Growth Factor and Placental Growth Factors During Retinal Vascular Development and Hyaloid Regression Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 839 - 847. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
