From the Department of Vascular Biology, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855 and
the § Department of Molecular Biology, Norris Cancer Center,
Topping Tower, University of Southern California,
Los Angeles, California 90033
Received for publication, August 21, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The process of angiogenesis is important in both
normal and pathologic physiology. However, the mechanisms whereby
factors such as basic fibroblast growth factor promote the
formation of new blood vessels are not known. In the present study, we
demonstrate that exogenously added plasminogen activator inhibitor-1
(PAI-1) at therapeutic concentrations is a potent inhibitor of basic
fibroblast growth factor-induced angiogenesis in the chicken
chorioallantoic membrane. By using specific PAI-1 mutants with either
their vitronectin binding or proteinase inhibitor activities ablated,
we show that the inhibition of angiogenesis appears to occur via two
distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.
PAI-11 is the primary
inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type
plasminogen activator (1, 2). In healthy individuals, PAI-1 expression
is low and generally confined to vascular smooth muscle cells and
megakaryocytes. However, its expression in many other cells, including
endothelial cells, can be induced by a number of stimuli such as
cytokines and growth factors (3). uPA and tissue-type plasminogen
activator both activate plasminogen to the broad spectrum enzyme
plasmin, which is thought to directly degrade basement membrane
proteins, and to activate latent metalloproteinases (4, 5). Plasmin
activity has been implicated in tissue remodeling (6), and migrating cells both in vitro and in vivo express elevated
levels of uPA, together with its receptor (7-9). Thus, PAs at the cell
surface are thought to promote cell migration through breakdown of the extracellular matrix. However, recent studies suggest that the role of
uPA as well as other proteinases may not be to cause generalized matrix
destruction but instead may be to expose cryptic cell attachment sites
necessary for cell migration (10-12).
PAI-1 interacts with a number of nonproteinase ligands, including the
cell adhesion protein vitronectin (13, 14). Vitronectin is a
multifunctional glycoprotein present in plasma, platelets, and the
extravascular matrix. Like PAI-1, vitronectin is deposited at sites of
disease or injury (15, 16), where it binds multiple ligands, including
collagens, fibrin, uPA receptor, integrins, and PAI-1 (17-21). Plasma
vitronectin is in a closed conformation that does not interact with
integrins. However, upon binding to a surface at a site of injury,
vitronectin undergoes a conformational change, which exposes the
integrin binding site (22). Similarly, active PAI-1 converts the closed
plasma form of vitronectin to the open integrin binding conformation
(22). Paradoxically, however, PAI-1 binding to vitronectin blocks the
adjacent RGD integrin binding site on vitronectin (10, 11, 14). Thus, the association of PAI-1 with vitronectin simultaneously induces the
cell binding conformation but blocks the cell binding site. However,
this block is likely to be transient, since reaction of PAI-1 with a
proteinase induces a rapid conformational change in PAI-1 that causes a
large reduction in its affinity for vitronectin. Likewise, complex
formation between PAI-1 and a proteinase induces an equally dramatic
increase in PAI-1 affinity for some members of the low density
lipoprotein receptor family, such as the low density lipoprotein
receptor-related protein, gp330/megalin, and the very low density
lipoprotein receptor (23-25). This leads to rapid repartitioning of
PAI-1 from vitronectin in the matrix to the endocytic receptors and
subsequent cellular degradation of the PAI-1-proteinase complex
(23). Thus, PAI-1 and vitronectin can be thought of as forming a
molecular switch (10, 19, 23, 26). When PAI-1 is bound to vitronectin,
the integrin adhesion site is cryptic, and cell migration is blocked.
However, association with a proteinase induces the conformational
change associated with the switch in PAI-1 affinity from vitronectin to
low density lipoprotein receptor-related protein, resulting in
clearance of the PAI-1 and allowing cellular integrins access to the
RGD adhesion site. Thus, PAI-1 bound to vitronectin within the
extracellular matrix may serve to regulate cell migration, both by
regulating specific proteolysis and by controlling access of cellular
integrins to specific adhesion sites within the matrix. Accordingly,
the present study examines the potential of exogenously added PAI-1 to
regulate vascular cell migration in vivo by testing the
hypothesis that PAI-1 can inhibit angiogenesis in vivo in
both a vitronectin-dependent and a
proteinase-dependent manner.
Proteins and Reagents--
To maintain PAI-1 functional activity
during the extended incubations required for these in vivo
assays, all recombinant PAI-1 proteins, unless otherwise noted, were
constructed on the constitutively active PAI-1 background (14-1b) (27).
This form of PAI-1 has been shown to be essentially identical to
wild-type PAI-1 with respect to inhibitory activity, as well as binding
to vitronectin, heparin, and low density lipoprotein receptor-related
protein but has a half-life nearly 75-fold longer than wild-type PAI-1 at 37 °C (145 versus 2 h) (23, 27). The 14-1b PAI-1
and latent wild type PAI-1 were from Molecular Innovations. All other
forms were constructed using the Transformer site-directed mutagenesis kit (CLONTECH) according to the manufacturer's
instructions and isolated as described (28), except that preparations
having significant detectable endotoxin levels following
phenyl-Sepharose chromatography were further purified by polymyxin B
chromatography (Detoxi-Gel; Pierce). Final endotoxin levels for all
proteins used were below the FDA limit for parenteral drugs of 5 endotoxin units/kg (29). The different forms of PAI-1 used were
as follows: 14-1b PAI-1 (PAI-1); latent PAI-1 (PAI-1L);
PAI-1 with mutation of Gln123 to Lys (PAI-1K),
which has been previously shown to have a specific defect in
vitronectin binding but retains full inhibitory activity (30); PAI-1
with mutation of Arg346 to Ala (PAI-1A), which
binds vitronectin with wild-type affinity but does not inhibit PAs
(10); and finally, a double mutant, Thr333 to Arg,
Ala335 to Arg (PAI-1R), which binds vitronectin
normally but has no inhibitory activity toward any proteinase. Cleaved
forms of PAI-1 were made by incubating PAI-1 with pancreatic elastase
at a molar ratio of 100:1 (PAI-1/elastase) for 30 min followed by
inactivation of the elastase with phenylmethylsulfonyl fluoride (1 mM) (31). This results in cleavage of the reactive center
loop at a single site and induces the conformational change in PAI-1
associated with reactive center loop insertion and loss of vitronectin
affinity (13, 31). Chicken vitronectin was purified from chicken plasma as described (32), and antiserum was raised in rabbits by standard techniques.
Binding Assays--
Competitive inhibition binding assays were
performed as described (23) using a form of 14-1b PAI-1 containing a
hexapeptide tag at the amino terminus (PAI-1HMK). This form
of PAI-1 can be labeled with 32P without loss of inhibitory
activity or binding affinity for vitronectin and is similar to a form
of PAI-1 previously described (23) that was constructed on the
wild-type background.
In Vivo Angiogenesis Assay--
The chicken CAM angiogenesis
assay was performed essentially as described (33). Briefly, filter
disks saturated with FGF-2 were applied to the chicken CAM. Next, 25 µl of PAI-1 variants or antibodies diluted in phosphate-buffered
saline to the concentrations indicated were then applied at times 0, 24, and 48 h, and the CAM tissues were harvested at 72 h.
Angiogenesis was evaluated as described (33) by a person unaware of the
codes for the different experimental groups. The data are calculated as
angiogenic index, which is determined by subtracting the mean branching
observed in the absence of FGF-2 treatment from the branch points in
samples treated with FGF-2 and presented as the percentage of the FGF-2 only-treated control.
Proteinase Assays--
SDS-PAGE zymography was used for analysis
of proteinase levels in CAM tissue. Briefly, tissues were harvested
after 72 h of treatment, rapidly frozen on dry ice, and stored at
The second order rate constant for the inhibition of chicken plasmin
(Molecular Innovations) by the constitutively active human PAI-1 was
determined as described (34). The relative inhibitory activity of the
PAI-1K mutant versus chicken plasmin was
compared with active PAI-1 by titration as described (35), except that the substrate Val-Leu-Lys-p-nitroanilide (Sigma) was
used. The relative inhibition of chicken uPA by the various forms of
PAI-1 was examined by incubation of PAI-1 with kidney extracts followed by SDS-PAGE zymography, and none of the forms of PAI-1 tested effectively blocked chicken uPA (data not shown). Immunoblot analysis of purified chicken plasminogen, plasmin, and CAM extracts was performed essentially as described (34). PAI-1 and plasmin complexes were formed by reacting equimolar amounts of PAI-1 and plasmin for 30 min at room temperature prior to the addition of SDS-PAGE sample buffer.
To determine whether PAI-1 was inhibiting plasmin within the CAM tissue
an ELISA specific for PAI-1-plasmin complexes was developed. Briefly, a
monoclonal antibody, MA33B8 (36) (Molecular Innovations), was coated
onto microtiter plates at 2 µg/ml in TBS for 1 h at 37 °C.
This antibody has previously been shown to bind reactive center loop
inserted forms of PAI-1, such as PAI-1 in complex with a proteinase,
with high affinity (37). Following coating, the plates were blocked for
1 h using 2% bovine serum albumin, 5% nonfat dried milk in TBS
(blocking buffer), after which samples of CAM tissue extracts in
blocking buffer were added to the plate and incubated at 37 °C for
1 h followed by washing with TBS, 0.01% Tween 20. The plate was
then incubated with a 1:10,000 dilution of rabbit anti-chicken
plasminogen antiserum in blocking buffer for 1 h at 37 °C,
followed by washing with TBS, 0.01% Tween 20. Next, goat anti-rabbit
IgG conjugated to alkaline phosphatase was added at a 1:5000 dilution
in blocking buffer and incubated for 1 h at 37 °C, followed by
washing. The plasmin-PAI-1 complexes were then detected using the
substrate p-nitrophenyl phosphate, disodium (Sigma) at a
concentration of 4 mg/ml in TBS. The values obtained for the
plasmin-PAI-1 complexes present in the CAM tissues were compared with a
standard curve of purified chicken plasmin-PAI-1 complexes. To ensure
the specificity of the ELISA, preliminary experiments were also
performed. These demonstrated that the constitutively active form of
PAI-1 (14-1b) used in our studies did not bind to MA33B8 coated onto
microtiter plates prior to complex formation with chicken plasmin when
detected with rabbit anti-human PAI-1 antiserum (data not shown).
Likewise, the ELISA was shown to be specific for PAI-1-plasmin
complexes when the rabbit anti-chicken plasminogen antiserum was used,
since purified PAI-1·uPA complexes were not detected in this
assay (data not shown).
Immunofluorescence--
For immunofluorescence analysis of
frozen samples, CAM tissues were embedded in OCT and snap-frozen in a
2-methylbutane dry ice slurry, and stored at PAI-1 Blocks Angiogenesis in the Chick CAM--
Previously, we
demonstrated that vitronectin enhanced the migration of smooth muscle
cells in vitro and that PAI-1 was an efficient inhibitor of
this enhancement (10). This inhibition was independent of PAI-1's
anti-proteinase activity and instead was dependent on PAI-1 binding to
matrix vitronectin, where it prevented the association of promigratory
integrins with vitronectin (10, 11). Similarly, vitronectin can also
stimulate the migration of endothelial cells in vitro, and
as with smooth muscle cells this stimulation is blocked by
PAI-1.2 Together, these data
suggest that vitronectin stimulates vascular cell migration and that
PAI-1 blocks this enhancement by preventing the association of
Since PAI-1 and vitronectin appear to act together in vitro
to regulate vascular cell migration, we tested the ability of PAI-1 to
inhibit angiogenesis in vivo. For these studies, we examined the effect of PAI-1 on FGF-2-induced angiogenesis in the chicken CAM
assay. Fig. 1 shows the results obtained
following treatment for 3 days with or without FGF-2 or FGF-2 plus
PAI-1. A and B show CAM tissue incubated with
either vehicle alone or with FGF-2, respectively. As expected, FGF-2
induced a strong angiogenic response in the CAM tissue, as is evident
from the increased number of branching vessels. C and
D show the effect of 1 µM latent-PAI-1 (PAI-1L; C) or active-PAI-1 (D) on
FGF-2-induced angiogenesis. These data indicate that only the active
form of PAI-1 is an effective inhibitor of FGF-2-mediated angiogenesis
in the CAM assay. Quantitation of these results demonstrated that
active PAI-1 completely blocks angiogenesis, whereas PAI-1L
has no significant effect (Fig.
2A). Moreover, the inhibition
of angiogenesis by active-PAI-1 is dose-dependent with an
IC50 value of 0.112 ± 0.005 µM (Fig.
2B). Remarkably, this value is nearly identical to the
reported Kd of 0.100-0.125 µM for
PAI-1 binding to native vitronectin in solution (13, 38), suggesting
that similar to vascular cell migration in vitro, PAI-1
inhibition of CAM angiogenesis might be at least in part vitronectin
dependent. However, since the active conformation of PAI-1 both binds
vitronectin with high affinity and inhibits proteinases, whereas
PAI-1L has neither property (13), these results do not
reveal whether the inhibition of angiogenesis is dependent on either
proteinase inhibition, vitronectin binding, or both.
Proteinase Inhibition by PAI-1 Inhibits FGF-2-induced Angiogenesis
in Vivo--
To dissect the contribution of PAI-1
proteinase-inhibitory activity away from its vitronectin binding
properties, on the inhibition of angiogenesis, a specific PAI-1 mutant
was constructed that binds vitronectin poorly but retains wild-type
inhibitory activity (30). This mutant, PAI-1K, has a
greater than 250-fold lower affinity for vitronectin than does
wild-type PAI-1 and should inhibit proteinase activity in the CAM
tissue without significantly blocking vascular cell adhesion to
vitronectin (13). Fig. 3 demonstrates
that this mutant, like fully functional PAI-1, significantly inhibits
vessel formation in the CAM in a dose-dependent manner with
an IC50 of 0.204 ± 0.017 µM. This value
is ~2-fold higher than that of fully functional PAI-1, which is 0.112 µM. This suggests that PAI-1's anti-proteinase activity
is critical for its inhibition of angiogenesis and that its binding to
vitronectin is likely to be less important for this inhibition.
However, it is also possible that these two activities overlap, such
that high levels of anti-proteinase activity may mask the importance of
vitronectin binding. This would be the case, for example, if
proteolytic remodeling of the matrix were required to expose cryptic
vitronectin adhesion sites, which then were able to promote vascular
cell migration.
Human PAI-1 Inhibits Plasmin Activity in the CAM--
To directly
examine proteinase activity during FGF-2-induced angiogenesis, CAM
extracts were analyzed by SDS-PAGE zymography. These results
demonstrate that there is a marked increase in uPA activity in CAM
tissue following 72 h of treatment with FGF-2 (Fig.
4A, lane
4), and PAI-1 does not appear to significantly reduce this
activity (Fig. 4A, lane 5). The lack
of inhibition of chicken uPA by active human PAI-1 is consistent with
previous findings (39) and suggests that human PAI-1's anti-proteinase activity in this system is probably directed toward another enzyme. Since the most obvious function of uPA is to activate plasminogen to
plasmin, and since PAI-1 has been reported to be a very efficient inhibitor of plasmin (40), we examined the ability of human PAI-1 to
directly inhibit chicken plasmin. These data indicated that human PAI-1
is a very efficient inhibitor of chicken plasmin, having a second-order
rate constant of ~7.6 × 105
M
To see if human PAI-1 actually does inhibit chicken plasmin in the CAM
tissue, a sensitive, PAI-1-plasmin complex-specific ELISA was developed
that utilizes a conformation-specific anti-PAI-1 monoclonal antibody
that does not bind to active PAI-1 but does bind PAI-1 in complex with
a proteinase (37). The captured PAI-1-plasmin complex is then detected
with a specific anti-chicken plasminogen antibody. Extracts from CAM
tissue after 72 h of treatment with FGF-2 or with FGF-2 and 1 µM active PAI-1 were examined in this assay. The results
of this analysis are shown in Table I and indicate that PAI-1 plasmin complexes are detected in all PAI-1-treated CAM samples tested but not in CAM tissues that were not treated with
human PAI-1. The levels ranged from 6.2 to 145 pg of complex per mg of
CAM tissue. Thus, these results demonstrate that exogenously added
human PAI-1 can inhibit chicken plasmin in vivo. Taken
together, these data suggest that PAI-1 can inhibit angiogenesis in the CAM in a proteinase-dependent manner but that the target
enzyme is unlikely to be chicken uPA, since it has been shown to be
resistant to inhibition by human PAI-1 (39). Instead, based on the rate constant of inhibition and the presence of PAI-1·plasmin complexes in vivo, human PAI-1 most likely acts in the CAM by
inhibiting the activity of chicken plasmin, thereby blocking
plasmin-mediated proangiogenic events, such as basement membrane and
fibrin matrix remodeling, and activation of matrix
metalloproteinases (5, 44, 45). Consistent with this interpretation,
several studies have demonstrated that specific plasmin inhibitors can
block angiogenesis in vitro. (46-48).
PAI-1 Binding to Vitronectin Inhibits FGF-2-induced Angiogenesis in
Vivo--
The results above suggest that the proteinase-inhibitory
properties of PAI-1 are important for its inhibition of angiogenesis in vivo. However, since PAI-1 inhibition of vascular cell
migration in vitro was shown to be dependent on vitronectin
binding (10, 11), and since the PAI-1K mutant was not as
effective an angiogenesis inhibitor as fully functional PAI-1, we also
examined whether or not PAI-1 binding to vitronectin in the absence of
proteinase inhibition plays a significant role in controlling
FGF-2-induced angiogenesis. For this analysis, it was first necessary
to determine whether human PAI-1 binds chicken vitronectin with high
affinity. Since the amino acid residues critical for the binding of
human PAI-1 to human vitronectin were recently identified (14), an alignment of the somatomedin B regions of all vitronectin sequences found in the GenBankTM data base to date was performed
(Table II). This analysis revealed that
all of the residues that have been identified as being essential for PAI-1 binding to human vitronectin are conserved across all species
thus far sequenced. This suggests that the structure of this region of
vitronectin has been well conserved and that human PAI-1 may bind
chicken vitronectin with high affinity. This was tested directly in a
competitive inhibition binding assay, which demonstrated that human
PAI-1 bound to chicken vitronectin with high affinity (Fig.
5A). Furthermore, just as with
human vitronectin (13), a PAI-1 mutant (PAI-1A) that does
not inhibit PA activity but has previously been shown to bind human
vitronectin, bound chicken vitronectin normally, whereas the
PAI-1K mutant did not (Fig. 5A). The
IC50 values calculated from these data were 17 ± 2.8 nM for fully active PAI-1 and 21 ± 2.5 nM
for PAI-1A. These values are essentially identical to
results obtained in similar experiments with human vitronectin, which
yielded an IC50 of 17 nM (data not shown).
Together, these results indicate that human PAI-1 binding to chicken
vitronectin is indistinguishable from its binding to human
vitronectin.
We next examined whether PAI-1 binding to vitronectin in the absence of
PA inhibition plays a significant role in its inhibition of
angiogenesis. In the first experiments, we examined the ability of the
PAI-1A mutant to block angiogenesis. This mutant has its P1 residue changed from Arg to Ala, which converts it from
an inhibitor of PAs to an inhibitor of
elastase3 but does not affect
its binding to vitronectin (Fig. 5A). These data are shown
in Fig. 5B and indicate that the PAI-1A mutant is able to block angiogenesis in the CAM assay but that it is ~50-fold less effective than the PAI-1K mutant
demonstrating ~50% inhibition of angiogenesis at 10 µM. These results suggest that PAI-1 binding to
vitronectin is less important for its inhibition of angiogenesis than
is PAI-1 inhibition of proteinase activity. However, since the
association of PAI-1A with a proteinase, either as an
inhibitor or a substrate, would result in its cleavage and therefore
loss of vitronectin affinity (13, 49), and since proteinase activity in
general is thought to be increased in areas of active angiogenesis,
then it is also possible that PAI-1A is being cleaved and
inactivated in the CAM. Such proteolytic inactivation of PAI-1 by both
neutrophil proteinases and matrix metalloproteinases has been
previously reported (50, 51). Therefore, to eliminate this possibility,
a new PAI-1 mutant was designed to have no inhibitory activity toward
any proteinase and to retain vitronectin binding even after cleavage by
a proteinase. This mutant was based on previous studies demonstrating
that proteinase inhibition and loss of vitronectin binding requires a
rapid conformational change in PAI-1 that involves insertion of its
reactive center loop into the interior of PAI-1
Detailed analysis of the IC50 values for the inhibition of
angiogenesis by PAI-1R, and comparison with fully active
PAI-1 appear to yield identical results (compare 0.115 ± 0.010 µM for PAI-1R and 0.115 ± 0.033 µM for PAI-1 in this experiment), suggesting that the
noninhibitory PAI-1R mutant is as effective an inhibitor of
angiogenesis as fully functional PAI-1. However, examination of the two
dose-response curves clearly indicates that despite very similar
IC50 values, the two fits are not the same, with the
PAI-1R mutant having a very different slope and range
compared with active PAI-1. This can be seen in Fig. 7A,
where the fit for active PAI-1 yields a range of 100% and clearly goes
to zero, while the range obtained with PAI-1R is only 85%
and instead of going to zero approaches an asymptotic value of ~85%
inhibition of angiogenesis. This suggests that, as in vitro
(52), vitronectin in the matrix may enhance vascular cell migration and
possibly vessel formation. In this case, it may be that the inhibition of angiogenesis by PAI-1R results from the capacity of this
noninhibitory mutant to block vitronectin's enhancement of vascular
cell migration and/or vessel formation in vivo. If this were
the mechanism, then the PAI-1R mutant should reduce
angiogenesis but be unable to block it completely, even in the presence
of excess PAI-1R. This appears to be exactly the result
obtained (Fig. 7A) and suggests that, in vivo,
vitronectin enhances angiogenesis, possibly by increasing the rate of
vascular cell migration. To test whether simply blocking access to
vitronectin could inhibit angiogenesis, CAMs were treated with FGF-2 in
the presence or absence of an affinity-purified polyclonal antibody to
chicken vitronectin. This is shown in Fig. 7B and
demonstrates that antibodies to vitronectin reduce angiogenesis by 57%
in the CAM. This confirms that blocking vitronectin is sufficient to
reduce angiogenesis and supports the conclusion that the
PAI-1R mutant inhibits angiogenesis by preventing cellular
access to vitronectin in the matrix. Finally, this proposition is
further supported by immunofluorescence analysis of FGF-2-treated CAM
tissue, which confirms that vitronectin is present in the vascular wall
in a diffuse pattern that suggests its association with the vascular
basement membrane (Fig. 7, C and D).
The inhibition of angiogenesis by PAI-1R and the
anti-vitronectin antibody complements previous studies demonstrating
that integrin antagonists such as synthetic RGD containing peptides and
function-blocking antibodies to the vitronectin receptor, integrin
In conclusion, the present study demonstrates that exogenously added
PAI-1 at supraphysiologic concentrations is a potent inhibitor of
angiogenesis in the CAM. We have also shown that this inhibition
appears to act via two separable but probably overlapping pathways, one
of which involves inhibition of proteinase activity, most likely
chicken plasmin, and another that is independent of PAI-1 proteinase
inhibitory activity but requires high affinity binding of PAI-1 to
vitronectin. That PAI-1 can regulate angiogenesis is consistent with
the reported regulation and expression of PAI-1 in vitro,
since angiogenic factors such as FGF and vascular endothelial cell
growth factor have both been shown to induce PAI-1 expression in
endothelial cells (55, 56). PAI-1 in turn has been shown to regulate
the expression of FGF in injured endothelial cells, suggesting a
possible feedback loop for these two proteins (57). PAI-1 has also been
shown to be specifically induced in cells surrounding angiogenic
vessels, increasing only in cells directly in contact with the
sprouting endothelium (58). However, previous in vivo
studies examining the role of PAI-1 in tumor growth and angiogenesis
have been more controversial, since different studies have yielded
contradictory results. In one study, tumor cells transfected with the
PAI-1 cDNA showed a slight reduction in tumor-associated angiogenesis (59), whereas two other studies using PAI-1-deficient mice
suggested alternatively that either tumor angiogenesis requires PAI-1
(60) or that PAI-1 levels have no effect on tumor growth (61). These
conflicting results might be due to the ability of PAI-1 to influence
angiogenesis in diverse ways. For example, an absence of PAI-1 could
lead to excessive proteolysis resulting in loss of cell adhesion sites
and inhibition of angiogenesis as was suggested by Bajou et
al. (60). Alternatively, our results indicate that inhibition of
proteolysis can also inhibit angiogenesis. This could result from an
inhibition of fibrinolysis in the provisional angiogenic matrix (45,
62), which could impact endothelial cell migration. In this model, the
effect would be similar to the reduced wound healing response
associated with plasminogen deficiency, which is thought to be due to
an inability of cells to migrate into the fibrin matrix in the absence
of plasmin (63, 64). Finally, our results suggest that PAI-1's
association with vitronectin can also regulate angiogenesis. It may be
that in some circumstances vitronectin and PAI-1 act together in
vivo to either promote or inhibit angiogenesis. In this model,
vitronectin present in the provisional matrix might enhance
angiogenesis by promoting vascular cell migration, and PAI-1, by
controlling access to the integrin adhesion site on vitronectin, might
regulate this enhancement.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
70 °C. Immediately prior to analysis, frozen tissues were weighed
and then extracted in nonreducing SDS-PAGE sample buffer, after which
equivalent amounts of tissue extract were subjected to SDS-PAGE on a
10% gel (Novex). Chicken kidney extracts containing uPA were prepared as described (34) and were included as positive controls. Following electrophoresis, gels were soaked in 2.5% Triton X-100 twice for 45 min each to remove the SDS. The gel was then applied to a 1% agarose gel containing 1% casein with or without 30 µg/ml of chicken plasminogen (Molecular Innovations) and incubated in a humid chamber at
37 °C. Samples were also analyzed on casein indicator gels containing 100 µg/ml of anti-human uPA (Chemicon).
80 °C until
processing. Sections were cut at 5 µm and fixed for 10 min in
methanol/acetone 1:1 and then rinsed in phosphate-buffered saline.
Samples were then stained with rabbit anti-chicken vitronectin
antibodies and analyzed by immunofluorescence using a rhodamine-labeled
goat anti-rabbit IgG (Pierce) for detection.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
v
3 or other promigratory integrins with
vitronectin in the matrix.
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Fig. 1.
Inhibition of FGF-2-induced angiogenesis in
the CAM by PAI-1. A, CAM treated with vehicle only;
B, CAM treated with FGF-2; C, CAM treated with
FGF-2 and 1 µM PAI-1L; D, CAM
treated with FGF-2 and 1 µM active PAI-1.
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Fig. 2.
Quantitation of angiogenesis in the CAM assay
with active and latent PAI-1. A, the angiogenic index
of CAM tissue treated with FGF-2 in the presence or absence of latent
or active PAI-1. None, CAMs treated with FGF-2 only;
p values of <0.05 relative to the FGF-2 only control are
shown. B, the inhibition of angiogenesis by increasing
concentrations of active PAI-1. The curve for active PAI-1 was
generated with a four-parameter fit as described (65). In both
A and B, the data are presented as the percentage
of the FGF-2 only control (100%), and n 6 for each
condition.
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Fig. 3.
PAI-1 inhibits angiogenesis in a proteinase
dependent manner. Inhibition of angiogenesis by increasing
concentrations of PAI-1 or PAI-1K is shown. The
inset shows an expanded view of the origin. The curves were
generated with a four-parameter fit as described (65), and the data are
presented as the percentage of the FGF-2-only control (100%). The
error bars indicate the S.E., and for all points
n 6.
1 s
1
(data not shown). This is essentially identical to the rate of 6.6 × 105 M
1
s
1 reported for bovine PAI-1 inhibition of
human plasmin (40). Human PAI-1 also formed SDS-stable high molecular
weight complexes with chicken plasmin as expected for inhibition of a
serine proteinase by a serpin (Fig. 4B, lane
4) (41). Similar to uPA, the levels of plasminogen present
in the CAM also increased upon FGF-2 treatment; however, unlike uPA,
the addition of PAI-1 to the CAM reduced the amount of plasminogen
present in 72-h extracts to levels similar to unstimulated CAM tissue
(Fig. 4B, lanes 5-7). This reduction most likely is a result of PAI-1 inhibition of angiogenesis, leading to
reduced blood volume in the tissue, as well as the expected rapid
clearance and degradation of PAI-1-plasmin complexes (23). The
observation that PAI-1 reduces plasminogen but not uPA in FGF-2-treated
tissue also suggests that the inhibition of angiogenesis is not due to
PAI-1 blocking FGF-2 signaling directly, since uPA, which should be
produced by cells locally in response to FGF-2 (42, 43), is not
significantly reduced. In contrast, plasminogen, which is produced in
the liver and secreted into the blood, would only be expected to
increase in the CAM when new vessels are established.
View larger version (60K):
[in a new window]
Fig. 4.
UPA and plasminogen in chicken CAM.
A, SDS-PAGE zymography of 72-h CAM extracts: chicken kidney
extract as a positive control for chicken uPA (lane
1); empty (lane 2); extract of
untreated CAM (lane 3); extract of FGF-2-treated
CAM (lane 4); extract of FGF-2 and 1 µM active PAI-1-treated CAM (lane
5). B, immunoblot analysis of purified chicken
plasminogen and CAM extracts: 1 ng of chicken plasminogen
(lane 1); 1 ng of chicken plasmin
(lane 2); 1 ng of active PAI-1 (lane
3); 1 ng of chicken plasmin pretreated with 1 ng of active
PAI-1 (lane 4); untreated CAM extract
(lane 5); extract from FGF-2 treated CAM
(lane 6); extract from FGF-2 and 1 µM active PAI-1-treated CAM (lane
7).
Concentration of PAI-1-plasmin complexes in FGF-2-treated CAM
tissue
Sequence comparison of the PAI-1 binding site in vitronectin from
different species
View larger version (19K):
[in a new window]
Fig. 5.
Human PAI-1 binds chicken vitronectin with
high affinity and inhibits angiogenesis. A, solid phase
competitive inhibition binding assays with increasing concentrations of
PAI-1 or PAI-1 mutants competing for the binding of
32P-labeled PAI-1 to chicken vitronectin. , active
PAI-1;
, PAI-1A;
, PAI-1K. The curves for
PAI-1 and PAI-1A were generated with a four-parameter fit
as described (65), and the error bars represent the S.E.
B, quantitation of FGF-2-induced angiogenesis in the CAM
assay in the presence of 0, 1, or 10 µM
PAI-1A. None, CAMs treated with FGF-2 only.
n
6 for each condition; p values of
<0.05 relative to the FGF-2 only control are shown.
-sheet A (41, 49).
The mutant, PAI-1R, was constructed on the wild-type PAI-1
background and is disabled by the introduction of two Arg residues at
positions 333 and 335 (residues P14 and P12 of
the reactive center loop). These residues are in the hinge region of
the reactive center loop, and upon reaction with a proteinase, they
normally fold into
-sheet A with their side chains oriented toward
the interior of PAI-1. Mutations in this region have been found to
greatly reduce the rate of conformational rearrangement associated with
proteinase inhibition, converting the inhibitor to a substrate (31,
49). Therefore, these two substitutions would be expected to greatly retard the insertion of the reactive center loop into
-sheet A upon
interaction with a proteinase and also to prevent the mutant from
adopting the latent conformation. Since loop insertion also results in
loss of vitronectin affinity (13), then the PAI-1R mutant
should not only fail to inhibit all proteinases; it should also retain
significant vitronectin affinity after reaction with a proteinase. This
is seen in Fig. 6, where the native forms
of both active-PAI-1 and PAI-1R are shown to inhibit the
binding of 32P-PAI-1 to vitronectin. However, following
cleavage in their reactive center loops and incubation for 24 h,
only the PAI-1R mutant retains the ability to compete for
vitronectin binding (compare the filled triangles
with the filled circles in Fig. 6). This
indicates that, as predicted, the conformational rearrangement of the
PAI-1R mutant is very slow, and thus it should retain high
affinity for vitronectin even in an environment that is high in
proteinase activity. Fig. 7A
demonstrates that PAI-1R is a very effective inhibitor of
FGF-mediated angiogenesis. Since the PAI-1R is devoid of
all proteinase-inhibitory activity, this indicates that angiogenesis
can be substantially blocked by PAI-1 in a proteinase-independent
manner. This inhibition is also dose-dependent, and
PAI-1R appears to be significantly more effective than
PAI-1A, inhibiting angiogenesis by ~41% at 0.1 µM (Fig. 7A), compared with only 26% for
PAI-1A at a 10-fold higher concentration (see 1 µM; Fig. 6B). This difference is probably due
to the ability of PAI-1R to retain vitronectin affinity
even in the presence of proteinase activity.
View larger version (21K):
[in a new window]
Fig. 6.
PAI-1R retains high affinity for
vitronectin after proteinase cleavage. Solid phase competitive
inhibition binding assays with increasing concentrations of PAI-1 or
PAI-1R, either native or cleaved, competing for the binding
of 32P-labeled PAI-1 to chicken vitronectin. , PAI-1;
, PAI-1R;
, cleaved PAI-1;
, cleaved
PAI-1R. The curves for PAI-1, PAI-1R, and
cleaved PAI-1R were generated with a four-parameter fit as
described (65), and the error bars represent the
S.E.
View larger version (54K):
[in a new window]
Fig. 7.
Inhibition of angiogenesis by noninhibitory
PAI-1R or vitronectin antibodies. A,
quantitation of the CAM assay with fully active PAI-1 ( ) and
PAI-1R (
). The inset shows an expanded
view of the origin. The curves for active PAI-1 and
PAI-1R were generated with a four-parameter fit as
described (65). B, quantitation of angiogenesis in the CAM
assay with or without treatment with either affinity-purified
anti-vitronectin antibodies or normal rabbit IgG at 15 µg/day.
p values of <0.05 relative to the FGF-2-only control
(None) are shown. In both A and B, the
data are presented as the percentage of the FGF-2-only control (100%),
and n
6 for each condition. C and
D show a vessel in a section of CAM tissue harvested after 3 days of treatment with FGF-2 and immediately frozen prior to analysis.
C is the light image, and D is the same section
stained with anti-chicken vitronectin antibodies and detected with
rhodamine labeled goat anti-rabbit IgG. The original magnification
was × 400.
v
3, can inhibit FGF-2-mediated
angiogenesis (53, 54). While these earlier studies indicated that
v
3 was required for angiogenesis, they
did not indicate whether or not there was a specific matrix component
that was necessary for this process, since
v
3 binding is not strictly limited to
vitronectin. The observation that a nonproteinase inhibitory PAI-1
mutant as well as antibodies to vitronectin can significantly reduce
angiogenesis suggests that the interaction of
v
3 with vitronectin is important for a
normal angiogenic response to FGF-2. Furthermore, since neither agent
can completely inhibit angiogenesis even at concentrations where
vitronectin is likely to be saturated, it would appear that other
matrix proteins can also facilitate vessel formation but that
angiogenesis is likely to be optimal when vitronectin is present in the
provisional angiogenic matrix. This is consistent with the in
vitro cell migration results, where the inclusion of vitronectin
in the matrix enhances the migration of both smooth muscle cells and
endothelial cells and where PAI-1 was able to block this enhancement
(10, 26).2 Of course, it is not possible to unambiguously
derive the molecular mechanism by which PAI-1 inhibits angiogenesis.
However, taken together, the data presented here suggest that both
PAI-1 and vitronectin play a role in angiogenesis and that both
PAI-1's proteinase inhibitory activity and its binding to vitronectin may be significant components of PAI-1's inhibition of angiogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Ingham, D. Strickland, and M. Sandkvist for helpful discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL55374, HL55747, and CA83090 (to D. A. L.) and CA74132 (to P. C. B.).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.
¶ Present address: Université Laval, CHUQ, Pav. Hôtel-Dieu de Québec, 9 rue McMahon, Québec, Québec G1R 2J6, Canada.
Present address: Dept. of Medicine, Division of
Hematology-Oncology, University of Pittsburgh Cancer Institute, 200 Lothrop St. Pittsburgh, PA 15237.
** Present address: Depts. of Radiation Oncology and Cell Biology, New York University School of Medicine, The Kaplan Cancer Center/Rusk Bldg., Rm. 812, 400 E. 34th St., New York, NY 10016.
To whom correspondence should be addressed: Dept. of Vascular
Biology, J. H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-517-0356; Fax: 301-738-0794; E-mail: Lawrenced@usa.redcross.org.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M007609200
2 G. A. McMahon and D. Lawrence, unpublished data.
3 S. Stefansson and D. A. Lawrence, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; ELISA, enzyme-linked immunosorbent assay; FGF-2, basic fibroblast growth factor; CAM, chicken chorioallantoic membrane; uPA, urokinase-type plasminogen activator; PAI-1L, latent PAI-1; PAI-1K, PAI-1 with mutation of Gln123 to Lys; PAI-1A, PAI-1 with mutation of Arg346 to Ala; PAI-1R, PAI-1 with mutation of Thr333 to Arg and Ala335 to Arg; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline.
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