Identification of the
v
3 Integrin-interacting Motif of
ig-h3 and Its Anti-angiogenic Effect*
Ju-Ock Nam
,
Jung-Eun Kim
,
Ha-Won Jeong
,
Sung-Jin Lee
,
Byung-Heon Lee
,
Je-Yong Choi
,
Rang-Woon Park
,
Jae Yong Park
and
In-San Kim
¶
From the
Cell and Matrix Biology National Research
Laboratory, Department of Biochemistry, and the
Department of Internal Medicine, Kyungpook
National University School of Medicine, Taegu 700-422, Korea
Received for publication, January 13, 2003
, and in revised form, April 11, 2003.
 |
ABSTRACT
|
---|
ig-h3 is an extracellular matrix protein that mediates adhesion and
migration of several cell types through interaction with integrins. In the
present study, we tested whether
ig-h3 mediates endothelial cell
adhesion and migration, thereby regulating angiogenesis. In this study, we
demonstrate that not only
ig-h3 itself but also all four fas-1 domains
of
ig-h3 mediate endothelial cell adhesion and migration through
interaction with the
v
3 integrin. We found
that the
v
3 integrin-interacting motif of
the four fas-1 domains of
ig-h3 is the same YH motif that we reported
previously to interact with
v
5 integrin.
The YH peptide inhibited endothelial cell adhesion and migration in a
dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic
activity in vitro and in vivo using an endothelial cell tube
formation assay and a Matrigel plug assay, respectively. Our results reveal
that
ig-h3 bears
v
3
integrin-interacting motifs that mediate endothelial cell adhesion and
migration and, therefore, may regulate angiogenesis.
 |
INTRODUCTION
|
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The
ig-h3 is an extracellular matrix protein whose expression is
induced by transforming growth factor-
in several cell types
(1). The
ig-h3 protein is
composed of 683 amino acids containing four homologous internal repeat
domains. These domains are homologous to similar motifs in the
Drosophila protein fasciclin-I and thus are denoted fas-1 domains.
The fas-1 domain has highly conserved sequences found in the secretory and
membrane proteins of many organisms, including mammals, insects, sea urchins,
plants, yeast, and bacteria. The fas-1 domain interacts with other matrix
proteins such as fibronectin, collagen, and laminin
(2) and mediates cell adhesion
and migration through interaction with integrins
(3,
4). We have reported previously
that it bears motifs interacting with the integrins
3
1
(3) and
v
5
(4). The fas-1 domain is also
known to be involved in cell growth, differentiation, tumorigenesis, wound
healing, and apoptosis
(58).
Although
ig-h3 mediates the adhesion of many different cell types,
including corneal epithelial cells, chondrocytes, and fibroblasts, it is not
known whether
ig-h3 mediates endothelial cell adhesion and migration,
thereby regulating angiogenesis. In this study, we demonstrate that
ig-h3 mediates endothelial cell adhesion and migration through
v
3 integrin and that the responsible motif
is the YH motif, which has been reported to interact with the
v
5 integrin. In addition, we show that the
YH peptide inhibits endothelial tube formation and reduces the number of blood
vessels in a Matrigel plug assay. Collectively, the YH motif of the fas-1
domains of the
ig-h3 protein interacts with
v
3 integrin and is an effective inhibitor
of angiogenesis. Our data suggest that the peptide fragment containing the YH
motif could be a drug candidate for the treatment of diseases dependent on
angiogenesis.
 |
EXPERIMENTAL PROCEDURES
|
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DNA Constructs and Synthetic PeptidesBacterial expression
vectors for wild-type
ig-h3, each fas-1 domain, and relevant mutants
have been described previously
(4). Recombinant
ig-h3
proteins were induced and purified as described previously
(4).
Cell CultureHuman umbilical vein endothelial cells
(HUVECs)1 were
cultured at 37 °C in 5% CO2 in EGM medium (Clontech)
supplemented with 2% fetal bovine serum. HEK293 cells stably transfected with
an empty vector (pcDNA3) or a human integrin
3 expression
vector were kindly provided by Dr. Jeffrey Smith (Burnham Institute, San
Diego). These stable cell lines were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum and antibiotics.
Cell Adhesion StudiesThe cell adhesion assay was performed
as described previously (4).
Briefly, flat-bottomed 96-well enzyme-linked immunosorbent assay plates
(Costar, Corning Inc., NY) were incubated overnight at 4 °C with 10
µg/ml of recombinant
ig-h3 proteins and then blocked for 1 h at room
temperature with phosphate-buffered saline (PBS) containing 2% bovine serum
albumin (BSA). Cells were suspended in medium at a density of 3 x
105 cells/ml, and 0.1 ml of the cell suspension was added to each
well of the coated plates. After incubation for 30 min at 37 °C,
unattached cells were removed by rinsing once with PBS. Attached cells were
then incubated for 1 h at 37 °C in 50 mM citrate buffer, pH
5.0, containing 3.75 mM
p-nitrophenyl-N-acetyl-D-glycosaminide and 0.25%
Triton X-100. Enzyme activity was blocked by adding 50 mM glycine
buffer, pH 10.4, containing 5 mM EDTA, and the absorbance was
measured at 405 nm in a Bio-Rad model 550 microplate reader.
Inhibition AssaySynthetic peptides purchased from AnyGen
Co. Ltd. (Kwangju, Korea) were tested for their ability to inhibit cells from
adhering to the protein substrates coating the wells. The cell adhesion assay
was performed in the presence or absence of the indicated concentrations of
each peptide. To identify the receptor for
ig-h3, monoclonal antibodies
specific to different types of integrins (Chemicon, Temecula, CA) were
preincubated (5 µg/ml) at 37 °C for 30 min with HUVEC in 0.1 ml of the
cell suspension (3 x 105 cells/ml). The cells were then
transferred onto plates precoated with recombinant
ig-h3 proteins and
incubated for 30 min at 37 °C. The attached cells were then quantified as
described above. Function-blocking monoclonal antibodies to the following
integrin subunits were used:
3 (P1B5),
5
(P1D6),
v (P3G8),
1 (6S6),
3 (B3A),
v
3 (LM609), and
v
5 (P1F6).
Migration AssayCell migration assays were performed in
transwell plates (8 µm pore size, Costar, Cambridge, MA). The undersurface
of the membrane was coated with 10 µg/ml recombinant
ig-h3 proteins
at 4 °C and then blocked for 1 h at room temperature with
phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA).
Cells were suspended in medium at a density of 3 x 105
cells/ml, and 0.1 ml of the cell suspension was added to the upper compartment
of the filter with or without the indicated concentrations of each peptide. In
some experiments, cells were preincubated at 37 °C for 30 min with
anti-
v
3 (LM609) or
v
5 (P1F6). Cells were allowed to migrate
for 68 h at 37 °C. Migration was terminated by removing the cells
from the upper compartment of the filter with a cotton swab, and the filters
were fixed with 8% glutaraldehyde and stained with crystal violet. The extent
of cell migration was determined by light microscopy, and within each well,
counting was done in nine randomly selected microscopic high power fields.
Flow Cytometric AnalysisCells were detached by gentle
treatment with 0.25% trypsin, 0.05% EDTA in PBS, washed, and incubated for 1 h
at 4 °C with antibodies to the
v
3
(LM609) or
v
5 integrins (P1F6). Cells were
then incubated for1hat4 °C with 10 µg/ml of the secondary antibody,
goat anti-mouse IgG conjugated with fluorescein isothiocyanate (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), and analyzed at 488 nm on the flow
cytometer FACScalibur system (BD Biosciences) equipped with a 5-watt
laser.
Binding Assay of
ig-h3A binding assay was
performed as described previously
(9). Cells were suspended in
medium at a density of 1 x 105 cells/ml, and 1 ml of the cell
suspension was preincubated with anti-
v
3
(LM609) or
v
5 (P1F6) for 30 min at 37
°C. The cells were incubated with biotinylated
ig-h3 in serum-free
media containing 0.1% BSA for 5 h at 4 °C. The cells were then washed
three times with phosphate-buffered saline, pH 7.4, before lysis at 4 °C
in ice-cold buffer A (10 mM Tris-Cl, pH 7.4, 150 mM
NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS, 0.02% sodium azide, 1
mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The
lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C.
Equal amounts of protein were then separated by SDS-PAGE, 8% gel. The amount
of biotinylated
ig-h3 was determined by immunoblotting.
To visualize the biotinylated
ig-h3, membranes were incubated with
streptavidin conjugated to horseradish peroxidase (Amersham Biosciences).
Binding of the peroxidase-labeled antibody was visualized using enhanced
chemiluminescence (ECL; Amersham Biosciences). Stripping and reprobing for
tubulin (Santa Cruz Biotechnology) immunoblotting as an internal control were
performed according to the manufacturer's instructions.
Endothelial Tube AssayMatrigel (Chemicon) was added (100
µl) to each well of a 96-well plate and allowed to polymerize. Cells were
suspended in medium at a density of 3 x 105 cells/ml, and 0.1
ml of the cell suspension was added to each well coated with Matrigel,
together with or without the indicated concentrations of each peptide. Cells
were incubated for 1618 h at 37 °C. The cells were then
photographed, and tubes were counted and averaged.
Matrigel Plug AssayAn in vivo Matrigel plug assay
was performed as described previously
(10). Five- to 6-week-old male
C57BL/6 mice were used. Matrigel (BD Biosciences) was mixed with 20 units/ml
heparin, 150 ng/ml basic fibroblast growth factor (R & D Systems), and
synthetic peptide (AnyGen Co. Ltd.). The Matrigel mixture was injected
subcutaneously, and after 7 days mice were sacrificed, and the Matrigel plugs
were removed and fixed in 4% paraformaldehyde. The plugs were embedded in
paraffin, sectioned, and H & E-stained. Sections were examined by light
microscopy, and the number of blood vessels from 4 to 6 high power fields
(x200) was counted and averaged. Each group consisted of three or four
Matrigel plugs.
 |
RESULTS
|
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ig-h3 Mediates HUVEC Adhesion through the
v
3
IntegrinRecently, we reported that
ig-h3 has two
3
1 integrin-interacting motifs mediating
epithelial cell adhesion (3)
and four
v
5 integrin-interacting motifs
mediating fibroblast cell adhesion
(4). In the present study, we
also found that
ig-h3 mediates endothelial cell adhesion and that each
fas-1 domain of
ig-h3 was equally active in mediating endothelial cell
adhesion (Fig. 1A). To
identify the integrin responsible for endothelial cell adhesion to
ig-h3, we used several integrin function blocking antibodies. As shown
in Fig. 1B,
endothelial cell adhesion to
ig-h3 was specifically inhibited by
antibodies to the
v
3 integrin and
3 integrin but not by antibodies against other integrins,
including the
v
5,
3, and
1 integrins. Endothelial cell adhesion to each fas-1 domain
was also blocked by a function-blocking antibody to the
v
3 integrin but not to the
v
5 integrin
(Fig. 1C). To confirm
that HUVECs express both the
v
3 and
v
5 integrin on their cell surface, we did
FACS analysis using specific monoclonal antibodies to both integrins. As shown
in Fig. 1D, HUVECs
express both integrins, but the expression level of the
v
5 integrin is far less than the
v
3 integrin. These results suggest that
each fas-1 domain bears a motif mediating endothelial cell adhesion through
the
v
3 integrin.

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FIG. 1. All fas-1 domains in ig-h3 mediate HUVEC adhesion by binding to
v 3 integrin. A, adhesion of
HUVEC to each fas-1 domain in ig-h3. 96-Well plates were coated with
ig-h3-WT or each fas-1 domain overnight at 4 °C. After seeding and
incubation, attached cells were quantified by measuring hexosaminidase, as
described under ``Experimental Procedures.'' B, identification of
integrins mediating the adhesion of HUVEC to ig-h3. HUVEC were
preincubated with the following function-blocking monoclonal antibodies to
integrin subunits and then added to the precoated wells:
3(P1B5), 5(P1D5),
v(P3G8), 1(6S6), 3(B3A),
v 3(LM609), and
v 5(P1F6). After seeding and incubation,
attached cells were quantified by measuring hexosaminidase. C, the
effect of the integrin v 3 function-blocking
antibody on HUVEC adhesion to each fas-1 domain. D, analysis of
integrins expressed on the HUVEC surface. Flow cytometric analysis was
performed on HUVEC stained with saturating concentration of the following
monoclonal antibodies: v 3(LM609) and
v 5(P1F6). The data are expressed as cell
number (y axis) plotted as a function of fluorescence intensity
(x axis) and are representative of three separate experiments.
Negative control cells were incubated with the secondary antibody alone.
|
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The YH Motif Is Necessary for
ig-h3-mediated Endothelial
Cell AdhesionTo identify the
v
3
integrin-interacting motif, we used proteins containing deletion mutations in
the 4th fas-1 domain, which have been described previously
(4). We found that the
v
3 integrin-interacting motif was present
within a fragment corresponding to amino acids 548614 because this is
the smallest fragment that still retains endothelial cell adhesion activity
(Fig. 2A). We reported
previously (4) that this
fragment contains the YH motif, which has been shown to bind the
v
5 integrin. We suspected that the YH motif
may also interact with the
v
3 integrin to
mediate endothelial cell adhesion. We used several substitution mutant 4th
fas-1 proteins as described previously
(4), whose tyrosine, histidine,
and flanking leucine/isoleucine residues were mutated in various combinations
(Fig. 2A). Neither
mutations on tyrosine and histidine nor mutations on leucine/isoleucine at
either side of YH residues abolished cell adhesion activity. On the contrary,
any combinatory mutations on YH and flanking leucine/isoleucine significantly
reduced cell adhesion activity (Fig.
2A). The results suggest that not only tyrosine and
histidine but also flanking leucine/isoleucine residues are required to
mediate HUVEC adhesion. To confirm further that the YH motif is responsible
for endothelial cell adhesion, we used YH18 synthetic peptides from each fas-1
domain, which have been shown to inhibit the adhesion of fibroblasts to
ig-h3 through the
v
5 integrin
(4), in a cell adhesion assay.
These peptides also inhibit endothelial cell adhesion to
ig-h3 in a
dose-dependent manner (Fig.
2B). These results suggest that the YH motif is also
responsible for mediating endothelial cell adhesion to
ig-h3 through the
v
3 integrin. Because the YH peptide was
reported previously to interact with the
v
5
integrin of fibroblasts (4) and
HUVECs express the
v
5 integrin, it is
expected that the
v
5 integrin of HUVECs
could also be involved in mediating HUVECs adhesion to
ig-h3. To
investigate whether
ig-h3-mediated HUVECs adhesion is specifically
dependent on the
v
3 integrin, we further
studied the binding affinity of
ig-h3 in the presence of a specific
function-blocking antibody to each integrin.
Fig. 3A shows that
ig-h3 binds to the HUVECs surface in a dose-dependent manner, and its
binding is specifically inhibited by an antibody to the
v
3 integrin but not by an antibody to the
v
5 integrin nor IgG
(Fig. 3B).
The
v
3
Integrin Is a Functional Receptor for
ig-h3To
confirm that the
v
3 integrin mediates
endothelial cell adhesion to
ig-h3, we used HEK293 cells stably
transfected with a human
3 integrin expression vector.
3/293 cells strongly adhered to
ig-h3, whereas pc/293 cells, which
were stably transfected with an empty vector, did not
(Fig. 4A).
3/293
cell adhesion to
ig-h3 was specifically inhibited by an antibody to the
v
3 integrin
(Fig. 4B). The YH18
peptides from each fas-1 domain also inhibited
3/293 cell adhesion to
the
ig-h3 protein (Fig.
4C). These results confirm that the YH motif of
ig-h3 mediates endothelial cell adhesion through interaction with the
v
3 integrin.
The YH Motif Mediates Endothelial Cell Migration through the
v
3
IntegrinWe tested whether
ig-h3 mediates endothelial
cell migration using a transwell system. We found that the migration of
endothelial cells was enhanced in those trans-wells whose undersurface was
coated with
ig-h3 and that this effect was inhibited by an antibody to
the
v
3 integrin but not by an antibody to
the
v
5 integrin
(Fig. 5, A and
B). The YH18 peptide also inhibited endothelial cell
migration toward
ig-h3 (Fig. 5,
C and D). These results suggest that the YH
motif of
ig-h3 mediates endothelial cell migration through the
v
3 integrin.
YH18 Peptides Inhibit Angiogenesis in Vitro and in VivoIn
the next experiment, we tested whether the YH18 peptide can inhibit
angiogenesis in vitro. The YH18 peptide selectively inhibited
endothelial tube formation on Matrigel in a dose-dependent manner
(Fig. 6, A and
B), whereas the control peptide did not. A
half-inhibition was observed at 500 µM. To test the in
vivo effect of the YH18 peptide, we performed a Matrigel plug assay in
mice. Matrigel was placed in the presence of bFGF with or without increasing
concentrations of the YH18 peptide. A significant reduction in the number of
blood vessels was observed at 500 µM
(Fig. 7, A and
C). These results suggest that the YH peptide inhibits
angiogenesis in both in vitro and in vivo assays at a
similar concentration.

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FIG. 6. The YH18 peptide inhibits HUVEC tube formation in vitro.
A and B, cells were preincubated with or without YH18 (500
µM or 1 mM) and then seeded in 96-well plates coated
with Matrigel. After 1618 h of culture, the number of tube branches in
a low power field was counted (three independent wells were counted and
averaged). con., control.
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FIG. 7. YH18 peptides inhibit angiogenesis in vivo. A, the
YH18 (500 µM or 1 mM) peptide was mixed with Matrigel
plus and then injected into the flank of a mouse. After 7 days, the animals
were sacrificed, and the plugs were removed and scanned at high resolution.
B and C, sections of each Matrigel plug stained by
hematoxylin and eosin were examined by light microscopy (x200
magnification), and the number of blood vessels from 4 to 7 high power fields
was counted and averaged. Inset in B, high magnification
view (x400) of blood vessels. Arrow indicates the position
magnified in insets. Arrowheads indicate the blood vessels.
con., control.
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 |
DISCUSSION
|
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ig-h3 has been known to mediate adhesion of several cell types,
including epithelial cells (3,
7), fibroblasts
(4), chondrocytes
(11), and vascular smooth
cells (12). We reported that
ig-h3 has two short motifs interacting with the
3
1 integrin for mediating adhesion of
corneal epithelial cells (3)
and keratinocytes (7).
Recently, we identified new motifs in the fas-1 domains of
ig-h3 that
mediate fibroblast adhesion
(4). This motif was designated
the YH motif because it contains highly conserved tyrosine and histidine
residues. In addition, the YH motif has a few more conserved leucines and
isoleucines flanking the tyrosine and histidine residues. At least 18 amino
acids, including these conserved residues, are required to mediate fibroblast
adhesion. The YH motif mediates the adhesion of fibroblasts by interacting
with the
v
5 integrin. In the present study,
we first demonstrated that
ig-h3 also mediates adhesion and migration of
endothelial cells acting through the
v
3
integrin. Interestingly, the
v
3
integrin-interacting motifs turned out to be the YH motif. The ability of the
YH peptide to interact with both the
v
3 and
v
5 integrins is not surprising because the
v
3 and
v
5 integrins are known to share ligands
(13), and both bind to the RGD
peptide and its peptidomimetics
(14). Because we found that
the interaction of the YH peptide with the
v
5
(4) and
v
3 integrins (data not shown) are
RGD-dependent, we hypothesize that the YH and RGD peptides interact with the
v
3 and
v
5 integrins in a similar manner. Because
the YH peptide is much larger than the RGD peptide, their binding sites in
each of the
v
3 and
v
5 integrins could be different.
Alternatively, their binding sites may overlap or the binding of one peptide
might cause a conformational change that affects the binding of the other
peptide. If the YH motif interacts with both the
v
3 and
v
5 integrins, why are the
v
5 integrins of HUVECs not involved in
ig-h3-mediated adhesion? Our FACS analysis shows that the amount of the
v
5 integrin of HUVECs is much lower than
that of the
v
3 integrin. Therefore, the
v
3 integrin may function predominantly in
mediating HUVECs adhesion to
ig-h3. Alternatively, the YH motif may have
different binding affinity with the
v
3 and
v
5 integrins, or its binding affinity
depends on the activation state of the
v
3
and
v
5 integrins. Our previous mutational
study shows mutations on either YH residues or leucine/isoleucine residues of
either side of YH partially inhibit an activity of
ig-h3 in mediating
fibroblasts adhesion (4),
whereas in the present study, they did not significantly affect an activity of
ig-h3 in mediating HUVECs. It suggests that amino acid residues of YH
motif may be required to interact differently with each of the
v
3 and
v
5 integrins. It is well known that RGD
peptides in different contexts or constraining their conformation with
cross-linkers show large differences in their affinity for different integrins
(1518).
However, it remains to be further studied how the YH motif interacts
differently with the
v
3 and
v
5 integrins and how each amino acid
residue such as tyrosine, histidine, and leucine/isoleucine affects the
structure and activity of the YH motif.
The
v
3 integrin and its closely related
v
5 integrin have been known to play a role
in angiogenesis (19). Thus,
numerous monoclonal antibodies and many RGD mimetics have been tested for
their ability to block angiogenesis by inhibiting the function of these
integrins. It is still unclear whether the
v
3 and
v
5 integrins are proangiogenic or not
(20). In fact, recently, these
two integrins were hypothesized to be negative regulators of angiogenesis
(20). Nevertheless, many
molecules targeting these integrins have been reported to have anti-angiogenic
effects. Several groups suggested that the
v
3 integrin is a receptor for various
proteolytic fragments of extracellular matrix proteins that can act as
anti-angiogenic factors
(2123).
For example, a fragment of the type IV collagen
3 chain acts
through the
v
3 integrin to induce apoptosis
of endothelial cells, thereby inhibiting angiogenesis
(10). Based on findings that
the YH peptide interacts with the
v
3
integrin, we believed that the YH peptide may act through the
v
3 integrin of endothelial cells to inhibit
angiogenesis. We found that the YH peptide inhibited endothelial cell adhesion
and migration. Because angiogenesis depends on specific endothelial cell
adhesion and migration processes mediated by the
v
3 integrin
(24,
25), the YH peptide may
disrupt the interaction of endothelial cells with substrates such as
fibronectin and vitronectin. In vitro angiogenesis assays using
Matrigel showed that the YH peptide significantly reduces the number of
capillary tubes formed. This anti-angiogenic effect was further confirmed by
an in vivo experiment in C57BL/6 mice using Matrigel plugs
demonstrating that the YH peptide effectively inhibits bFGF-induced
neovascularization. The anti-angiogenic activity of the YH peptide does not
seem to be associated with cell proliferation because the YH peptide does not
significantly affect endothelial cell growth in the presence of serum or
growth factors such as bFGF and VEGF at a concentration of 500
µM (data not shown), which clearly shows anti-angiogenic
activity in both in vitro and in vivo assays.
We reported previously (4)
that the YH motif consisting of 18 amino acids is minimally required to
mediate cell adhesion and hence is less active than the fas-1 domain fragment
in inhibiting cell adhesion to
ig-h3. Expectedly, the ability of the YH
peptide to inhibit endothelial cell adhesion and migration is less than that
of the 4th fas-1 domain of
ig-h3 (data not shown). Accordingly, its
anti-angiogenic activity is weaker than that of the fas-1 domain fragment
(data not shown). For this reason, we are currently using the fas-1 domain
protein rather than the YH peptide in in vivo experiments to test its
anti-tumor effects.
The YH motif is highly conserved in many fas-1 domains of several proteins.
Recently, the human protein FEEL-1 (stabilin-1), which has seven fas-1 domains
and is expressed in endothelial cells, was suggested to play a role in
angiogenesis (26). Although
there is no direct in vivo evidence showing that the fas-1
domain-containing proteins are involved in angiogenesis, our results together
with the above report suggest that the fas-1 domain-containing proteins or
their degradation products may regulate angiogenesis and therefore could be
used in the development of drugs targeting angiogenesis.
 |
FOOTNOTES
|
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* This work was supported by National Research Laboratory Program
M10104
[GenBank]
00036-01J0000-01610. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
¶
To whom correspondence should be addressed: Dept. of Biochemistry, Kyungpook
National University School of Medicine, 101 Dongin-dong, Jung-gu, Taegu,
700-422, Korea. Tel.: 82-53-420-6933; Fax: 82-53-422-1466; E-mail:
iskim{at}knu.ac.kr.
1 The abbreviations used are: HUVECs, human umbilical vein endothelial cells;
FACS, fluorescence-activated cell sorter; BSA, bovine serum albumin; PBS,
phosphate-buffered saline; HEK, human embryonic kidney; WT, wild type; bFGF,
basic fibroblast growth factor. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Jeffrey Smith (Burnham Institute, San Diego) for providing the
3/293 cells.
 |
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