Potentiation of Endothelial Cell Proliferation by
Fibrin(ogen)-bound Fibroblast Growth Factor-2*
Abha
Sahni,
Lee Ann
Sporn, and
Charles W.
Francis
From the Department of Medicine, Vascular Medicine Unit, University
of Rochester School of Medicine & Dentistry, Rochester, New York
14642
 |
ABSTRACT |
Endothelial cell growth is stimulated by
fibroblast growth factor-2 (FGF-2), and both adhesion and proliferation
are modulated by interactions with fibrinogen and fibrin. Previous
evidence indicates that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin, suggesting that their effects on endothelial cells may be coordinated. In this study, we have, therefore,
investigated the ability of FGF-2 bound to fibrinogen and fibrin to
stimulate proliferation of endothelial cells. Human umbilical vein
endothelial cells were cultured in the presence of FGF-2 with or
without fibrinogen, and proliferation was assessed by microscopic
examination of cultures, incorporation of
[3H]thymidine and by cell counting. Cells cultured
in the presence of both FGF-2 and fibrinogen proliferated more rapidly
than those with FGF-2 alone and exhibited a decreased population
doubling time. At concentrations of FGF-2 up to 150 ng/ml, there was
greater endothelial cell proliferation in the presence of fibrinogen
than in its absence with the most pronounced effect below 1 ng/ml. The
maximum effect of fibrinogen was observed at a molar ratio of
fibrinogen to FGF-2 of 2:1, corresponding to the maximum molar binding
ratio. Endothelial cells proliferated when plated on fibrin or
surface-immobilized fibrinogen with FGF-2, indicating that FGF-2 bound
to surface-associated fibrin(ogen) retained activity. We conclude that
fibrinogen- or fibrin-bound FGF-2 is able to support endothelial cell
proliferation and that fibrinogen potentiates the proliferative
capacity of FGF-2.
 |
INTRODUCTION |
Endothelial cells normally have a low rate of proliferation in the
adult with a life span of 100-10,000 days (1), but the endothelium
retains its capacity for proliferation, which occurs physiologically in
the corpus luteum and uterus and also during wound healing. Endothelial
cell proliferation, differentiation and migration are also needed for
angiogenesis, an important process in many pathologic conditions
including tumor growth, diabetic retinopathy, inflammation, and
ischemic thrombotic diseases. Polypeptide growth factors play an
important regulatory role in angiogenesis, and several stimulatory and
inhibitory molecules have been identified (2, 3) including fibroblast
growth factor-2 (FGF-2,1
basic fibroblast growth factor), an 18-kDa polypeptide of the FGF
family, which exerts a variety of effects on many cells and organ
systems (4, 5).
Endothelial cells in culture require an FGF to support proliferation
and to prevent apoptosis (6). In addition, FGFs promote endothelial
cell migration (7, 8) and increase synthesis of several proteins that
are important in degradation of the extracellular matrix during cell
migration or angiogenesis including collagenase (8, 9), urokinase
plasminogen activator, urokinase plasminogen activator receptor
(8-11), and plasminogen activator inhibitor-1 (12, 13). FGFs also
regulate endothelial cell adhesion by modulating expression of integrin
receptors (14), and they can increase expression of vascular
endothelial growth factor, another angiogenic peptide (15). Although
FGF-1 and FGF-2 lack signal peptides, they are both active in the
pericellular environment and bind with high affinity to specific
receptor tyrosine kinases (16). FGFs are released from vascular cells
following injury, and FGF-2 mRNA is up-regulated in atherosclerotic
arteries (17) and following vessel injury (18).
The hemostatic system is activated at sites of tissue injury, and this
results in local formation of fibrin, which also plays a role in
regulating the endothelial cell responses required for healing and
angiogenesis. Endothelial cells adhere, spread and proliferate on both
fibrinogen and fibrin in vitro through binding to integrins
v
3 and
5
1
(19, 20), and fibrinogen supports leukocyte tethering to endothelial
cells at sites of inflammation through ICAM-1 (21). Although
endothelial cells are physiologically stable, when exposed normally to
a high concentration of fibrinogen in vivo, they interact
with fibrin uncommonly, and marked phenotypic change results.
Specifically, fibrin exposure in vitro results in loss of
monolayer organization, cell retraction, and migration (22, 23). Fibrin
also stimulates synthesis and secretion of tissue plasminogen activator
and prostacyclin (24, 25), induces interleukin-8 expression (26),
suppresses plasminogen activator inhibitor-1 release (13, 27), and
causes rapid mobilization of high molecular weight von Willebrand
factor from Weibel-Palade bodies (28). These responses require the
thrombin-induced cleavage of fibrinopeptide B from the fibrinogen B
chain, which results in exposure of a reactive site at the new amino
terminus of the fibrin
chain (29). An endothelial cell receptor
interacting with a site within the 15-42 region of the
chain has
been identified (30) and recently shown to be VE-cadherin (31).
We have recently demonstrated specific high-affinity binding of FGF-2
to fibrinogen and fibrin (32). This suggests a mechanism by which
fibrin can localize FGF-2 at sites of inflammation or tissue injury to
coordinate endothelial cell responses. Previous studies have shown that
FGF-2 specifically binds to extracellular matrix and is released by
heparinase and by heparin, and the proliferative activity of FGF-2 is
enhanced by binding to heparin (33-35). In this study we have
investigated the ability of fibrinogen- and fibrin-associated FGF-2 to
stimulate proliferation of endothelial cells in vitro. The
results indicate that FGF-2 associated with surface-immobilized
fibrinogen or fibrin retains its mitogenic activity, and that the
endothelial cell proliferative response to FGF-2 is potentiated by fibrinogen.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary endothelial cells were obtained from
human umbilical veins as described previously (36), seeded on 0.2% w/v
gelatin-coated 25-cm2 tissue culture flasks and cultured in
McCoy's 5A medium (Flow Laboratories, McLean, VA) containing 20%
fetal bovine serum (FBS), 50 µg/ml endothelial cell growth supplement
(ECGS) (Collaborative Research, Inc., Bedford, MA) and 100 µg/ml
heparin (Sigma) until they reached confluence, typically within 4-5
days. The cells were passaged up to two times before use and then
placed in suspension by trypsinization of monolayers. Cells were
suspended by rinsing in Hanks' balanced salt solution followed by
brief incubation with trypsin-EDTA (Life Technologies, Inc.). The cells
were pelleted by centrifugation for 10 min at 500 × g
and resuspended in McCoy's 5A medium. This wash procedure was repeated
twice before use in experimental protocols.
[3H]Thymidine Incorporation--
Cell
proliferation using [3H]thymidine incorporation was
quantitated as described previously (37). Briefly, approximately 2 × 104 endothelial cells suspended in McCoy's 5A medium
supplemented with 20% FBS, 50 µg/ml ECGS, and 100 µg/ml heparin
were plated in gelatin-coated 24-well, nontissue culture-treated plates
(Becton Dickinson & Co., NJ) and allowed to adhere for 6 h. The
medium was then removed, and the cells were washed twice with
serum-free McCoy's 5A medium. Serum-free medium was then added
containing 1% Nutridoma® (Roche Molecular Biochemicals),
25 ng/ml human recombinant FGF-2 (R&D Systems, Inc., Minneapolis, MN)
and 1 µCi/ml [3H]thymidine (NEN Life Science Products)
in the presence or absence of 10 µg/ml fibrinogen. After incubation
at 37 °C for 24 h, nonadherent cells were removed by washing
twice with ice-cold phosphate-buffered saline (PBS). To each well was
then added 500 µl of 10% ice-cold trichloroacetic acid, and
precipitates were collected on a filter using a manifold. Filters were
washed twice with ice-cold 5% trichloroacetic acid, followed by 95%
ethanol, allowed to air dry, and then suspended in scintillation fluid.
Acid precipitable counts per min (cpm) were quantitated using a
scintillation counter.
Fibrinogen and Fibrin Preparation--
Human fibrinogen was
obtained from American Diagnostica (Greenwich, CT), and copurifying
fibronectin was removed by gelatin-Sepharose chromatography (38).
Residual fibronectin remaining was further depleted by immunoaffinity
chromatography as described elsewhere (39). The fibronectin
concentration was determined by enzyme-linked immunosorbent assay
(American Diagnostica) and represented less than 0.02% of the total
protein. Cell culture wells were coated by incubation for 1 h at
25 °C with 0.4 ml of 10 µg/ml fibrinogen in McCoy's 5A medium.
Excess fibrinogen was aspirated, and the wells were washed twice with
McCoy's 5A medium before the cells were plated. Fibrin-coated wells
were prepared using 1 mg/ml fibrinogen in McCoy's 5A medium to which 1 unit/ml thrombin (Calbiochem-Novabiochem Corp.) was added, mixed, and
rapidly pipetted into 24-well cell culture plates. The solution was
aspirated after 45 s and before polymerization, leaving a thin
coating of fibrin on the surface. Wells coated with fibrinogen or
fibrin with FGF-2 were prepared in the same way except that 25 ng/ml
FGF-2 was added to the fibrinogen solution and incubated for 20 min at
37 °C before coating wells. Fibrin-coated wells were treated with 1 µg/ml
D-phenylalanyl-L-prolyl-L-arginylchloromethyl ketone (Bachem, Torrance, CA), a synthetic specific thrombin inhibitor, for 30 min to inhibit any remaining thrombin, and this was followed by
two washes with McCoy's 5A medium before plating the cells.
Measurement of Apoptotic Nuclei by Terminal Deoxynucleotidyl
Transferase in Situ Labeling (TUNEL)--
Endothelial cells cultured
on Thermanox® coverslips (Marsh Biomedical Company,
Rochester, NY) were fixed in 3.7% formaldehyde in PBS for 20 min,
postfixed in ethanol-acetic acid (2:1) at
20 °C for 5 min and
rinsed three times in PBS. Coverslips were then stained using the TUNEL
method (40) using the Apoptag® kit (Oncor®,
Gaithersburg, MD). Coverslips were then incubated with terminal deoxynucleotidyl transferase and digoxigenin-dUTP and stained with
fluorescein isothiocyanate-anti-digoxigenin antibody according to the
manufacturer's instructions. They were then mounted (cells facing up)
on glass microscope slides using Gel/Mount® (Biømedia
Corp., Foster City, CA). Propidium iodide counterstain (3 µg/ml in
PBS) was applied, and the slide was covered with a glass coverslip; the
edges were sealed using rubber cement, and the slides were stored at
20 °C. Cells were viewed using a Nikon Eclipse E-800 fluorescence
microscope equipped with a dual wavelength filter cube. Normal nuclei
exhibit orange fluorescence due to propidium iodide staining, whereas
apoptotic nuclei, which have incorporated the digoxigenin labeled
nucleotides, exhibit green fluorescence.
Determination of Population Doubling Time--
Endothelial cells
were grown in McCoy's 5A medium containing 20% FBS, 25 ng/ml FGF-2,
and 1 unit/ml hirudin with or without 10 µg/ml fibrinogen. Cells were
passaged at a ratio of 1:3 and plated in the presence or absence of
fibrinogen up to passage 10. Before each passage, photographs were
taken, and the number of cells per field was counted to determine the
fold increase. Population doubling time was calculated using the
following formula: population doubling time = days in culture/fold
increase in cell number. The fold increase = 2n, where
n = number of doublings.
Statistical Analysis--
Each experiment was performed at least
three times, and either triplicate or quadruplicate wells were used in
each experiment. The significance of differences in means was
determined using a two-tailed Student's t test. Variance is
described as ±S.E.
 |
RESULTS |
FGF-2 is needed to support endothelial cell growth and survival in
culture. To determine whether it retains this activity when bound to
fibrinogen, cells were cultured in medium containing 1.5-3
nM FGF-2 in the presence or absence of 30 nM
fibrinogen. Because our previous studies demonstrated that FGF-2 binds
to fibrinogen with an apparent kD of 1.3 nM, the concentration of free FGF-2 under these conditions
would be less than 0.01 nM and insufficient to support cell
growth. Cells proliferated well in the presence of fibrinogen and FGF-2
and appeared normal microscopically (Fig.
1A). Apoptosis, which is known
to be a consequence of growth factor deprivation, did not occur when
cells were cultured with fibrinogen and FGF-2 (Fig. 1B) but
was observed with fibrinogen but no FGF-2 (Fig. 1C). These
initial microscopic observations indicated that the presence of
fibrinogen did not block the biologic activity of FGF-2.

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Fig. 1.
Growth and survival of endothelial cells in
the presence of FGF-2 and fibrinogen. Endothelial cells were
plated on gelatin-coated wells and incubated in McCoy's 5A medium with
20% FBS and 100 µg/ml heparin and allowed to adhere for 6 h.
The medium was then removed, cells were washed twice, and then
serum-free medium was replaced with 1% Nutridoma and 10 µg/ml
fibrinogen in the presence (panels A and
B) and absence (panel C) of 25 ng/ml
FGF-2. The cells were then cultured for an additional 24 h and
photographed live using phase contrast (panel A).
For panels B and C, the cells were
fixed and stained using the TUNEL method, counterstained with propidium
iodide, and viewed under dual wavelength excitation. Normal nuclei
exhibit orange fluorescence (propidium iodide;
panel B), and apoptotic nuclei typically appear
condensed with green fluorescence (panel
C). Bar in panel A = 1 mm and bar in
panels B and C = 100 µm.
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The effect of fibrinogen on FGF-2-induced proliferation of endothelial
cells was further examined by cell counting and determination of
population doubling times. Cells were grown in the presence or FGF-2
with or without fibrinogen in the medium. Hirudin was included to
inhibit the low levels of thrombin present in fetal bovine serum and
prevent conversion of fibrinogen to fibrin. Population doubling times
were calculated using endothelial cells between passages 2 and 8, which
were cultured using 25 ng/ml FGF-2 in the presence or absence of 10 µg/ml fibrinogen. A population doubling time of 5.6 ± 0.2 days
(n = 7) was determined in the absence of fibrinogen,
whereas it was shortened to 2.8 ± 0.3 days (n = 7) (p < 0.0001) when fibrinogen was present in the
culture medium. Passage 1 was not used because cells in primary culture
and first passage retain the capacity for proliferation independently
of exogenously added growth factors. Doubling times were prolonged at
later passages probably due to the onset of senescence. The maximum
cell density achieved at confluence decreased with time in culture (not
shown), but was not influenced by presence or absence of fibrinogen. No
morphologic differences were observed microscopically between cultures
with or without fibrinogen.
The effect of fibrinogen on FGF-2-mediated endothelial cell
proliferation was also evaluated by [3H]thymidine
incorporation (Fig. 2). In the absence of
FGF-2, there was no increase in [3H]thymidine uptake
between 6 and 48 h, indicating little or no cell proliferation.
Also, little incorporation resulted from the addition of 10 µg/ml
fibrinogen to the medium in the absence of FGF-2. As expected, FGF-2
alone stimulated proliferation, and 3H-thymidine
incorporation was increased over control at all times points. Addition
of fibrinogen potentiated proliferation mediated by FGF-2, with the
greatest effect evident at 24 h (p < 0.04). There
was little additional proliferation at later times, reflecting contact
inhibition (not shown). Microscopic examination confirmed slower growth
in the absence of fibrinogen, and a longer time was needed to reach
confluence.

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Fig. 2.
Time-dependent endothelial cell
proliferation in the presence or absence of FGF-2 and fibrinogen.
Endothelial cells were plated on gelatin-coated wells in McCoy's 5A
medium supplemented with 20% FBS, 50 µg/ml ECGS, and 100 µg/ml
heparin and allowed to adhere for 6 h. The cells were then washed
twice with McCoy's medium and incubated in serum-free medium
containing 1% Nutridoma, 1 µCi/ml [3H]thymidine with
or without FGF-2 and fibrinogen for varying time intervals. Isotope
incorporated into DNA was precipitated with trichloroacetic acid,
collected by vacuum filtration, and measured by scintillation counting.
At each time point, data are shown for cells incubated in the absence
of FGF-2 or fibrinogen (gray bars), with 25 ng/ml FGF-2, and
no fibrinogen (open bars), with 25 ng/ml FGF-2 and 10 µg/ml fibrinogen (hatched bars) or 10 µg/ml fibrinogen
and no FGF-2 (black bars).
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Cell proliferation was dependent on FGF-2 concentration in the presence
and absence of fibrinogen (Fig. 3), with
a maximum 6.4-fold increase in [3H]thymidine
incorporation at 25 ng/ml in the absence of fibrinogen. There was,
however, greater proliferation at all concentrations of FGF-2 in the
presence of fibrinogen than in its absence. The effect of fibrinogen
was particularly evident at FGF-2 concentrations below 1 ng/ml (Fig. 3,
inset). For example, at 0.1 ng/ml FGF-2, there was 1.2 ± 0.5-fold increase over baseline in the absence of fibrinogen but
2.8 ± 0.5-fold in its presence (p < 0.001). The
FGF-2 concentration dependence of endothelial cell proliferation was
more complex in the presence of fibrinogen than in its absence. Maximum
proliferation in the presence of both fibrinogen and FGF-2 occurred at
25 ng/ml, with a 13.7 ± 2.7-fold increase in comparison with
5.5 ± 0.2-fold in the absence of fibrinogen at the same FGF-2 concentration (p < 0.03). In the presence of
fibrinogen, proliferation decreased at FGF-2 concentrations over 25 ng/ml, declining to 8.8 ± 0.6-fold over baseline at 100 ng/ml and
8.1 ± 0.5-fold at 150 ng/ml. At both latter concentrations,
however, the [3H]thymidine incorporation remained higher
than in the absence of fibrinogen.

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Fig. 3.
Effect of fibrinogen on the FGF-2
concentration dependence of endothelial cell proliferation.
Endothelial cells were plated on gelatin-coated wells in McCoy's 5A
medium supplemented with 20% FBS, 50 µg/ml ECGS, and 100 µg/ml
heparin and allowed to adhere for 6 h. The medium was then
removed, the cells washed twice, and then cells were overlaid with
serum-free medium containing 1% Nutridoma, and 25 ng/ml FGF-2 and 1 µCi/ml [3H]thymidine in the presence (solid
line) or absence (dotted line) of 10 µg/ml
fibrinogen. After 24 h of incubation, nonadherent cells were
removed, and the isotope incorporated into DNA was extracted with
trichloroacetic acid. Precipitates were collected by vacuum filtration,
and incorporated isotope was measured by scintillation counting; cpm
per well was determined and normalized to the results obtained in the
absence of FGF-2 and fibrinogen.
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The capacity to potentiate FGF-2 stimulated cell proliferation was also
characterized over a range of fibrinogen concentrations (Fig.
4). An increase in cell proliferation was
observed at 0.25 µg/ml (0.75 nM), and there was
progressive enhancement of activity to a maximum of 2.9-fold over
baseline at 5 µg/ml (15 nM), representing a molar ratio
of fibrinogen to FGF-2 of 2:1. At higher concentrations of fibrinogen,
no increased effect on cell proliferation was observed.

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Fig. 4.
Fibrinogen concentration dependence of
FGF-2-mediated endothelial cell proliferation. Endothelial cells
were plated on gelatin-coated wells in McCoy's 5A medium supplemented
with 20% FBS, 50 µg/ml ECGS, and 100 µg/ml heparin and allowed to
adhere for 6 h. The medium was then removed. Cells were washed
twice, and then serum-free medium was added containing 1% Nutridoma,
and 25 ng/ml FGF-2, and 1 µCi/ml [3H]thymidine in the
presence of different concentrations of fibrinogen The molar ratios of
FGF-2 to fibrinogen varied from 0.1-2. After 24 h of incubation,
nonadherent cells were removed, and isotope incorporated into DNA was
extracted with trichloroacetic acid. Precipitates were collected by
vacuum filtration, and incorporated isotope was measured by
scintillation counting.
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Following tissue injury, thrombin converts fibrinogen to fibrin, an
insoluble polymer that forms the initial matrix required for cell
adhesion and wound healing. To determine whether FGF-2 was active when
bound to fibrinogen or fibrin presented as an adhesive substrate, we
prepared surfaces coated with either fibrin or fibrinogen with or
without added FGF-2. Endothelial cells were cultured on these surfaces
and viewed microscopically. Cells grown on surfaces of fibrinogen (Fig.
5A) or fibrin (Fig.
5B) in the absence of FGF-2 were consistently sparse, but
incorporation of FGF-2 into the matrix resulted in marked proliferation
(Fig. 5, C and D). Gelatin was used as an
alternative adhesive substrate, and wells coated with a solution of
gelatin to which FGF-2 had been added did not adequately support
proliferation (Fig. 5E). In control wells, however,
endothelial cells grew well on a coating of gelatin if FGF-2 was
included in soluble form in the culture medium (Fig. 5F). As
quantitated by [3H]thymidine incorporation, proliferation
was minimal on a surface of either fibrinogen or fibrin in the absence
of FGF-2 (Fig. 6), but was significantly
enhanced with FGF-2 immobilized with either fibrinogen or fibrin
(p < 0.04 for both).

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Fig. 5.
Endothelial cells cultured on fibrinogen or
fibrin in the presence or absence of FGF-2. Culture wells
containing coverslips were coated with fibrinogen (A and
C) or fibrin (B and D) with no FGF-2
(A and B) or with 25 ng/ml FGF-2 (C
and D). In control experiments, cells were plated on tissue
culture wells coated with gelatin and 25 ng/ml FGF-2 (E) or
gelatin alone (F). The cells were cultured in McCoy's
medium containing no FGF-2 (A-E) or containing
25 ng/ml FGF-2 (F). After 24 h, the cells were fixed,
permeabilized in 0.5% Triton X-100, washed with PBS twice, stained
with propidium iodide, and the coverslips were viewed with a
fluorescence microscope. Bar = 100 µm.
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Fig. 6.
Endothelial cell proliferation on fibrinogen
or fibrin coated surfaces in the presence or absence of FGF-2.
Endothelial cells were plated in wells coated with fibrinogen
(open bars) or fibrin (filled bars) in the
absence or presence of 25 ng/ml FGF-2. After 6 h the medium was
removed and replaced with serum-free medium containing 1 µCi/ml
[3H]thymidine, and the cultures were incubated for an
additional 24 h.
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Samples of culture medium containing fibrinogen were collected
following the 24 h incubation and analyzed by SDS-polyacrylamide gel electrophoresis. The migration pattern of A
, B
, and
chains was unchanged and showed no fibrinopeptide A or fibrinopeptide B
cleavage, indicating that there was no proteolytic degradation or
conversion of fibrinogen into fibrin (data not shown). To determine whether other adhesive glycoproteins also stimulated cell proliferation with FGF-2, endothelial cells were also cultured in the presence of
vitronectin (10 µg/ml) or fibronectin (10 µg/ml). Neither
fibronectin nor vitronectin increased proliferation significantly,
indicating the specificity of fibrinogen in enhancing the effect of
FGF-2.
 |
DISCUSSION |
The findings presented demonstrate that fibrin- or
fibrinogen-bound FGF-2 retains biological activity. Cells cultured in
medium containing 1.5 nM FGF-2 and 30 nM
fibrinogen supported proliferation and did not undergo apoptosis, which
is known to occur under conditions of growth factor deprivation (6). At
the concentrations of fibrinogen and FGF-2 used in these experiments,
the amount of free FGF-2 would be insufficient to support cell growth.
FGF-2 associated with surface-immobilized fibrinogen or fibrin was also active as indicated by its ability to support growth (Fig. 5). Cell
proliferation was quantitated by [3H]thymidine
incorporation, determination of population doubling times, and cell
counting. The findings with each of these methods indicated that the
proliferative potential of FGF-2 is enhanced in the presence of
fibrinogen. The population doubling time was shorter with fibrinogen,
the proliferative rate was greater (Fig. 2), and the cells responded to
a lower concentration of FGF-2 when presented in combination with
fibrinogen (Fig. 3). At all FGF-2 concentrations, fibrinogen increased
its proliferative capacity (Fig. 3), and the maximum effect was
observed at a molar ratio of fibrinogen to FGF-2 of 2:1 (Fig. 4),
corresponding to the maximum molar binding ratio (32).
Fibrinogen is an adhesive substrate for endothelial cells, but it is
unlikely that the enhanced proliferation observed was due to an effect
of fibrinogen on adhesive properties. Cells were fully spread before
FGF-2 exposure, and no change in cell spreading was observed during
incubation with fibrinogen. No significant increase in FGF-2-induced
cell proliferation was observed with fibronectin or vitronectin,
indicating the fibrinogen effect was specific. Also, enhanced cell
proliferation was observed with fibrinogen even in medium containing
20% fetal bovine serum, which is rich in adhesive proteins, confirming
the specificity of the enhancement by fibrinogen. Proliferation was
evaluated using several methods because of the experimental limitations
of each. Cultures were examined microscopically to evaluate morphologic
characteristics including spreading and apoptosis in addition to
proliferation. [3H]Thymidine incorporation was measured
as an overall index of DNA synthesis, recognizing that proliferation
may be underestimated with prolonged exposures. Population doubling
time was determined as a direct measure of cell proliferation during
prolonged exposure to FGF-2 and fibrinogen with multiple cell passages.
Each of these methods indicated significant enhancement of FGF-2
proliferative capacity in the presence of fibrinogen.
The mechanism by which fibrinogen potentiates FGF-2 activity is not
known but may involve receptor clustering or coordination of cell
signaling. Because fibrinogen is a dimeric molecule, binding of two or
more FGF-2 molecules could increase cell activation through receptor
dimerization analogous to that observed with heparin (41). Also,
binding of a complex of fibrinogen and FGF-2 could result in
co-localization of integrin and FGF receptors at the focal adhesion
complex, contributing to signal integration (42). Fibrinogen may
protect FGF-2 from inactivation by serum or cell-associated proteases,
thereby prolonging and increasing its activity. Such protection from
proteolytic degradation has been observed for FGF-2 bound to
extracellular matrix (43, 44). Additionally, a recent report indicates
that FGF-2 can bind to
v
3 (45), an
endothelial cell integrin receptor that also binds fibrinogen,
suggesting that the adhesive and proliferative activities of FGF-2 and
fibrinogen may be coordinated through a single receptor.
Endothelial cell responses to injury and angiogenesis are dependent on
both growth factor stimulation and interactions with matrix components.
The importance of endothelial cell-matrix interactions in angiogenesis
is evident from the binding of FGF-2 to extracellular matrix heparan
sulfate proteoglycans. Although of lower affinity than the binding to
specific tyrosine kinase receptors, the association with heparan
sulfates is physiologically important in protecting FGFs from
proteolytic degradation (43, 44, 46) and by providing a local reservoir
of growth factor that can be released by enzymes that degrade
proteoglycans (47, 48). Additionally, heparan sulfates increase the
binding affinity of FGFs for specific receptors and facilitate
presentation to transmembrane signaling receptors (41). The mechanisms
by which heparan sulfate proteoglycans modulate FGF function remain
under investigation, but they may act to reduce the dimensionality of
ligand diffusion to a plane from three dimensions (48). Endothelial
cells are exposed to fibrin both pathologically and in response to
vessel injury where fibrin forms the initial matrix necessary for cell
organization and healing. As such, fibrin could play a role similar to
that of the extracellular matrix in binding FGF-2, which both localizes and prolongs its action.
The potential to manipulate angiogenesis therapeutically is now being
realized in initial clinical trials, and strategies to either inhibit
or stimulate new vessel growth appear promising (49, 50). The
association of FGF-2 with fibrin(ogen) is relevant to the development
of these therapeutic strategies. Because of its high plasma
concentration, binding to fibrinogen will affect distribution of FGF-2
if administered systemically. Fibrin binding may also be important
therapeutically. For example, in the successful initial trial in
coronary artery disease (51), FGF-1 was injected locally near the site
of vascular anastomosis where fibrin formation would be expected.
Binding of the growth factor to fibrin would serve to localize and
possibly increase its effect, contributing to the observed
neovascularization. The binding of FGF-2 to fibrin(ogen) and the
effects on endothelial cell proliferation suggest a new level of
coordination between the hemostatic system and cell regulatory growth
factors in the vascular response to injury and angiogenesis.
 |
ACKNOWLEDGEMENT |
The assistance of Carol Weed in preparing this
manuscript is acknowledged gratefully.
 |
FOOTNOTES |
*
This work was supported in part by Grants HL-30616 and
HL-07152 from the NHLBI, National Institutes of Health, Bethesda,
Maryland.The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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 Medicine,
Vascular Medicine Unit, Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-3762; Fax:
716-473-4314; E-mail: charles_francis{at}URMC.rochester.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
FGF-2, fibroblast
growth factor-2;
PBS, phosphate-buffered saline;
ECGS, endothelial cell
growth supplement;
FBS, fetal bovine serum;
TUNEL, terminal
deoxynucleotidyl transferase in situ labeling.
 |
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