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Tumor Biology |
Departments of Genetics [S. H., Y. M., M. Sh.] and Cancer Cell Research [A. O., M. Se.], Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, and Cancer Institute, Tokyo 170-0012 [T. N.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flt-1 is expressed as a full-length tyrosine kinase receptor and in
some cases as a soluble form, which carries only the extracellular
domain (9, 10, 11, 12, 13)
. Biochemically, Flt-1 shows an affinity to
VEGF that is at least 10-fold higher than that of KDR/Flk-1, but its TK
activity is 
10-fold weaker than that of KDR/Flk-1 (2, 3, 4
, 14, 15, 16, 17, 18, 19)
, suggesting a function different from KDR/Flk-1. Flt-1
and KDR/Flk-1 are specifically expressed on vascular endothelial cells
(20, 21, 22, 23, 24)
, and as an exception, the flt-1 mRNA
has been shown to be expressed in monocyte/macrophages (25
, 26)
.
Recently, gene targeting studies were carried out on flt-1 and KDR/flk-1 in mice (7 , 8) . The KDR/flk-1 (-/-) homozygous mice died at embryonic day 8.5 (E8.5) because of a severe deficiency in vascular formation associated with a strong hematopoietic impairment (7) . The flt-1 (-/-) homozygous mice also showed embryonic lethality at almost the same stage (E8.5–9.0); however, the phenotype was quite different. The blood vessels in flt-1 (-/-) mice were disorganized, and the endothelial-like abnormal cells were overgrowing within the vascular lumens (8) . These results suggest that Flt-1 has a negative role in the early angiogenesis in embryo. More recently, we have shown that the Flt-1 TK domain-deficient [flt-1 TK (-/-)] mice developed basically normal blood vessels and survived with an impairment of macrophage migration toward VEGF (27) . Thus, Flt-1 is considered to play a negative function in embryogenesis by trapping the endogenous VEGF and adjusting the levels of VEGF to an appropriate range.
Although one important function of Flt-1 was found to be a VEGF-absorbing activity, it is still not clear whether the Flt-1 has a positive role in pathological angiogenesis, such as tumor angiogenesis. To answer this question, we introduced an Flt-1-specific ligand into a tumor cell line and examined the tumor growth and tumor angiogenesis in both the wild-type flt-1 TK (+/+) and flt-1 TK (-/-) mice. Our results indicate clearly that the TK of Flt-1, although it does not have strong enzymatic activity, is important for angiogenesis under certain pathological conditions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells Overexpressing Flt-1 Ligand.
Human PlGF2 or human VEGF165 cDNA was inserted
into murine pSR
MSVtk-neo retroviral vector, and the high titer
retroviruses were used to infect murine endothelial cell-derived
F2 (kindly provided by Dr. K. Toda, Department of
Dermatology, Kyoto University, Kyoto, Japan; Ref. 28
) or
LLC cell line by the Polybrene method. G418-resistant cells, designated
as F2-PlGF, LLC-PlGF, or LLC-VEGF, were harvested as a mixed
population, and the production of human PlGF2 or
VEGF165 from these cells into culture medium was
examined by Western blotting with antibody against PlGF or VEGF.
Injection of Ligand-containing Matrigels and Ligand-overexpressed
LLC Cells into Mice.
F2-PlGF (5 x 106 cells) or 200
ng/ml of PlGF purified from F2-PlGF culture medium using HiTrap Heparin
Affinity Column (1 ml; Pharmacia, Uppsala, Sweden) as described
previously (27)
were mixed with 1 ml of Matrigel (Becton
Dickinson Laboratory) and s.c. injected into the back of anesthetized
mice. Mouse VEGF164 and antimouse
VEGF164 neutralizing antibody were purchased from
R&D Systems (Minneapolis, MN). Parental LLC, LLC-PlGF, or LLC-VEGF
cells were collected by centrifugation, resuspended in DMEM at
5 x 106 cells/ml, and injected
into the backs of mice.
Preparation of Proteins and Western and Northern Blot Analyses.
LLCs were lysed in an ice-cold buffer containing 150 mM
NaCl, 50 mM HEPES (pH 7.4), 10 mM EDTA, 1%
Triton X-100, 10% glycerol, 2% aprotinin, and 1 mM
phenylmethylsulfonyl fluoride and incubated with heparin beads for
2 h. The protein-bound beads were collected by centrifugation,
washed in PBS, and analyzed by SDS-PAGE and Western blotting. For
Northern blot analysis, 0.2 kb of mouse PlGF cDNA and 0.5 kb of mouse
VEGF-B cDNA were used for hybridization probes.
Immunohistochemistry.
Tumor tissue sections were immunohistochemically stained with an
antiserum against human vWF (Dako, Carpinteria, CA) as an endothelial
cell-specific marker or antiserum against mouse F4/80 antibody (BMA) as
a macrophage marker. Tissue sections (8-µm thick) were fixed with
acetone at -20°C for 5 min and rehydrated in PBS. For inhibition of
endogenous peroxidase, sections were incubated in 0.1%
H2O2 methanol solution for
10 min at room temperature and washed three times with PBS.
Nonspecific binding of antibody was blocked by incubation with 10%
normal goat serum-containing PBS for 30 min. Subsequently, sections
were incubated with primary antibody for 2 h, washed three times
with PBS, incubated with biotinylated goat antirabbit IgG (Vector,
Burlingame, CA) for vWF, washed again with PBS, and then incubated with
ABC kit (Vector). Horseradish peroxidase-conjugated sheep antirabbit
IgG was used for F4/80. After washing with PBS, vWF and F4/80 were
detected by 3-amino-9-ethyl carbazole (Vector). The smooth muscle cells
in the thoracic aorta and tumor tissues were stained with a
FITC-conjugated anti--smooth muscle actin antibody (Sigma) at 4°C
overnight.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To continuously stimulate the Flt-1 TK, at the first step, we used
F2-PlGF cells, a murine endothelial-derived cell line (28)
that expresses relatively low levels of endogenous VEGF but high levels
of exogenously introduced Flt-1-specific ligand, PlGF-2 (Refs.
13
, 15,
and 29, 30, 31, 32
; see "Materials and
Methods"). F2-PlGF cells were mixed with Matrigel and s.c. injected
into flt-1 TK (+/+) or flt-1 TK (-/-) mice
(Fig. 1)
.
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. Microscopically, the Matrigel/F2-PlGF mixture in
flt-1 TK (+/+) mice showed a clear angiogenic response with
several dilated vessels but not in flt-1 TK (-/-) mice.
The original F2 cells without the exogenous PlGF expression showed no
detectable angiogenic response either in flt-1 TK (+/+) or
in TK (-/-) mice (Fig. 1, A and B)
.
During this incubation period, most of the F2 or F2-PlGF cells died
within the Matrigel because of its weak transforming potential (Fig. 1)
. However, these results strongly suggest that Flt-1 TK is directly
involved in angiogenesis under certain conditions, i.e.,
when the Flt-1-specific ligand is highly expressed.
Purified PlGF-2 Induces an Angiogenic Response in flt-1
TK (+/+) Mice
To avoid any effects produced by F2 cells on the angiogenesis
assay, we next used the PlGF-2 purified from the culture medium of
cells overexpressing PlGF-2 (see "Materials and Methods") and mixed
it with Matrigel for in vivo analysis. The same amounts (200
ng) of PlGF-2 or VEGF164 mixed with Matrigel were
inoculated s.c. for 4 days. The Matrigel containing PlGF-2 showed a
mild angiogenic response with dilated blood vessels in flt-1
TK (+/+) mice but not in flt-1 TK (-/-) mice (Fig. 2, A and B)
. On the other hand, the Matrigel
containing VEGF instead of PlGF induced small new blood vessels to form
both in flt-1 TK (+/+) and TK (-/-) mice (Fig. 2, C and D)
.
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, indicating that the major angiogenic factor in this
system is PlGF itself. In total, however, the responses to purified
ligands were mild, even in the wild-type mice, possibly because of a
rapid inactivation and degradation of ligands in Matrigels.
Comparison of Tumor Angiogenesis in flt-1 TK (+/+)
Mice and in flt-1 TK (-/-) Mice
Expression of PlGF-2 or VEGF165 in LLC Cells Does Not
Modify the in Vitro Cell Growth.
Because F2 or F2-PlGF cells could not make tumors in vivo,
we used the murine LLC cell line for an in vivo
tumorigenicity assay. Before introducing human PlGF or VEGF cDNA to
LLCs, we examined the endogenous VEGF-related ligands in LLCs. We found
that after overnight culture, LLCs secreted 40 ng/ml of
VEGF164 (Fig. 3A)
, less than the ascites-generating sarcoma cell lines (170
to 850 ng/ml; Ref. 33
). LLCs also expressed VEGF-B mRNA
(34, 35, 36)
at levels similar to a melanoma but higher than
normal placenta and lung (Fig. 3C)
. LLCs did not express
detectable levels of PlGF mRNA (Fig. 3B)
.
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200
ng/ml of PlGF, and LLC-VEGF secreted 40 ng/ml of exogenous
VEGF165 (total, 80 ng/ml) per 1 x 107 cells in overnight culture (data not shown).
The growth rate of these LLCs overexpressing either PlGF or
VEGF165 was essentially the same as that of the
parental LLCs in in vitro culture (Fig. 4)
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. PlGF protein secreted from LLC-PlGF could be detected in tumor
tissues 3 weeks after inoculation (data not shown).
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300% that in flt-1 TK (-/-) mice (Fig. 5A)
.
On the other hand, LLC-VEGF cells induced tumors of almost the same
size in the two genotypes of mice (Fig. 5C)
. This is to be
expected because the overexpressed VEGF could stimulate KDR/Flk-1 in
these genotypes, which carries potent TK activity and mediates a strong
angiogenic signal.
The tumor growth rate of the original LLCs was basically low but
slightly better in flt-1 TK (+/+) mice when compared with
flt-1 TK (-/-) mice (Fig. 5B)
. This minor
difference could be attributable to an endogenous VEGF-B expression in
LLCs. The LLC-PlGF-induced tumors showed a clear increase in tumor
vessel density, particularly small- to middle-sized blood vessels, as
indicated in Table 1
and Fig. 6
.
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. However, LLC-VEGF cells induced many
small vessels not >50 µm in diameter (Fig. 6, I and J
; Table 1
). The large vessels induced by LLC-PlGF, as well
as small vessels induced by LLC-VEGF, carried a single layer of
vWF-positive endothelial cells (Fig. 7
, upper), but smooth muscle cells positive for -smooth
muscle actin staining were almost undetectable (Fig. 7
,
lower). These results suggest that the processes of
angiogenesis stimulated by PlGF and by VEGF are not identical.
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. Because LLC-PlGF tumors are growing faster even at day 8 in
flt-1 TK (+/+) mice when compared with those in flt-1
TK (-/-) mice, we do not think that the macrophages infiltrated
into tumors play a major role in LLC-PlGF-induced tumor angiogenesis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, using LLC-PlGF and both wild-type flt-1 TK
(+/+) and flt-1 TK (-/-) mice, we have shown that the
tumor growth was 3-fold higher in the wild-type compared with
flt-1 TK (-/-) mice. The increase in the tumor growth in
the wild-type mice correlated well with the increase in tumor
angiogenesis. These results strongly suggest that the TK domain of
Flt-1 is required for a certain pathological angiogenesis, such as
Flt-1-specific, ligand-induced tumor angiogenesis in vivo.
Because Flt-1 bears weak stimulatory activity for endothelial cell proliferation and because angiogenesis is a multistep process involving matrix proteolysis, migration, proliferation, and tube formation, several steps other than direct cell proliferation are also considered to be stimulated by Flt-1.
Recently, our preliminary results suggested that MMP-2 was activated in blood vessel-enriched regions in tumor tissues in wild-type but not in the flt-1 TK (-/-) mice.4 MMP is known to be important for tumor growth because MMP-2 null mutation in mice resulted in the reduction of tumor growth via a decrease in angiogenesis (39, 40, 41) . In addition, batimastat (BB94), an inhibitor for MMPs, disturbed the growth of a transplanted LLC-induced tumor by 25% (42) .
Precursor forms of MMP-2 and MMP-1, as well as tissue inhibitor of MMP-1 and tissue inhibitor of MMP-2 are known to be produced in endothelial cells (39) . Therefore, signaling via the TK domain of Flt-1 appears to activate MMP-2, leading to a proteolytic digestion of matrix in tumor tissue to facilitate migration and tube formation of endothelial cells.
It is also possible that some angiogenic factors, such as VEGF secreted
from the macrophages that migrated in tumor tissues, induce
angiogenesis. However, this seems unlikely, because approximately the
same number of macrophages have migrated into tumor tissues at day 8,
when a difference in tumor angiogenesis between the two strains of mice
was already observed (Tables 1
and 2)
. Moreover, a partially purified
PlGF-induced angiogenesis in wild-type mice, even in the presence of
anti-VEGF neutralizing antibody, suggested a minor contribution of VEGF
secreted from infiltrating inflammatory cells (Fig. 2E)
.
Taken together, a simple explanation for the rapid growth of LLC-PlGF in wild-type mice might be direct stimulation of both the activation of proteases and the proliferation of endothelial cells via a positive signal from the Flt-1 tyrosine kinase.
Possible Mechanisms for Morphological Differences in Blood Vessels
Induced by PlGF and VEGF.
We found a difference in the formation of blood vessels between
LLC-PlGF- and LLC-VEGF-induced angiogenesis. The vessels induced by
LLC-PlGF were often larger in diameter than those induced by LLC-VEGF.
In our preliminary experiments, the proteolysis surrounding the newly
formed blood vessels was increased in LLC-PlGF-induced tumor tissues
compared with LLC-VEGF-induced tumor.4
Dvorak’s
group reported previously that, in addition to the sprouting mechanism,
angiogenesis occurred via an increase in the diameter of blood vessels
because of a proliferation of endothelial cells, followed by
segregation of an enlarged vessel to several small ones
(43)
. Therefore, the morphological difference induced by
PlGF and by VEGF might be attributable to a difference in the
remodeling process of angiogenesis. PlGF may induce less sprouting-type
vessels but rather large-diameter-type vessels.
Another difference between PlGF- and VEGF-induced angiogenesis may be
in the cells surrounding endothelial cells, such as smooth muscle cells
and pericytes. However, this seems unlikely because no vessels induced
by either PlGF or VEGF were stained with anti--smooth muscle actin
antibody, except for preexisting arteries, indicating that in both
cases the association of smooth muscle cells with vessels was equally
low.
Differential Roles of Flt-1 during Embryonic and Pathological
Angiogenesis.
We have shown previously that the extracellular domain of Flt-1 plays a
negative role in embryogenesis. Although null mutation of the
flt-1 gene results in embryonic lethality because of an
overgrowth of endothelial-like cells, the flt-1 TK (-/-)
mice developed an almost normal vascular system and could be mated as
wild-type mice. Because the loss of a single allele of the
VEGF gene in mice strongly disturbed angiogenesis and was
lethal between the stage of E11–E12, a tight regulation of VEGF levels
in vivo is thought to be necessary for normal angiogenesis
in embryogenesis. Thus, the extracellular domain of Flt-1 may function
as a ligand-trapping molecule and negatively regulate the levels of
VEGF, decreasing the signals from KDR/Flk-1.
Recently, elevations of human PlGF mRNA in renal cell carcinoma as well as some brain tumors (44 , 45) and an increase in human VEGF-B mRNA in melanoma, lung carcinoma, and colon cancer have been reported (46) . These observations, as well as the results shown here, suggest that not only KDR/Flk-1 but also Flt-1 is involved in tumor angiogenesis in a positive manner. Therefore, in addition to the blocking of VEGF and KDR/Flk-1, suppression of the Flt-1-PlGF/VEGF-B pathway may be required to decrease certain types of pathological angiogenesis in vivo.
On the basis of these findings, Flt-1 is considered to have dual functions in endothelial cells, either negative or positive, depending on the biological conditions. Because Flt-1 can bind VEGF, PlGF, and VEGF-B with different affinity and because the gene regulation of each of these three ligands is different, further characterization is required to elucidate the network of the VEGF ligand family and Flt-1 in each pathological condition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant-in-Aid for
Special Project Research on Cancer-Bioscience 04253204 from the
Ministry of Education, Science, Sports and Culture in Japan and by
research grants for the program "Research for the Future" of the
Japan Society for Promotion of Science. 
2 To whom requests for reprints should be
addressed, at Department of Genetics, Institute of Medical Science,
University of Tokyo, Minato-ku, Tokyo 108-8639 Japan. Phone:
81-3-5449-5550; Fax: 81-3-5449-5425.
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; VEGFR, vascular endothelial growth factor
receptor; Flt-1, Fms-like tyrosine kinase 1; PlGF, placenta growth
factor; TK, tyrosine kinase; vWF, von Willebrand factor; MMP, matrix
metalloproteinase; LLC, Lewis lung carcinoma.
4 S. Hiratsuka and M. Shibuya, unpublished
observation.
Received 4/24/00. Accepted 11/30/00.
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D.-G. Bae, T.-D. Kim, G. Li, W.-H. Yoon, and C.-B. Chae Anti-Flt1 Peptide, a Vascular Endothelial Growth Factor Receptor 1-Specific Hexapeptide, Inhibits Tumor Growth and Metastasis Clin. Cancer Res., April 1, 2005; 11(7): 2651 - 2661. [Abstract] [Full Text] [PDF]
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 T. Tammela, B. Enholm, K. Alitalo, and K. Paavonen The biology of vascular endothelial growth factors Cardiovasc Res, February 15, 2005; 65(3): 550 - 563. [Abstract] [Full Text] [PDF]
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 D. J. Hicklin and L. M. Ellis Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis J. Clin. Oncol., February 10, 2005; 23(5): 1011 - 1027. [Abstract] [Full Text] [PDF]
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E. S. Wang, J. Teruya-Feldstein, Y. Wu, Z. Zhu, D. J. Hicklin, and M. A. S. Moore Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo Blood, November 1, 2004; 104(9): 2893 - 2902. [Abstract] [Full Text] [PDF]
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H. Takahashi, S. Hattori, A. Iwamatsu, H. Takizawa, and M. Shibuya A Novel Snake Venom Vascular Endothelial Growth Factor (VEGF) Predominantly Induces Vascular Permeability through Preferential Signaling via VEGF Receptor-1 J. Biol. Chem., October 29, 2004; 279(44): 46304 - 46314. [Abstract] [Full Text] [PDF]
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 I. Shiojima and K. Walsh The Role of Vascular Endothelial Growth Factor in Restenosis: The Controversy Continues Circulation, October 19, 2004; 110(16): 2283 - 2286. [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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 S. Emanuel, R. H. Gruninger, A. Fuentes-Pesquera, P. J. Connolly, J. A. Seamon, S. Hazel, R. Tominovich, B. Hollister, C. Napier, M. R. D'Andrea, et al. A Vascular Endothelial Growth Factor Receptor-2 Kinase Inhibitor Potentiates the Activity of the Conventional Chemotherapeutic Agents Paclitaxel and Doxorubicin in Tumor Xenograft Models Mol. Pharmacol., September 1, 2004; 66(3): 635 - 647. [Abstract] [Full Text] [PDF]
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J.-C. Tille and M.S. Pepper Hereditary Vascular Anomalies: New Insights Into Their Pathogenesis Arterioscler Thromb Vasc Biol, September 1, 2004; 24(9): 1578 - 1590. [Abstract] [Full Text] [PDF]
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 N. Ferrara Vascular Endothelial Growth Factor: Basic Science and Clinical Progress Endocr. Rev., August 1, 2004; 25(4): 581 - 611. [Abstract] [Full Text] [PDF]
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M. Bjorndahl, R. Cao, A. Eriksson, and Y. Cao Blockage of VEGF-Induced Angiogenesis by Preventing VEGF Secretion Circ. Res., June 11, 2004; 94(11): 1443 - 1450. [Abstract] [Full Text] [PDF]
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C. Calvi, P. Dentelli, M. Pagano, A. Rosso, M. Pegoraro, S. Giunti, G. Garbarino, G. Camussi, L. Pegoraro, and M. F. Brizzi Angiopoietin 2 Induces Cell Cycle Arrest in Endothelial Cells: A Possible Mechanism Involved in Advanced Plaque Neovascularization Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 511 - 518. [Abstract] [Full Text]
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A. Babiak, A.-M. Schumm, C. Wangler, M. Loukas, J. Wu, S. Dombrowski, C. Matuschek, J. Kotzerke, C. Dehio, and J. Waltenberger Coordinated activation of VEGFR-1 and VEGFR-2 is a potent arteriogenic stimulus leading to enhancement of regional perfusion Cardiovasc Res, March 1, 2004; 61(4): 789 - 795. [Abstract] [Full Text] [PDF]
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 F. Mac Gabhann and A. S. Popel Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H153 - H164. [Abstract] [Full Text] [PDF]
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B. Ruggeri, J. Singh, D. Gingrich, T. Angeles, M. Albom, H. Chang, C. Robinson, K. Hunter, P. Dobrzanski, S. Jones-Bolin, et al. CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases with Potent Antiangiogenic Activity and Antitumor Efficacy in Preclinical Models Cancer Res., September 15, 2003; 63(18): 5978 - 5991. [Abstract] [Full Text] [PDF]
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 H Yoshiji, S Kuriyama, J Yoshii, Y Ikenaka, R Noguchi, D J Hicklin, Y Wu, K Yanase, T Namisaki, M Yamazaki, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis Gut, September 1, 2003; 52(9): 1347 - 1354. [Abstract] [Full Text]
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J.-S. Silvestre, R. Tamarat, T. G. Ebrahimian, A. Le-Roux, M. Clergue, F. Emmanuel, M. Duriez, B. Schwartz, D. Branellec, and B. I. Levy Vascular Endothelial Growth Factor-B Promotes In Vivo Angiogenesis Circ. Res., July 25, 2003; 93(2): 114 - 123. [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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V. Louzier, B. Raffestin, A. Leroux, D. Branellec, J. M. Caillaud, M. Levame, S. Eddahibi, and S. Adnot Role of VEGF-B in the lung during development of chronic hypoxic pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L926 - L937. [Abstract] [Full Text] [PDF]
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 D. S. Torry, D. Mukherjea, J. Arroyo, and R. J. Torry Expression and Function of Placenta Growth Factor: Implications for Abnormal Placentation Reproductive Sciences, May 1, 2003; 10(4): 178 - 188. [Abstract] [PDF]
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H. Oura, J. Bertoncini, P. Velasco, L. F. Brown, P. Carmeliet, and M. Detmar A critical role of placental growth factor in the induction of inflammation and edema formation Blood, January 15, 2003; 101(2): 560 - 567. [Abstract] [Full Text] [PDF]
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E. Geimonen, S. Neff, T. Raymond, S. S. Kocer, I. N. Gavrilovskaya, and E. R. Mackow Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses PNAS, October 15, 2002; 99(21): 13837 - 13842. [Abstract] [Full Text] [PDF]
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 T. Itokawa, H. Nokihara, Y. Nishioka, S. Sone, Y. Iwamoto, Y. Yamada, J. Cherrington, G. McMahon, M. Shibuya, M. Kuwano, et al. Antiangiogenic Effect by SU5416 Is Partly Attributable to Inhibition of Flt-1 Receptor Signaling Mol. Cancer Ther., March 1, 2002; 1(5): 295 - 302. [Abstract] [Full Text] [PDF]
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E. I. Deryugina, L. Soroceanu, and A. Y. Strongin Up-Regulation of Vascular Endothelial Growth Factor by Membrane-type 1 Matrix Metalloproteinase Stimulates Human Glioma Xenograft Growth and Angiogenesis Cancer Res., January 1, 2002; 62(2): 580 - 588. [Abstract] [Full Text] [PDF]
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D. Lu, X. Jimenez, H. Zhang, Y. Wu, P. Bohlen, L. Witte, and Z. Zhu Complete Inhibition of Vascular Endothelial Growth Factor (VEGF) Activities with a Bifunctional Diabody Directed against Both VEGF Kinase Receptors, fms-like Tyrosine Kinase Receptor and Kinase Insert Domain-containing Receptor Cancer Res., October 1, 2001; 61(19): 7002 - 7008. [Abstract] [Full Text] [PDF]
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 B. Bussolati, C. Dunk, M. Grohman, C. D. Kontos, J. Mason, and A. Ahmed Vascular Endothelial Growth Factor Receptor-1 Modulates Vascular Endothelial Growth Factor-Mediated Angiogenesis via Nitric Oxide Am. J. Pathol., September 1, 2001; 159(3): 993 - 1008. [Abstract] [Full Text] [PDF]
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