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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Vascular Medicine Unit, Department of
Medicine, University of Rochester School of Medicine and Dentistry,
Rochester, NY.
Vascular development and response to injury are regulated by
several cytokines and growth factors including the members of the
fibroblast growth factor and vascular endothelial cell growth factor
(VEGF) families. Fibrinogen and fibrin are also important in these
processes and affect many endothelial cell properties. Possible
specific interactions between VEGF and fibrinogen that could play a
role in coordinating vascular responses to injury are investigated.
Binding studies using the 165 amino acid form of VEGF immobilized on
Sepharose beads and soluble iodine 125 (125I)-labeled
fibrinogen demonstrated saturable and specific binding. Scatchard
analysis indicated 2 classes of binding sites with dissociation constants (Kds) of 5.9 and 462 nmol/L.
The maximum molar binding ratio of VEGF:fibrinogen was 3.8:1. Further
studies characterized binding to fibrin using 125I-labeled
VEGF- and Sepharose-immobilized fibrin monomer. These also demonstrated
specific and saturable binding with 2 classes of sites having
Kds of 0.13 and 97 nmol/L and a molar binding ratio of 3.6:1. Binding to polymerized fibrin demonstrated one binding
site with a Kd of 9.3 nmol/L. Binding of VEGF
to fibrin(ogen) was independent of FGF-2, indicating that there are
distinct binding sites for each angiogenic peptide. VEGF bound to
soluble fibrinogen in medium and to surface immobilized fibrinogen or
fibrin retained its capacity to support endothelial cell proliferation.
VEGF binds specifically and saturably to fibrinogen and fibrin with
high affinity, and this may affect the localization and activity of VEGF at sites of tissue injury.
(Blood. 2000;96:3772-3778) Activation of the coagulation system leads to
fibrin formation, which both stabilizes the hemostatic plug and
provides the temporary matrix required for subsequent cellular
responses of wound and vessel repair. In these processes the role of
fibrin is not passive, but rather it actively directs cellular
responses through specific receptor-mediated interactions with cells of the blood and vessel wall. These result in fibrin-specific responses of
endothelial cells including adhesion and spreading,1
proliferation,2 protein synthesis3 and
secretion,4 and angiogenesis.5 Fibrin is also
a component of the stroma of many tumors and is deposited as a result
of increased vascular permeability, extravasation of plasma protein,
and the presence of tissue factor and other procoagulants associated
with tumors.6,7 Fibrinogen and fibrin are also implicated
in the development, progression, and thrombotic complications of
atherosclerosis.8
The angiogenic peptide vascular endothelial growth factor (VEGF), also
known as vascular permeability factor, stimulates both increased
microvascular permeability, which leads to local fibrin formation, and
also endothelial cell proliferation needed for angiogenesis.9 There are 5 different human VEGF isoforms
consisting of monomers of 121, 145, 165, 189, and 206 amino acids that
are derived from a single gene by alternative splicing.10
The active forms of VEGF are homodimers, and the best characterized
VEGF species is the heparin-binding 165 amino acid
form.10,11 VEGF is a multifunctional cytokine for
endothelial cells expressing VEGF receptor-1 (flt-1)12 and
receptor-2 (flk-1/KDR),13,14 and it exerts several
effects including von Willebrand factor release,15
induction of the expression of integrins,16,17 interstitial collagenase,18 plasminogen activators,
plasminogen activator receptor, plasminogen activator
inhibitor-1,19-21 and stimulating migration of endothelial
cells.22 VEGF also induces enhanced tissue factor
expression23 and stimulates migration of monocytes
expressing the VEGF receptor-1.24 The role of VEGF in
vessel injury and repair is further supported by evidence that VEGF
synthesis and release from vessel wall cells increase after injury25 and that VEGF messenger RNA (mRNA) is upregulated
in atherosclerotic arteries.26 Recent in vitro and in vivo
studies have revealed that the transformation of endothelial cells to an angiogenic phenotype is induced by several factors, including VEGF,
fibroblast growth factors (FGFs), hepatocyte growth factor, interleukin-8, and others,27 but the interaction between
VEGF and its receptors is the most important angiogenic event in the physiological and pathological angiogenesis.
The need for fibrin to support endothelial cell spreading, migration,
and angiogenesis and the potent stimulation of similar angiogenic
responses by VEGF suggest that these processes may be interrelated.
This concept is supported by evidence that fibrin clots provide a good
matrix to support VEGF-stimulated angiogenesis in vitro28
and that angiogenic peptide FGF-2 binds specifically to fibrinogen and
fibrin.29 We have, therefore, investigated the association
of VEGF with fibrinogen and fibrin. The results demonstrate specific
and saturable high-affinity binding of functionally active
VEGF with implications for colocalization of angiogenic peptides at
sites of injury and for distribution of pharmacologically administered VEGF.
Protein preparation
Binding of fibrinogen to immobilized VEGF
To characterize the protein that was bound to immobilized VEGF, 1 mg/mL 125I-fibrinogen was passed through a column of immobilized VEGF. The column was washed with 0.1 mol/L SPB (pH 7.4) containing 0.25 mol/L NaCl to remove unbound radioactivity. Bound protein was then eluted by the addition of 2 mg/mL unlabeled fibrinogen or ovalbumin as a control, and 200 µL aliquots were collected and counted. Aliquots of selected fractions were electrophoresed on sodium dodecyl sulfate (SDS) 7% polyacrylamide gels after disulfide bond reduction, dried, and used to prepare autoradiograms. Binding of 125I-VEGF to fibrin monomer A similar approach was used with incubation of 125I-VEGF with fibrin monomer immobilized on Sepharose beads. Affigel-15 beads were first incubated with 1 mg/mL purified monoclonal antibody J88B, which is directed against a site within the sequence arganine 63-methionine 78 (Arg63-Met78) of the human fibrinogen -chain,32 in 0.2 mol/L sodium bicarbonate
buffer (pH 8.3) and gently mixed at 25°C for 2 hours. Residual sites
were blocked by incubation in 1 mol/L ethanolamine (pH 8.0), and
suspension was washed several times with 0.2 mol/L sodium bicarbonate
buffer (pH 8.3) containing 0.25 mol/L NaCl. Gel containing bound
antibody was then incubated with 200 µg/mL fibrinogen in SPB (pH 7.4)
containing 0.25 mol/L NaCl to remove unbound fibrinogen. This was
continued until no further fibrinogen was removed, as determined by
monitoring the optical density at 280 nmol/L. To convert bound
fibrinogen to fibrin monomer, beads were incubated with 0.5 U/mL
thrombin at 37°C for 90 minutes. Characterization of binding of
125I-VEGF to fibrin monomer was performed in the same way
as 125I-fibrinogen binding to immobilized VEGF (above).
125I-VEGF at concentrations from 0.1-100 nmol/L was
incubated with a 0.02-mL suspension of beads containing 0.1 µg fibrin
in a final volume of 0.1 mL. Non-specific binding was determined in
parallel experiments using a 10-fold molar excess of unlabeled VEGF.
Specificity of the binding of VEGF to fibrin was confirmed by
competition experiments in which 0.1 nmol/L 125I-VEGF was
incubated with 1 µg/mL immobilized fibrin monomer in a final volume
of 0.1 mL, and the binding was competitively inhibited by unlabeled
VEGF at concentrations from 0.1-100 nmol/L.
Binding of VEGF to polymerized fibrin 125I-VEGF at concentrations of 0.1-100 nmol/L was added to 100 µg/mL fibrinogen in 0.1 mol/L Tris (tris[hydroxymethyl] aminomethane) buffer containing 0.25 mol/L NaCl. Thrombin was then added to a final concentration of 0.5 U/mL, which resulted in clotting of the solution. Following incubation at 37°C for 30 minutes, the clot and supernatant were separated by vacuum filtration using GF/C glass micro fiber filters (Sigma Chemical, St Louis, MO) previously soaked overnight in a solution of 0.5% polyvinylpyrolidone and 0.1% Tween-20 to reduce non-specific binding. The clot on the filter was washed quickly with cold 0.1 mol/L Tris buffer containing 0.25 mol/L NaCl, and associated radio-label was measured. Non-specific binding was determined in parallel experiments incorporating a 10-fold molar excess of unlabeled VEGF.Preparation of fibrinogen- and fibrin-coated surfaces Cell culture wells were coated by incubation for 1 hour at 25°C with 0.4 mL of 10 µg/mL fibrinogen in McCoy 5A medium (Flow Laboratories, McLean, VA). Excess fibrinogen solution was aspirated, and the wells were washed twice with McCoy 5A medium before the cells were plated. Fibrin-coated wells were prepared using 1 mg/mL fibrinogen in McCoy 5A medium to which 1 U/mL thrombin (Calbiochem-Novabiochem) was added, mixed, and using a pipette, rapidly added to 12-well nontissue culture-treated cell culture plates. The solution was aspirated after 45 seconds and before polymerization, thereby leaving a thin coating of fibrin on the surface. Wells coated with fibrinogen or fibrin with VEGF were prepared in the same way except that 20 ng/mL VEGF was added to the fibrinogen solution and incubated for 20 minutes at 37°C prior to 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 minutes to inhibit any remaining thrombin, and this was followed by 2 washes with McCoy 5A medium before plating the cells.Cell culture Primary endothelial cells were obtained from human umbilical veins as described previously;33 seeded on 0.2% wt/vol gelatin-coated 25-cm2 tissue culture flasks; and cultured in McCoy 5A medium 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 to 5 days. The cells were passaged up to 2 times before use and then placed in suspension by trypsinization of monolayers. Cells were suspended by rinsing in Hanks balanced salt solution (HBSS) followed by brief incubation with trypsin-EDTA (ethylenediamine tetraacetic acid) (Gibco Life Technologies, Inc, Grand Island, NY). The cells were pelleted by centrifugation for 10 minutes at 500g and resuspended in McCoy 5A medium in the absence of serum. This wash procedure was repeated twice prior to use in experimental protocols.Hydrogen 3-thymidine incorporation Approximately 2 × 104 human umbilical vein endothelial cells suspended in McCoy 5A medium supplemented with 20% FBS, 50 µg/mL endothelial cell growth supplement, and 100 µg/mL heparin were plated in gelatin-coated 12-well plates (Becton Dickinson & Company, Rutherford, NJ) and allowed to adhere for 6 hours. The medium was then removed, and the cells were washed twice with McCoy 5A medium. We then added serum-free medium containing 1% Nutridoma (Boehringer Mannheim, Indianapolis, IN), 20 ng/mL VEGF, or 25 ng/mL FGF-2 and 0.037 MBq/mL (1 µCi/mL) hydrogen 3 (3H)-thymidine (New England Nuclear, Boston, MA) in the presence or absence of 10 µg/mL fibrinogen. After incubation at 37°C for 24 hours, nonadherent cells were removed by washing twice with ice-cold phosphate-buffered saline. Then 500 µL 10% ice-cold trichloroacetic acid was added to each well, and precipitates were collected on a filter using a filtration manifold. Filters were washed twice with ice-cold 5% trichloroacetic acid (TCA) followed by 95% ethanol, allowed to air dry, and then suspended in scintillation fluid. Acid precipitable counts per minute were quantitated using a scintillation counter.Data analysis Unless otherwise indicated, the data are expressed as the mean ± SD. Scatchard analysis of the data was performed using the Ligand program34 (Biosoft, Ferguson, MO). Each experiment was performed at least 3 times, and either triplicate or quadruplicate wells were used in each experiment. The SD in means was determined using a 2-tailed Student t test.
Binding of fibrinogen to immobilized VEGF was saturable and
specific, with non-specific binding representing less than 20% of the
total (Figure 1A). Saturation of specific
binding occurred at a fibrinogen concentration of 150 nmol/L, and only
an increase in non-specific binding was observed at higher
concentrations. In control experiments there was a maximum of 5%
binding of 125I-fibrinogen over the same range of
concentrations to beads with immobilized anti-VEGF immunoglobulin only
or to beads with no protein-bound and active sites blocked with
ethanolamine. A plot of bound versus bound-free fibrinogen (Figure 1B)
was nonlinear, thereby suggesting the presence of more than one binding
site. This was confirmed by Scatchard analysis, which indicated that binding was best described by a 2-site model with apparent
Kds of 5.9 and 462 nmol/L. Bmax was
12 and 53 nmol/L for the high- and low-affinity sites, respectively,
and the maximum molar binding ratio of VEGF:fibrinogen was 3.8:1.
To further characterize the protein that bound to VEGF,
125I-fibrinogen was passed over a column of immobilized
VEGF. Following washing, the bound protein was eluted with 2 mg/mL
unlabeled fibrinogen (Figure 2) and
approximately 90% of bound radio-labeled protein rapidly eluted in 2 fractions. SDS-PGE (polyacrylamide gel electrophoresis) of the eluted
protein showed bands consistent with the A
Conversion of fibrinogen to fibrin is mediated by thrombin that cleaves
fibrinopeptides A and B from the A
Characterization of binding to polymerized fibrin presents technical
and interpretive problems because transport of VEGF into the gel may be
slow or incomplete, and access to potential binding sites within
individual fibrin fibers may also be restricted. We chose, therefore,
to add 125I-VEGF to a solution of fibrinogen, which was
then clotted with thrombin to limit problems of transport of VEGF into
a preformed gel. Total binding was measured with this clotting system
in the absence of an unlabeled competitor, whereas non-specific binding was measured in the presence of 10-fold molar access of unlabeled VEGF
(Figure 4A). Non-specific binding
represented up to 50% of the total (Figure 4A), and this was higher
than seen with binding to fibrinogen or fibrin monomer (Figures 1A and
3A), which possibly reflects entrapment of radio-label within the
fibrin gel. A plot of bound versus bound-free 125I-VEGF was
linear (Figure 4B), and Scatchard analysis identified a single binding
site with an apparent Kd of 9.3 nmol/L. The
maximum molar binding ratio of VEGF:polymerized fibrin was
0.1:1.
It has been shown previously that VEGF is a potent mitogen for
endothelial cells.35 To determine whether VEGF retains its activity when bound to fibrinogen, human umbilical vein endothelial cells were cultured in medium containing 20 ng/mL VEGF in the presence
or absence of 10 µg/mL fibrinogen, and proliferation of cells was
measured by 3H-thymidine incorporation after 24 hours. In
the presence of VEGF alone there was a 3.6 ± 1.3-fold increase in
3H-thymidine incorporation with medium alone containing no
VEGF (Figure 5). A similar
4.3 ± 1.8-fold increase in proliferation was observed with
fibrinogen-bound VEGF, which demonstrated no loss of activity with
fibrinogen binding. It has been demonstrated previously that a
combination of VEGF and FGF-2 stimulates greater endothelial cell
proliferation than either alone.36 We confirmed this
finding with a 6.1-fold increase with VEGF plus FGF-2 compared to a
3.3-fold increase with VEGF or a 4.1-fold increase with FGF-2 alone
(P < .05 and P < .03, respectively). The
addition of fibrinogen resulted in no additional proliferation compared
to VEGF plus FGF-2. Of note, endothelial cell proliferation was almost
the same with FGF-2 in combination with fibrinogen (6.5-fold), which was similar to FGF-2 plus VEGF.
Thrombin converts fibrinogen to fibrin, which provides the
initial matrix required for cell adhesion and wound healing after tissue injury. To determine whether VEGF was active when bound to
fibrinogen or fibrin as adhesive substrate, surfaces were coated with
fibrinogen or fibrin with or without VEGF and FGF-2. As assessed visually, cells grown on the surface of fibrinogen or fibrin in the
absence of growth factors were very sparse, but incorporation of VEGF
into the matrix resulted in more robust growth. As quantitated by
3H-thymidine incorporation, proliferation was minimal on
fibrinogen or fibrin without VEGF or FGF-2 (Figure
6). Proliferation was increased 4.8-fold
by the addition of FGF-2 on fibrinogen-coated surfaces
(P < .001) and 4.5-fold on fibrin-coated surfaces
(P < .005). Similarly, VEGF increased endothelial cell
proliferation 3.1 ± 0.9-fold on fibrinogen-coated surfaces
(P < .02) and 2.3 ± 0.5-fold on fibrin-coated surfaces
(P < .04). The combination of FGF-2 and VEGF did not
increase proliferation over FGF-2 alone on either surface.
The results presented demonstrate that VEGF binds specifically and saturably to both fibrinogen and fibrin and that fibrinogen-bound VEGF retains functional activity. Two distinct binding sites for VEGF were identified with both Sepharose-immobilized fibrinogen and fibrin monomer with similar Kds, and the maximum molar-binding ratios were 3.8 and 3.6, respectively. Considering that fibrinogen is a dimerically symmetric molecule37 and that 2 binding sites with different Kds were identified, the ratio of 4 VEGF to 1 fibrinogen would be expected and consistent with the presence of 2 structurally distinct and independent sites on each half-molecule. Caution is needed in interpreting curvilinear Scatchard plots as indicative of receptor heterogeneity, as low-affinity or non-specific binding can cause artifact.38 This problem was approached by estimating non-specific binding with experiments using an excess of unlabeled ligand and by computer modeling of the data. However, the relatively high amounts of non-specific binding could affect estimates of Kd for low-affinity sites. Binding of VEGF to polymerized fibrin differed from fibrinogen or fibrin monomer, as only a single binding site was identified, and the maximum molar-binding ratio of VEGF:fibrin was 0.1:1, indicating that VEGF bound to only 1 in 10 monomeric units. This is most likely explained by structural changes in fibrin after polymerization, which results in formation of highly organized fibers composed of multiple protofibril units aggregated laterally.39 This polymerization may prevent VEGF from binding to sites near to those involved in intermolecular association and may also restrict binding to less accessible molecules within aggregated fibers. FGF-2, another angiogenic peptide, also binds with high affinity to fibrin and fibrinogen,29 and this binding increases the capacity of FGF-2 to stimulate endothelial cell proliferation.40 The current results demonstrate that the binding of VEGF to fibrinogen is not inhibited by FGF-2, thereby indicating that the binding sites on fibrinogen for these 2 angiogenic peptides are independent and separate. This is further supported by differences in the maximum molar binding ratios for fibrin, which were 3.8 for VEGF but 2.0 for FGF-2.29 The binding affinity for both FGF-2 and VEGF increased after conversion of Sepharose-immobilized fibrinogen to fibrin monomer, suggesting that thrombin-mediated conformational changes affect the binding sites and result in increased affinity for both angiogenic peptides. Insulin-like growth factor-1, another peptide important in wound healing, also binds to fibrinogen and fibrin through insulin-like growth factor-1 binding protein-3 with Kd values of 0.67 and 0.7 nmol/L, respectively.41 These high-affinity interactions support a role for fibrinogen and fibrin as a reservoir of growth factors at the site of injury. The significance of VEGF binding to fibrinogen and fibrin must be
considered in relation to both the tissue distribution of VEGF and the
availability of other sites for binding within the vasculature. VEGF is
present in normal serum at a concentration of 0.9 pmol/L, and patients
with cancer may have elevated levels of up to 12 pmol/L.42
At a normal fibrinogen concentration of 7 µmol/L, essentially all
VEGF in plasma should be bound to fibrinogen considering the
Kds in the nmol/L range. VEGF also binds to
Vessel wall cells, including endothelial cells, synthesize and secrete VEGF,45,46 which then remains closely associated and bound to the cell membrane or matrix.47 Membrane binding is mediated in part by specific surface receptors of the tyrosine kinase family including flt-1 with a Kd of 10 to 12 pmol/L12 and flk-1/KDR, which has a Kd of 75 to 125 pmol/L.14 VEGF also binds specifically and with high affinity to intact bovine endothelial cells with Kds of 10 pmol/L and 100 pmol/L.48 The higher affinities for specific receptors compared to fibrin would result in preferential binding to cell receptors at a site of injury, although the total capacity of fibrin binding could be important because of the variable but potentially large capacity of the fibrin deposit. There are some parallels in this interaction between VEGF and heparan sulfate,49 which is also considered important in determining its association with vascular cells and extracellular matrix. VEGF binds to heparan sulfate and to heparin as an inactive complex that resists proteolytic degradation. The interaction of VEGF with abundant cell surface heparan sulfate may promote its binding to high-affinity transmembrane signaling receptors,50 and the affinity of VEGF for specific receptors on endothelial cells is increased 8-fold in the presence of heparin.50 Gitay-Goren et al50 have shown that cell-associated heparan sulfate enhances the binding of VEGF to high-affinity receptors in a manner similar to that reported for FGF-2. The binding of VEGF to heparan sulfate proteoglycans in the extracellular matrix may provide a protected reservoir of biologically active VEGF that is available following enzymatic release. Houck et al47 have demonstrated in vitro that plasmin can release VEGF, which is biologically active both as an endothelial cell mitogen and as a vascular permeability enhancing agent.47 Integration of cell responses to VEGF and fibrinogen may occur at the
receptor and the signal transduction level. Binding of endothelial
cells to matrix glycoproteins, including fibrinogen and fibrin, occurs
through integrin receptors, and this alters their sensitivity to growth
factor-induced signaling.51 In turn, VEGF stimulates
endothelial cell surface integrin expression,16,17 thereby
regulating the cell response to fibrinogen and fibrin. Both fibrinogen
and fibrin can support endothelial cell attachment through occupancy of
VEGF and FGF-2, both of which bind to fibrinogen and fibrin, have distinct and complementary actions in angiogenesis. VEGF is a direct-acting secreted polypeptide45 that can serve as an initiator of angiogenesis. This is in contrast to FGF-2, which lacks a signal peptide and requires prolonged exposure to cells for maximum effect. Pepper et al55 have shown that the greatest proliferative and angiogenic effects of VEGF were observed with FGF-2, and the combination was synergistic. Optimum angiogenesis requires the presence of both. The binding of both VEGF and FGF-2 to fibrinogen or fibrin may provide needed colocalization and spatial organization of both angiogenic peptides for maximum effect at sites of inflammation or injury.
Submitted May 24, 2000; accepted July 26, 2000.
Supported in part by grants HL-30616 and HL-07152 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
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: Charles W. Francis, Vascular Medicine Unit, PO Box 610, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642; e-mail: charles_francis{at}urmc.rochester.edu.
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Abstract
Introduction
, B
, and
