© 1999 American Heart Association, Inc.
Original Contributions |
Cooperation Between VEGF and TNF-
Is Necessary for Exposure of Active Tissue Factor on the Surface of Human Endothelial Cells
From the Institute of Pharmacological Sciences, University of Milan, Milan, Italy (M.C., E.T.); the Division of Thrombosis Research, Department of Medicine (P.L.A.G., Y.N.), the Departments of Medicine and Pathology (J.F.), the Ruttenberg Cancer Center (B.M.A.), and The Cardiovascular Institute, Department of Medicine, Mount Sinai School of Medicine (M.T.), New York, NY.
Correspondence to Marina Camera, PhD, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti, 9, 20133 Milan, Italy. E-mail Marina.Camera{at}unimi.it
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Abstract |
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Abstract—This study was undertaken to characterize tissue factor (TF) induction, localization, and functional activity in cultured human umbilical vein endothelial cells (HUVECs) exposed to recombinant vascular endothelial growth factor (rVEGF) and recombinant tumor necrosis factor-
(rTNF-). rVEGF (1 nmol/L) and
rTNF- (500 U/mL) synergistically increased TF mRNA, protein, and
total activity, as measured in cell lysates. To examine surface TF
expression, living cells were treated with antibody to TF and examined
microscopically. Almost no staining was seen in control cells or cells
treated with a single agent. In contrast, cells treated with both
agonists showed intense membrane staining with surface patches,
appearing as buds by confocal microscopy. To determine surface TF
activity, studies were performed using a parallel-plate flow chamber,
which allows detection of factor Xa generation on living cells. rVEGF
and rTNF- induced little surface TF activity (0.032±0.008 and
0.014±0.008 fmol/cm2, respectively). In combination, they
significantly increased TF expression on the cell surface (0.429±0.094
fmol/cm2, P<0.05). These data indicate that
the synergistic effect of rVEGF and rTNF- is necessary to generate
functional TF on the surface of endothelial cells. The
requirement for multiple agonists to expose active TF may serve to
protect endothelial cells from acting as a procoagulant
surface, even under conditions of cell perturbation.
Key Words: endothelium • factor Xa generation • procoagulant activity • cytokines • growth factors
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Introduction |
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Tissue factor (TF) is a 47-kDa transmembrane glycoprotein that activates the coagulation cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis.1 TF expression is regulated in a cell-specific manner. The protein is constitutively expressed by a number of extravascular cell types,2 3 4 whereas normally it is not expressed by cells that are in direct contact with blood, such as the endothelium lining normal arteries and circulating monocytes. However, numerous agents, eg, cytokines and growth factors, induce TF in blood monocytes and cultured endothelial cells.5 6 7 8 9 10 11 12 13 14 Functional TF is present at high levels on the surface of stimulated human monocytes.2 15 16 Studies with endothelial cells, on the contrary, indicate that little or no TF is present on the surface of agonist-induced endothelial cells, suggesting that these cells handle newly synthesized TF differently from monocytes or other cells constitutively expressing the protein.7 17 18 19 Regulation of TF synthesis is predominantly at the level of transcription and mRNA stabilization.11 20 21 22 After its synthesis, TF protein can be translocated to the cell surface, where it binds factor VII. The TF/factor VIIa complex activates factors IX and X, thus triggering coagulation. In this context, the expression of TF may be relevant to pathophysiological conditions only if it results in increased surface expression of TF.
The relationship between total TF synthesis and surface TF expression
has not been fully elucidated. Data suggest that exposure of
endothelial cells to recombinant tumor necrosis
factor- (rTNF-), though inducing substantial amounts of total TF,
results in limited expression of active protein on the cell
surface.18 Several agonists can potentiate the effect of
rTNF-, inducing endothelial cells to synthesize very
high levels of TF.7 13 No information, however, is
available concerning the effect of this potentiation on the surface
expression of functional TF.
In this study, we examined the localization of TF antigen and activity
on human umbilical vein endothelial cells (HUVECs)
in response to recombinant vascular endothelial growth
factor (rVEGF) and rTNF- by employing immunohistochemical techniques
and a parallel-plate perfusion system. Whereas either agonist alone
induced little surface TF activity, the combination of the 2 agonists
resulted in a >100-fold increase in surface TF activity. These data
were confirmed by immunolocalization of TF: almost no staining was seen
in control cells or in cells treated with a single agent, whereas cells
treated with both agonists showed intense membrane staining.
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Methods |
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Materials
PBS, medium 199, L-glutamine, penicillin, and streptomycin were from GIBCO Laboratories. FCS was from Mascia Brunelli. rTNF-
was obtained from Genentech. rVEGF, a 165–amino
acid variant of human VEGF, was from R&D Systems. All cell culture
reagents and solutions were checked for endotoxin contamination in the
Limulus lysate chromogenic assay (Coatest
endotoxin, Kabi Diagnostica). All reagents and media
contained <50 pg/mL lipopolysaccharide. Human coagulation factor VIIa was purchased from Novo Nordisk A/S 2880. Human coagulation factor X was purified as previously described.23 The chromogenic substrate for activated factor X, Ile-Pro-Arg-p-nitroaniline (IPR-pNA) was synthesized in our laboratory. PMSF, benzamidine, and aprotinin were from Sigma Chemical Co. Immunohistochemical-grade 3,3'-diaminobenzidine was purchased from Biogenex.
Cell Culture
HUVECs were isolated and characterized as previously
described.24 The cells used for these studies were between
the second and the fourth passage and had been derived from single
cords and cultured in medium 199 supplemented with 2 mmol/L
L-glutamine, 50 UI/mL penicillin, 50 µg/mL streptomycin,
20% heat-inactivated FCS, 50 µg/mL porcine intestinal
heparin, and 50 µg/mL crude extract of endothelial
cell growth factor obtained from bovine
hypothalamus.25
Determination of TF Activity by 1-Stage Clotting Assay
Confluent monolayers of HUVECs seeded in 12-well plates were
washed 3 times with Hanks' balanced salt solution and incubated at
37°C for various times up to 24 hours in incubation medium (culture
medium lacking FCS, heparin, and endothelial cell
growth factor but containing the appropriate concentration of the test
compounds or stock solvent; final concentration, maximally 0.1%
vol/vol). Immediately before the assay, cells were lysed with 15
mmol/L octyl-ß-D-glycopyranoside at 37°C for 10 minutes
and diluted with 25 mmol/L HEPES–saline. TF activity was
determined as procoagulant activity by a one-stage plasma
recalcification assay.26 Clotting times were quantified by
a standard curve obtained by serial dilution of a standard human
thromboplastin (Thromborel, Behring) preparation. Preincubation of
cells with a monoclonal antibody against TF (American
Diagnostica Inc, Greenwich, Conn) blocked procoagulant
activity by >90%, thus demonstrating the specificity of the assay. In
addition, an assay performed with plasma from donors congenitally
deficient in factor VII had no effect on clotting times. Data are
expressed as units of TF activity per microgram of cell protein, as
determined by the Bradford method.27
TF Staining
Immunostaining was performed using an antibody
that specifically recognizes the extracellular domain (residues 1-218)
of human TF (soluble TF antibody).28 29 30 For staining of
living HUVECs, cells grown on 9.4-cm2 chamber
slides were either left untreated or were treated with rVEGF, rTNF-,
or both as previously described. At the end of the incubation time,
HUVECs were exposed to 1 µg/mL soluble TF antibody for 30 minutes,
washed, and fixed in PBS–4% p-formaldehyde, pH 7.4.
Primary antibody was detected using a biotin-streptavidin–amplified
detection system (SuperSensitive kit, Biogenex) and developed with
3,3'-diaminobenzidine. Slides were counterstained with hematoxylin,
coverslipped, and examined by light microscopy. In most other studies,
agonist-treated HUVECs were first fixed in PBS–4%
p-formaldehyde, pH 7.4, and then incubated with 1 µg/mL
soluble TF antibody for 2 hours at 37°C. Primary antibody to soluble
TF was detected as previously described. For confocal microscopy, the
primary antibody was detected with fluoresceinated
anti-rabbit IgG antibody (Sigma). Slides were examined using a Leica
confocal laser scanning microscope. Positive control slides, nonimmune
negative controls, and processing controls were performed for each
antigen stain.
Determination of TF Activity by Monitoring Factor Xa
Generation
TF activity was also assessed by monitoring hydrolysis of factor
X. Confluent monolayers of HUVECs seeded on gelatin-coated,
9.4-cm2 plastic chamber slides (Nunc) were
incubated for various times in incubation medium containing the
indicated agents. Duplicate sets of chamber slides were stimulated in
each single experiment to allow for simultaneous
determination of total (cell lysates) and surface-associated TF. To
measure factor Xa generation in cell lysates, monolayers were lysed by
incubation with 15 mmol/L octyl-ß-D-glycopyranoside
for 15 minutes at 37°C. Factor VIIa (1 nmol/L) and factor X (150
nmol/L) were added sequentially. Aliquots of 40 µL were taken every
minute and added to 96-well plates, and the wells were filled with 100
µL EDTA buffer (bicine buffer, pH 8.5: 25 mmol/L EDTA, 1 g/L
BSA) to stop factor Xa production. Twenty-five microliters of a
15 mmol/L solution of IPR-pNA was added to each well, and
absorption at the 405-nm wavelength was measured in a kinetic plate
reader (Tmax, Molecular Devices). The concentration of factor Xa was
calculated from the slope of the absorption curve. To measure factor Xa
generation on the cell surface, the slides were mounted in a
parallel-plate flow chamber (1 mL/min volumetric flow rate, which
corresponds to a shear rate of 97
s-1).31 Factors X and VIIa were
continuously circulated for 15 minutes through the chamber by using a
peristaltic pump. Every 3 minutes, a 40-µL sample was taken and
assayed for factor Xa as described above. All experiments were
performed at room temperature. Femtomoles of TF per square centimeter
were obtained by assuming a Kcat of the
TF/factor VIIa complex of 300 min-1. This value
is based on factor X titration to lysed HUVECs and a fixed amount (1
pmol/L) of factor VIIa.
Determination of TF Antigen Levels
TF antigen was determined by an ELISA with the Imubind tissue
factor kit (American Diagnostica Inc). For this assay,
cells were solubilized with PBS containing 1% Triton X-100, 1
mmol/L PMSF, 100 U/mL aprotinin, and 5 mmol/L benzamidine. Values
are expressed as nanograms of TF antigen per microgram of protein.
Western Blot Analysis
Cell lysates separated by SDS–polyacrylamide gel
electrophoresis without reduction were transferred electrophoretically
to nitrocellulose membranes (Schleicher & Schuell). The membranes were
incubated overnight at 4°C in blocking buffer (20 mmol/L Tris
hydroxide, pH 7.4; 137 mmol/L NaCl; 0.1% Triton X-100; and
5% dry skim milk) and then with an anti-TF monoclonal antibody at room
temperature for 1 hour in blocking buffer. Membranes were washed in the
same buffer without milk and incubated at room temperature for 1 hour
with horseradish peroxidase–conjugated IgG sheep anti-mouse IgG.
Immunoblots were developed using enhanced chemiluminescence
(Amersham Corp).
[3H]Leucine Incorporation
Protein synthesis in HUVECs was quantified by determination of
the incorporation of [3H]leucine into
trichloroacetic acid (TCA) –precipitable radioactivity. In brief,
cells grown to confluence in 12-well plates were incubated with 5
µCi/well of [3H]leucine in the experimental
medium in the presence or absence of the agents to be studied. FCS
(20%) was used as the positive control. After 6 hours the cells were
washed 3 times with cold PBS, and the proteins were precipitated by
addition of 10% ice-cold TCA to each well for 30 minutes at 4°C.
Wells were then washed twice with 95% ethanol. All TCA-precipitated
material was solubilized in 0.1 mol/L NaOH and transferred to vials
containing scintillation fluid, and radioactivity was counted in a beta
counter.
RNA Purification and Northern Blot Analysis
Total cellular RNA was obtained according to Chomczynski and
Sacchi.32 RNA blot analysis was performed as
described.33 Ten micrograms of total RNA was loaded on
each lane, and Northern blotting was performed by capillary transfer of
RNA from the agarose gel to nitrocellulose membranes (Schleicher &
Schuell BA-S NC; pore size, 0.45 µm) with 20x SSPE (0.75 mol/L
NaCl, 0.05 mol/L
NaH2PO4 ·
H2O). Filters were prehybridized at 42°C for at
least 2 hours in 50% deionized formamide, 5x Denhardt's solution
(0.1% Ficoll type 400, 0.1% polyvinylpyrrolidone, and 0.1% BSA), 5x
SSPE, 0.1% SDS, and 100 µg/mL denatured salmon sperm DNA and
hybridized at the same temperature and in the same solution with
heat-denatured 32P-labeled DNA probe for 16 to 24
hours. Blots were then washed once at room temperature and twice at
55°C for 20 minutes in 0.1% SDS, 2x SSPE, followed by 2 washes at
55°C in 0.1% SDS, 0.1x SSPE. Filters were exposed to Kodak XAR film
at -80°C with intensifying screens. The level of GAPDH was used to
normalize densitometric values for variations in RNA loads. Each
experiment was performed in triplicate.
DNA Probes
The human TF cDNA probe was a 500-bp EcoRI fragment
cloned into pUC19 obtained from the American Type Culture Collection,
Manassas, Va. The human GAPDH probe was a 1469-bp BamHI
fragment cloned into pBR322 kindly provided by Dr P. Castelli
(Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy). The
probes were labeled with [-32P]dCTP
(DuPont-NEN) by the random priming technique34 to a
specific activity of 5x108 to
5x109 counts per minute per microgram of
DNA.
Statistical Analysis
The results are reported as mean±SEM. ANOVA (1-way) followed by
Tukey's test was used for determination of significance levels. A
value of P<0.05 was considered significant.
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Results |
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Effect of rVEGF and rTNF-
on TF Activity in HUVECsTotal TF activity was determined in cell lysates by using a one-stage clotting assay. Confluent HUVECs did not express detectable amounts of TF under basal conditions. In accord with previous reports,7 8 500 U/mL rTNF-
markedly stimulated TF
expression at 6 hours (Table 1
).
rVEGF (0.001 to 1 nmol/L) also caused a concentration-dependent
increase in TF activity (data not shown) with a >10-fold stimulation,
compared with control, observed at 1.0 nmol/L (Table 1). The
potential interaction between rVEGF and rTNF- was studied at
concentrations of each agent that by themselves induced substantial
amounts of TF activity (1 nmol/L for rVEGF and 500 U/mL for rTNF-).
Concomitant treatment of HUVECs with rVEGF and rTNF- for 6 hours
induced 10-fold more TF activity than did rTNF- alone (Table 1). Only concentrations of rVEGF (0.1 to 1.0 nmol/L) that were
able to induce TF activity by themselves were synergistic with rTNF-
(Figure 1). Total TF activity in
HUVECs treated with rVEGF and rTNF- used alone or in combination
peaked at 6 hours (Figure 2). By
12 hours, the activity was reduced by half in all conditions, though
remaining >6- or 20-fold higher in rVEGF+rTNF-–treated cells than
in cells treated with rTNF- or rVEGF alone, respectively (Figure 2).
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) or incubated with rVEGF
(1 nmol/L, 
), rTNF-
), or rVEGF in combination with
rTNF-
) for different times up to 12 hours and assayed for TF
activity by a one-stage clotting assay. Data are the mean of duplicate
determinations, and a representative of 3 experiment is
shown.
The synergistic effect of rVEGF+rTNF- on TF activity was
paralleled by a marked increase in immunologically detectable TF
protein, both by ELISA (Figure 3A)
and by Western blot analysis (Figure 3B). This was not
due to a generalized synergistic increase in protein synthesis.
Treatment of HUVECs with rVEGF+rTNF- did not significantly enhance
total protein synthesis as measured by incorporation of
[3H]leucine (25±3.8%, 10±3.6%, and
19±3.6% increase over control in rVEGF+rTNF-–, rVEGF-, and
rTNF-–treated HUVECs, respectively, n=3; P=NS). mRNA
levels determined at 2 hours after stimulation with rVEGF+rTNF- were
also markedly higher compared with single agent–treated cells
(TF/GAPDH ratio in densitometric arbitrary units: control, 0.04; rVEGF,
0.5; rTNF-, 1.2; and rVEGF+rTNF-, 1.6; Figure 3C).
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To assess whether the potentiating effect of rVEGF was specific for
this growth factor, experiments with basic fibroblast growth factor
(bFGF) and acidic FGF (aFGF)/heparin, alone or in combination with
rTNF-, were performed. bFGF and aFGF/heparin alone did not affect TF
activity in HUVECs (0.01±0.003 and 0.02±0.001 TF U/µg cell
protein, respectively). The combination of bFGF with rTNF- resulted
in a significant increase in TF activity that was lower than that
exerted by the combination of rVEGF+rTNF- (4.1±0.1 versus
10.54±2.3 TF U/µg cell protein, respectively, n=3;
P<0.01). Similar results were obtained with
aFGF/heparin+rTNF- (2.25±0.3 TF U/µg cell protein). Although the
presence of FCS in the experimental system amplified the response of
HUVECs to the single agent, TF activity in rVEGF+rTNF-–treated
samples was 2- and 10-fold greater compared with rTNF- and rVEGF
alone, respectively (TF U/µg cell protein: control, 0.09±0.02;
rVEGF, 3.78±1.11; rTNF-, 15.96±4.65; and rVEGF+ rTNF-,
30.3±0.1; n=3).
Immunolocalization of TF in HUVECs
To identify TF accessible on the cell surface of HUVECs,
monolayers of living cells were stimulated for 6 hours with rVEGF,
rTNF-, or both and then incubated for 30 minutes with soluble TF
antibody before fixation. Minimal staining was observed in untreated
(Figure 4A) and in single
agent–treated (Figure 4B and 4C) cells, whereas the majority of
the cells treated with both agonists showed intense membrane staining
with some patchy cell surface distribution of TF (Figure 4D).
Patches were further characterized on cells that were fixed 6 hours
after treatment with rVEGF+rTNF- and then stained with the soluble
TF antibody. By light (Figure 4F and 4G) and confocal (Figure 4H) microscopy, the patches were detected as "buds" on the
cell surface.
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Effect of rVEGF and rTNF- on Cell Surface–Associated
TF
To determine the amount of TF expressed on the cell surface in
response to agonists, HUVECs were grown on slides; incubated for 6
hours with rVEGF (1 nmol/L), rTNF- (500 U/mL), or both; and then
perfused with purified clotting factors in a parallel-plate flow
chamber. To assess total TF activity, monolayers of HUVECs treated in
the same manner were lysed, and factor Xa generation was measured. This
confirmed the results obtained with the one-stage clotting assay
(Figure 5A). Unstimulated cells
had virtually no surface TF activity; rVEGF- or rTNF-–treated
HUVECs also generated minimal amounts of surface TF activity (0.032 or
0.014 fmol of surface TF per cm2, respectively;
Figure 5B). On the contrary, the combination of agonists induced
surface expression of 
0.429 fmol of TF per cm2
of confluent cell monolayer (Figure 5B). The peak of surface TF
activity was observed 6 hours after stimulation (Figure 6B), similar to that of lysed
samples (Figure 6A). Whereas lysed samples displayed a
time-dependent accumulation of TF beginning at 2 hours (Figure 6A), increased expression on the cell surface was not seen until
6 hours (Figure 6B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The data presented in this study demonstrate that concomitant stimulation of HUVECs with rVEGF and rTNF-
induces
endothelial cells to become highly thrombogenic due to
a synergistic induction of surface TF activity. On the contrary,
treatment of HUVECs with either agonist alone has minimal effect on
surface TF activity expression.
TF is a transmembrane glycoprotein that serves as the
initiator of coagulation. On its synthesis, TF is translocated to the
cell surface, where it binds factor VIIa, thus triggering the
coagulation cascade. Although TF protein has been found on the plasma
membrane of a variety of cells35 and on shed membrane
vesicles in conditioned medium of cultured human
monocytes15 36 and tumor cells,37 studies
attempting to localize sites of TF expression on
endothelial cells have yielded controversial results.
TF is located mainly on the basolateral side of the
endothelial cell membrane38 as well as on
the apical surface.19 39 A predominant matrix-associated
TF expression has also been reported.30 40 A finding
common to these studies, however, is that stimulation of HUVECs with
rTNF-, a strong inducer of TF mRNA and activity, results in minimal
TF expression on the cell surface.
In this study, we have shown that rTNF- and rVEGF increased TF mRNA,
protein, and total activity as determined by the one-stage clotting
assay or by factor Xa generation. In addition, the 2 agents had
synergistic effects on the same parameters, in accordance
with previous reports.14 By using a parallel-plate flow
chamber, which measures factor Xa generation on the cell surface of
endothelial monolayers under dynamic conditions, we
were able to detect only minimal surface TF activity on HUVECs exposed
to concentrations of rTNF- that markedly increased total TF mRNA,
protein, and activity. Similarly rVEGF, which also induces TF mRNA,
protein, and activity, failed to significantly increase TF activity on
the cell surface. Minimal expression of TF on the cell surface in
response to either agonist was confirmed by immunolocalization of the
protein on living cells. These data, together with the results of total
TF activity measured in cell lysates, indicate that the majority of TF
induced by rTNF- or rVEGF appears not to be readily accessible on
the cell surface.
This report also shows that stimulation of HUVECs by the combination of
rTNF- and rVEGF induced a marked increase in TF surface activity
(30- and 15-fold compared with rTNF- and rVEGF alone, respectively),
which is out of proportion to the rise in total TF activity (4- and
8-fold, respectively). In this context it is interesting to note that
doubling the concentration of rTNF- resulted in a doubling of TF
activity in the cell lysates but failed to increase the amount of
surface TF synthesis (data not shown). These data suggest that only
certain agonists or groups of agonists may be able to reveal surface TF
activity.
The expression of active TF on the cell surface in response to
rTNF-+rVEGF occurred with a time course distinct from that found for
the induction of total TF activity measured in cell lysates. Of
particular note, the increase in surface activity was transient and
seen only between 5 and 6 hours after treatment. A similar kinetic
pattern has been observed in smooth muscle cells.29 The
fate of surface TF is presently the object of several
investigations. It has been recently proposed that in cells
constitutively producing TF, the translocation of the protein into
caveolae may represent a mechanism leading to downregulation of
TF-initiated proteolytic function.41 42 This might explain
the rapid decay in surface TF activity observed in this study.
The synergistic increase in endothelial cell TF
activity induced by rVEGF+rTNF- was also observed by staining the
surface of living cells with an anti-TF antibody. HUVECs showed a
diffuse pattern of TF expression interspersed with patches of more
densely staining material. By confocal microscopy, these patches
appeared as buds on the cell surface. The buds might represent
clumping of TF molecules, similar to that shown for a variety of
receptors.43 These clumps may ultimately be extruded from
the cells into the culture medium or may be resorbed and degraded in
lysosomes.42 Indeed, the presence of small
vesicles containing TF in the medium of stimulated
endothelial cells has recently been
described.44 As previously mentioned, caveolae-associated
TF has also been proposed to contribute to endothelial
regulation of hemostasis, owing to its colocalization with
thrombomodulin and urokinase receptor.42
The expression of TF is thought to be responsible for the thrombotic complications associated with septic shock, cancer, and atherosclerosis.45 46 47 Our data suggest that endothelial cells process TF differently from monocytes, in that the surface expression of active TF appears to be more tightly regulated. This is not surprising, given the strategic position of endothelium as the lining of the luminal vessel surface, and it may have important implications in the regulation of thrombosis by endothelial cells. The ability of endothelial cells to generate active TF on their surfaces may require multiple agonists, and this mechanism may protect endothelial cells from acting as a procoagulant surface, even under stress conditions.
VEGF has been used to induce angiogenesis in a rabbit ischemic
hindlimb model48 and to promote
re-endothelialization in a model of carotid artery
balloon injury, thereby attenuating neointimal
thickening.49 These studies have provided the impetus for
the use of VEGF as a new therapeutic modality in the management of
arterial insufficiency and intimal
hyperplasia.49 It should be noted that increased levels of
TNF- may also be present under these conditions. The synergistic
effects of rVEGF and rTNF- on endothelial cell
surface TF expression may need to be taken into account in evaluating
VEGF therapy.
Received July 20, 1998; accepted August 1, 1998.
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