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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 543-550
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Domain 5 of high molecular weight kininogen (kininostatin)
down-regulates endothelial cell proliferation and migration and
inhibits angiogenesis
Robert W. Colman,
Bradford A. Jameson,
Yingzhang Lin,
Donald Johnson, and
Shaker A. Mousa
From the Sol Sherry Thrombosis Research Center, Temple University
School of Medicine, Philadelphia, PA; Center for Neurovirology,
MCP-Hahnemann Medical School, Philadelphia, PA; and Cardiovascular
Division, DuPont Pharmaceuticals, Wilmington, DE.
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Abstract |
We have demonstrated that high molecular weight kininogen (HK) binds
specifically on endothelial cells to domain 2/3 of the urokinase
receptor (uPAR). Inhibition by vitronectin suggests that
kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to
HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit
angiogenesis. We found that recombinant domain 5 (D5) inhibited
endothelial cell migration toward vitronectin 85% at 0.27 µM with an
IC50 (concentration to yield 50% inhibition) = 0.12
µM. A D5 peptide, G486-K502, showed an IC50 = 0.2
µM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 µM (IC50 > 50 µM). D6
exhibited weaker inhibitory activity (IC50 = 0.50
µM). D5 also potently inhibited endothelial cell proliferation with
an IC50 = 30 nM, while D3 and D6 were inactive. Using
deletion mutants of D5, we localized the smallest region for full
activity to H441-D474. To further map the active region, we created a
molecular homology model of D5 and designed a series of peptides
displaying surface loops. Peptide 440-455 was the most potent
(IC50 = 100 nM) in inhibiting proliferation but did not
inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast
growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay
at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for
which we suggest the designation, "kininostatin") is a potent
inhibitor of endothelial cell migration and proliferation in
vitro and of angiogenesis in vivo.
(Blood. 2000;95:543-550)
© 2000 by The American Society of Hematology.
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Introduction |
Angiogenesis is the formation of new capillaries from
preexisting blood vessels. Many solid tumors are critically dependent on this new blood vessel formation to provide nutrients and oxygen and
to support growth; thus, antiangiogenic therapy is an important goal
for cancer therapy. Angiogenesis consists of the following steps: (1)
endothelial cell detachment from adhesive proteins; (2) enzymatic
degradation of the basement membrane by plasmin or plasmin-activated
matrix metalloproteinases; (3) endothelial cell migration into
perivascular spaces; (4) proliferation; and (5) tube
formation. These steps result in new vessels. Stimuli include basic fibroblast growth factor FGF2, vascular endothelial cell
growth factors (VEGF), other growth factors, and cytokines. Endogenous
inhibitors of angiogenesis include plasma proteins such as
thrombospondin and fragments of plasma proteins such as angiostatin,
which is a proteolytic degradation product of plasminogen. Inhibition
of angiogenesis can be mediated at 1 or more of the critical steps in
the process.
The urokinase receptor (uPAR) plays a critical part in initiating
cellular migration, regulating adhesion, and enhancing proteolysis, which are of central importance in the angiogenesis
required for tumor growth and metastasis. This receptor binds
prourokinase (proUK) with high affinity,1 thereby
concentrating the expression of plasmin activity on the cell
surface.2 Furthermore, uPAR is expressed on migrating
cells, both in vivo and in vitro, indicating that uPAR enhances cell
translocation.3 The urokinase receptor participates in the
control of cell adhesion because it binds vitronectin with high
affinity at a site within domains 2 and 3 of uPAR.4 In
contrast, uPA is bound to uPAR within domain 1.5
Plasma HK (high molecular weight kininogen) is coded for by a single
gene.6 The first 9 exons code for the heavy chain, and exon
10 codes for bradykinin (BK) domain 4 and the long light chain. The
heavy chain of HK consists of domains 1, 2, and 3, and the light chain
consists of domains 5 and 6. Mediation of the biologic effects of HK
requires prior cell binding. Domain 3 (D3) inhibits thrombin action on
platelets7 and cell binding to platelets8 and
endothelial cells.9 Using recombinant fusion proteins and
constrained peptides designed by homology modeling, we have mapped the
region responsible for inhibiting thrombin binding to platelets to a
heptapeptide within D38 coded for by exon 7 and found that
it also blocks the binding of HK to neutrophils. Herwald et
al9 have mapped the endothelial cell binding site on D3 and
demonstrated that a peptide from D3 coded for by exon 9 inhibited
thrombin-mediated tissue-type plasminogen activator release and
prostacyclin synthesis. Following the cleavage of BK from HK, the
resulting active cofactor, HKa, acquires the ability to bind to anionic
surfaces.10 We have shown that within D5 of the light
chain, critical amino acids (residues 441-457) in a
histidine-glycine-rich region are responsible for binding to an
artificial negatively charged surface.11 Using deletion mutagenesis, we have further defined a second subdomain a
histidine-glycine-lysine-rich region (residues 475-502) that also
supports binding to an anionic surface.12 Our studies with
recombinant polypeptides and monoclonal antibodies13 indicate that on neutrophils, D5 contains a
binding site for HK, and we have mapped the site to residues
440-478,14 a site overlapping with the binding site for
anionic surfaces. Hasan et al15 have mapped the endothelial
cell binding domain on D5 to the histidine-glycine-lysine-rich region,
specifically residues 471-496, which could represent a second region
for cell interaction. The binding of D5 to cells appears to be
responsible for the "antiadhesive" action of HKa. We showed that
HKa can displace 125I-fibrinogen from both neutrophils and
platelets.16 Asakura et al17 have confirmed and
extended these results by showing that HKa inhibits the adhesion and
spreading of human osteosarcoma cells on vitronectin-coated polystyrene
plates and the spread of bovine aortic endothelial cells on
vitronectin.17 The C-terminus of the light chain of HK in
D6, which contains 31 amino acid residues 556-595, is required for the
binding of prekallikrein (PK)18 and is exposed in
HK. Because HK binds to cells via D3 or D5, D6 remains
free to bind PK to endothelial cells.
We have recently observed that HKa binds to uPAR on endothelial
cells.19 Domains 2 + 3 of uPAR are involved because both an antibody to these domains and vitronectin can inhibit its binding. HKa binds to purified soluble suPAR, while HK under the same conditions does not. In addition, we found that endothelial cell-bound HKa can
bind PK, which, after activation to kallikrein by a cellular cysteine
protease, can activate pro-uPA to uPA on uPAR.20 Hence, the
interaction of kallikrein-cleaved HK (HKa) with uPAR may be important
in both the ability of uPAR to catalyze the formation of cell surface
plasmin as well as in the regulation of endothelial cell adhesion to
extracellular matrix. Both of these activities are centrally involved
in angiogenesis.21,22 We therefore tested HKa, D5, and 5 selected peptides from D5 for their ability to inhibit 2 other critical
components of angiogenesis, endothelial proliferation and migration to
vitronectin. We evaluated selected components in in vivo angiogenesis
in a chicken chorioallantoic membrane (CAM) model.
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Materials and methods |
Materials
HK and HKa were purchased from Enzyme Research Laboratories, South
Bend, IN. HK was more than 95% of a single band of 110 kd on
both nonreduced and reduced SDS electrophoresis. HK had been digested
with plasma kallikrein (molar ratio of 100:1 of HK to plasma
kallikrein) for 20 minutes at 37°C. The resulting HKa was composed
of 2 bands of 62 kd and 46 kd when analyzed by reduced SDS
electrophoresis. Vitronectin (0.25 mg per vial) was purchased from
Collaborative Biochemical Products, Bedford, MA.
Recombinant proteins
Deletion mutants of the light chain of HK were prepared as
previously described.12 These mutants have been constructed
in pGE × 2T vectors using a complementary DNA
coding for the light chain of human HK as study material. Each has
glutathione-S-transferase (GST) at the N-terminal of the polypeptide
fused to the HK peptide. The amino acid sequence linked to GST is
indicated. The mutants were purified on a glutathione column to more
than 90% purity as previously described.12
Synthetic peptides
Peptide synthesis and high-performance-liquid-chromatography
purification to more than 95% purity were performed by Dr John Lambris
of the University of Pennsylvania, Philadelphia, PA. The cysteine-containing peptides were folded by dissolving each in a buffer
containing 50 mM ammonium bicarbonate, pH 8.5, at a final concentration
of 100 µg/mL and air oxidized for 3 days at 4°C with continuous
agitation, then frozen and lyophilized. Additionally, 5,5-dithiobis
(2-nitrobenzoic acid) was used to ensure that there were no detectable
free-sulfhydryl groups in the peptide, and size exclusion on FPLC was
used to ensure that the major population of peptide has intramolecular
disulfide bonds and runs as a monomer.
Endothelial cell migration assay
Endothelial cells from human umbilical cords (HUVEC) were cultured
as previously described.23 Endothelial cell adhesion to
vitronectin was measured as described previously.24 We used a unique assay24 to measure the migration of endothelial
cells to vitronectin or fibronectin. These assays were performed using a Neuroprobe 96-well disposable chemotaxis chamber with an 8-µm pore
size. This chamber allows for quantitation of cellular migration toward
a gradient of either vitronectin or fibronectin. Cultured cells were
removed following a standardized method using
ethylenediaminetetraacetic aicd (EDTA)/trypsin (0.01%/0.025%).
Following removal, the cells were washed twice and resuspended
(2 × 106/mL) in EBM (endothelial cell basal media;
Clonetics, Inc, Walkersville, MD). The cell suspension (45 µL) containing 25 000 endothelial cells was added to a polypropylene
plate containing 5 mL of test agent at different concentrations and
incubated for 10 minutes at 22°C. Either vitronectin or fibronectin
(28 µL) at 0.0125 to 100 µg/mL was added to the lower wells of a
disposable chemotaxis chamber; the chamber was then assembled using the
preframed filter. We added 25 µL of cell/test agent suspension to the
upper filter wells and then incubated overnight (22 hours at 37°C,
5% CO2) in a humidified cell culture incubator. After the
overnight incubation, nonmigrated cells and excess media were gently
removed using a 12-channel pipette and a cell scraper. The filters were
then washed twice in phosphate-buffered saline (PBS) (no
Ca++ or Mg2+) and fixed with 1% formaldehyde
in PBS (0.05 mol/L NaPO4 buffer containing 0.15 mol/L NaCl,
pH 7.4). Membranes of migrated cells were permeated with Triton X-100
(0.2%) and then washed 2 to 3 times with PBS. The actin filaments of
migrated cells were stained with rhodamine phalloidin (12.8 IU/mL) for
30 minutes (22°C). Rhodamine phalloidin was made fresh weekly and
reused for up to 3 days when stored protected from light at 4°C.
Chemotaxis was quantitatively determined by fluorescence detection
using a Cytofluor II microfilter fluorimeter (530 excitation/590
emission). All cell treatments and subsequent washings were carried out
using a uniquely designed treatment/wash station. This station consists of 6 individual reagent units, each with a 30-mL volume capacity. Individual units were filled with 1 of the following reagents: PBS,
formaldehyde, Triton X-100, or rhodamine phalloidin. Using this
technique, filters were gently dipped into the appropriate solution,
thus minimizing migrated cell loss. This technique allows for maximum
quantitation of cell migration and provides reproducible results with
minimal inter- and intra-assay variability. The results presented are
the mean of 3 separate experiments.
Endothelial cell proliferation assay
Endothelial cell proliferation was measured by using CyQUANT cell
proliferation assay kit from Molecular Probes (Eugene,
OR). The basis for the CyQUANT assay is the use of a proprietary green fluorescent dye (CyQUANT GR dye) that exhibits strong fluorescence enhancement when bound to cellular nucleic acids. HUVEC cells (50 000/well) in a 96-well microtiter plate were stimulated with 10 ng/mL FGF2 (Life Technologies, Gaithersburg, MD) in serum-free, M199
growth medium with or without PK or HK peptides for 48 hours at
37°C in a CO2 incubator. Cells were washed with
serum-free medium and frozen at  40°C. Frozen cells were
thawed and lysed with a buffer containing the CyQUANT GR dye.
Fluorescence was measured using Cytofluor II fluorescence multi-well
plate reader with excitation 485 nm and emission 530 nm.25
The results presented are the mean of 3 separate experiments. In a
48-hour assay, there could be some contribution of an effect of these
peptides in inhibiting apoptosis, which might enhance the stimulation
or proliferation. The results are presented as a mean of 6 experiments
±SEM.
Neovascularization on the chicken chorioallantoic membrane
Ten-day-old embryos were purchased from Spafas, Inc (Preston, CT)
and were incubated at 37°C with 55% humidity. A small hole was
punctured in the shell concealing the air sac with a hypodermic needle.
A second hole was punctured in the shell on the broad side of the egg
directly over an avascular portion of the embryonic membrane, as
observed during candling. A false air sac was created beneath the
second hole by the application of negative pressure to the first hole,
which caused the chicken CAM to separate from the shell. A window,
approximately 1.0 cm2, was cut in the shell over the
dropped CAM with the use of a small crafts grinding wheel (Dremel,
Division of Emerson Electric Co, Racine, WI), which allowed direct
access to the underlying CAM.
Filter disks of No. 1 filter paper (Whatman Inc,
Clifton,NJ) were soaked in 3 mg/mL cortisone acetate
(Sigma, St Louis, MO) in a solution of 95% ethanol and water and
subsequently air-dried under sterile conditions. FGF2 was used to grow
vessels on the CAMs of 10-day-old chick embryos. Sterile filter disks
adsorbed with FGF2 dissolved in PBS at 1 µg/mL were placed on growing
CAMs. At 24 hours, control vehicle or test compounds were added to CAMs topically or by intravenous injection.
A range of 0 to 25 µg in 25 µL of HK recombinant domains or
synthetic HK peptides 10 to 200 µM or 25 µL of saline was applied to the saturated filter 24 hours later and was applied to the stimulated CAMs. At least 10 embryos were used per treatment group. Each experiment was performed at least 3 times. Thus, the mean ±SEM
is based on 30 separate observations.
Microscopic analysis of CAM sections
CAM tissue directly beneath FGF2-saturated filter disk was resected
from embryos treated 48 hours prior with compounds or controls. Tissues
were washed 3 times with PBS. Sections were placed in a 35-mm Petri
dish (Nalge Nunc, Rochester, NY) and examined under an SV6
stereomicroscope (Karl Zeiss, Thornwood, NY) at
50 × magnification. Digital images of CAM sections adjacent to
filters were collected using a 3-CCD color video camera system (Toshiba America, New York, NY) and analyzed with Image-Pro Plus software (Media
Cybernetics, Silver Spring, MD). The number of vessel branch points
contained in a circular region equal to the area of a filter disk
(angiogenesis index) was counted for each section. Percent inhibition
data are expressed as the quotient of the experimental value minus the
negative control value divided by the difference between the positive
control value and the negative control value.
Molecular modeling
We modeled domain 5 after the homologous protein hisactophilin. The
atomic coordinates of the hisactophilin protein (Brookhaven Database
File: 1HCE) were obtained and, based on the sequence alignment shown
below, used to model the D5 domain of HK. None of the gaps shown in the
alignment violate the hydrophobic core of the protein. In fact, all of
the sequence deletions are easily accommodated by the surface-exposed
loops of the protein. The HK-specific side chain replacement on the
hisactophilin template were used to create the new model of D5, as
previously described by Jameson.26,27 After replacing the
side chains, the model was subjected to alternating rounds of
molecular mechanics (energy minimizations) and molecular dynamics
(energy-dependent stimulations of molecular motion). The modeling was
performed using the Biopolymer module from the Sybyl
computational chemistry suite of programs (Tripos and Assoc, St
Louis, MO) on a Silicon Graphics OCTANE computer. A Connolly Surface
was calculated for the nuclear magnetic resonance
(NMR)-based structure using a hypothetical sphere with a
radius of 0.28 nm (twice the radius of a water molecule).
An electropotential gradient has been superimposed on the surface of
the protein and the surface exposure in terms of solvent accessibility calculated.
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Results |
To test the hypothesis that HK fragments could modulate
angiogenesis, we tested sequences from domains 3 and 6 and the entire domain 5 for their ability to inhibit endothelial cell proliferation (Table 1). A peptide from domain 3 (T255-Y280) was selected because it bound to platelets8 and
contained a binding sequence to neutrophils.14 Domain 5 (GST-K420-S513) was selected because it contains sites for binding to
platelets, neutrophils, and endothelial cells. A 30-amino acid
sequence from domain 6 (S565-P594) was used because it contains the
information for binding to PK. GST-D5 gave 100% inhibition of
proliferation at 0.27 µM, while the peptide from D3 and D6 showed no
inhibition at concentrations in excess of 100-fold.
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Table 1.
Percent inhibition by high molecular weight kininogen
(Hk) peptides of recombinant fusion proteins of human umbilical vein
endothelial cell proliferation
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To further delineate the amino acid sequence(s) responsible for
inhibition of endothelial cell proliferation, we used 6 deletion mutants of the light chain previously constructed,12 shown
in Figure 1. All are fused to GST at the
N-terminal end. All were tested at the same concentration (0.35 µM).
GST domain 5 (K420-S513) inhibited 100 ± 0%, as previously
shown. We found that the mutant 441-626 gave 100 + 0% inhibition of
endothelial proliferation at 0.35 µM (Figure 1). Removal of amino
acids 442-447 from the N-terminal end in mutant 448-626 resulted in
less inhibitory activity (65 + 8%), while mutant 465-626 showed
0 + 0% inhibition and mutant 475-626 displayed 0 + 0% inhibition.
GST420-474 (94 ± 6%) and GST420-513 (GST-D5) (100 ± 0%)
showed virtually complete inhibition. Thus, the sequence H441-D474 is
the smallest deduced sequence containing the full inhibitory activity
of HK D5. Neither GST (0 + 0%) nor a peptide from D6
565-594 (0 + 0%) had inhibitory activity at 100-fold
the concentration of GST-D5.
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| Fig 1.
Schematic structures of the HK deletion mutants of D5 and
D6 and inhibition of endothelial proliferation.
The HG-rich (light gray) and HGK-rich (black) regions of D5
and D6 (dark gray) are shown. Deletions are represented by a
line. Mutants are fused to glutathione-S-transferase (GST) at the
N-terminal. GST without HK sequence is indicated by a line. The bottom
construct is a synthetic peptide from D6.
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Because of the potential involvement of the D5 domain of HK in
angiogenesis, we searched the known databases for a suitable template
on which to base a homology-modeled structure. The D5 domain of HK
shows 30.6% identity and 40.5% homology (identity + similarity) with
a histidine-rich actin binding protein, hisactophilin 1, from
Dicytostellium discoideum (Figure
2). The NMR solution structure for the
hisactophilin 1 was determined several years ago28,29 and
found to display a 3-dimensional structure very similar to
interleukin-1 and FGF2. By analogy, the D5 of HK might also resemble
these proteins. The structure of the homology model of HK D5 is shown
in Figure 3. The ribbon representation of
HK D5 shown in Figure 4A indicates that the
core is composed of sheets ending in turns, which represent
surface-exposed loops. The solvent accessibility parameters were
calculated as a measure of buried and exposed residues. Figure 4 is a
plot of the surface exposure of each of the residues in the D5 domain.
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| Fig 2.
Alignment of HK D5 and hisactophilin.
A vertical line equals identity; 2 dots represent similarity.
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| Fig 3.
Homology model of HK D5.
(A) Ribbon diagram of model. The amino acid residues are labeled. (B)
Surface properties of model. The red area, which subsumes the amino
acids 440-455, is adjacent to the green area, amino acids 483-485.
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| Fig 4.
Surface exposure plot of the amino acid residues of D5.
Solvent accessibility is plotted against the amino acid.
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Five peptides were selected, each of which displayed surface loops that
could potentially participate in protein-protein interactions (Figure
5). Each was studied as a function of
concentration from 0.01 µM to 100 µM for its ability to inhibit
endothelial cell proliferation, and the IC50 (concentration
to yield 50% inhibition) ± SEM was calculated and compared to
GST-D5 (amino acids 420-513). Peptide 440-455 (IC50 = 0.11 ± 0.08 µM) was almost as potent as GST-D5 (IC50 = 0.05 ± 0.02 µM). The sequence
shows the highest electropotential of the Connolly surface (Figure 4B).
Because it is unlikely that buried residues partake in the binding
activities, it seems probable, from the surface exposure plot (Figure
4), that the observed activity in peptide 440-455 maps to its
carboxy-terminal end. Two of the other active D5 peptides, 475-485 (IC50 = 1.1 ± 0.5 µM) and 483-497 (IC50 = 2.8 ± 0.9 µM) overlap between residues 483-485 (Figure 4B). This conclusion is consistent with the finding that peptide 486-502 has an IC50 of 55 ± 15 µM. As
can be seen from the surface exposure plot in Figure 5, residue 486 is
relatively buried. The residues 483-485 form part of a surface patch
that is adjacent to the surface presented by residues 440-455 (Figure 4B) and may represent a secondary interaction site.
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| Fig 5.
Inhibition of endothelial cell proliferation by synthetic
peptides derived from D5 by molecular modeling.
The concentration to yield 50% inhibition (IC50) is
plotted on a log scale for each peptide and GST-D5.
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We also tested the peptides from D3, GST-D5, the peptides derived from
molecular modeling of D5, as well as the D6 peptide for their ability
to inhibit endothelial migration to a higher concentration of
vitronectin in the lower compartment of a Boyden chamber (Table
2). HK D3 is a region that inhibits the
binding of HK to neutrophils14 and platelets.30
Even at 139 µM, it only inhibits migration to vitronectin 10% with
an IC50 > 50 µM. It should be noted that the D3
peptide T255-Y280 is a linear, nonoptimized peptide of 26 amino acids,
which may account for the relatively high concentrations needed. A
control peptide (D3-control) with random amino acids on each side of a
small binding region inhibited 5% and thus did not differ from the D3
peptide. In contrast, HK D5 is highly inhibitory of migration to
vitronectin (85%) at 0.20 µM, with an IC50 = 0.12
µM. In contrast to the results for proliferation, in which peptide
440-455 was the most active and peptides 475-485 and 483-497 had more
modest activity, only peptide G486-K502 gave inhibition of migration.
This finding suggests that more than 1 region accounts for the
inhibitory effect of D5 (Table 2). In D6, a 30-mer peptide from the
binding site for PK gave 31% inhibition at 0.2 µM with an
IC50 = 0.5 µM.
To ascertain whether HK peptides inhibited angiogenesis in vivo, we
used CAM. We had tested the HK D5 (GST-K420-S513), which inhibited both
endothelial cell migration (85%) and proliferation (100%) at 0.20 µM. At the same concentration, it inhibited new vessel formation
after CAM stimulation by FGF2 97% (Figure
6). In contrast, GST failed to inhibit
FGF2-stimulated angiogenesis (Figure 6). Importantly, HK D5 also
inhibited a CAM stimulated with both FGF2 and VEGF 95% (data not
shown). In Figure 7, representative photomicrographs of neovascularization by FGF2 and its inhibition by
GST-D5, but not GST, are shown. Interestingly, HKa but not HK was
equally potent to GST-D5 (data not shown).
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| Fig 6.
Inhibition of angiogenesis in CAM stimulated by FGF2 by
GST D5.
The difference between control (PBS)- and FGF2-stimulated angiogenesis
index in CAM is highly significant (P < .01). The
difference between FGF2 + GST-D5 and FGF2 + GST D5 is highly
significant (P < .01).
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| Fig 7.
Photomicrograph of CAM: inhibition by HK D5 and HKa.
Few vessels are seen when CAM is incubated with a disk containing
control (PBS). When the CAM is incubated with a disk containing GST
(0.2 µM), no differences from control are observed (not shown). Many
new vessels are seen with stimulation by FGF2. When the CAM
is incubated with FGF2 and GST (0.2 µM), no differences from FGF2
alone are observed. The formation of new vessels by FGF2 + GST-D5 (0.2 µM) is markedly decreased compared to FGF2 alone and does not differ
from control (original magnification × 40).
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Finally, we tested the ability of the most potent peptide in inhibiting
endothelial proliferation to inhibit angiogenesis in vivo, and we
compared it to GST-D5. GST-D5 (50.0 µg) inhibited 85.2 ± 12.9%, and peptide 440-455 (41.2 µg)
inhibited 79 ± 6.9%. To define the specificity of the
inhibition, we prepared 2 altered peptides. The first peptide, 440 mutant, had the sequence scrambled to change the relative positions of
the histidine residues with an amino acid sequence of
HGGLGHGHGHEQHQGLGHG-amide. The second peptide, 440AA, had 2 alanines
replacing the 2 glutamines, 449 and 450, in 440-455. Peptide 440 mutant
(21.5 µg) only inhibited 29.4 ± 16.9%, while peptide 440AA
(21.5 µg) showed no detectable inhibition. Thus, the sequence in
440-455 seems to be specific not only for proliferation but for in vivo angiogenesis.
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Discussion |
We have shown that domain 5 (GST420-513) of HK inhibits endothelial
cell proliferation at nanomolar concentrations, while sequences from D3
that bind to endothelial cells and domain 6 that bind PK fail to
inhibit cell proliferation when tested at concentrations over 100-fold
higher. Using deletion mutants of GST-D5, we mapped the required region
to H441-D474. To design peptides, we used a molecular model based on
the NMR structure of hisactophilin 1, which has a 3-dimensional
structure similar to the angiogenic protein FGF. We selected 5 peptides
that subsumed surface residues and obtained a peptide 440-455 with an
IC50 of 100 nM, which was only 3-fold less potent than the
entire domain 5 (IC50 = 30 nM) in inhibiting
proliferation. These concentrations are in the same range of potency as
that reported for angiostatin (IC50 = 10-100
nM).31 The antiangiogenic activity of a fragment of a
proangiogenic plasma protein is a common feature of both kininostatin
(HK D5)/HK and angiostatin/plasminogen and suggests a similar negative
feedback mechanism. Although D5 was also a potent inhibitor of
migration toward vitronectin (IC50 = 120 nM), only 1 peptide from the 5 we chose could reproduce this effect, and this
required a higher concentration than GST-D5, suggesting that a
different region was required for migration than for proliferation. The
ability of different peptides from D5 to inhibit migration and
proliferation suggests that D5 could inhibit angiogenesis by more than
1 mechanism. Further, we showed that GST-D5, which inhibits both
proliferation and migration, inhibited neovascularization of the
chicken CAM induced by FGF2. GST had no effect, but HKa was equally as
potent as GST-D5. Finally, we showed that peptide 440-455 was not only
potent but specific in inhibiting angiogenesis, because a scrambled
peptide had little inhibitory action and a substituted peptide showed
no inhibition.
Our initial hypothesis that HKa was involved in regulating angiogenesis
stemmed from our observation that HKa, but not HK, formed a complex
with soluble uPAR19 and that the binding of HKa to
endothelial cells was blocked by soluble uPAR. The penetration of cells
through extracellular matrix is a requirement for
angiogenesis.21,22 The expression of cell surface plasmin
activity is essential for promoting cell migration through
extracellular matrix32 and depends on proUK, uPAR,
plasminogen, and plasminogen-activator inhibitor type-1 (PAI-1). The
expression of cell surface urokinase activity requires initial binding
to a specific, high-affinity cellular receptor, uPAR,1 a
55 000-kd protein5 expressed on the surfaces of monocytes,
endothelial cells,33 and most tumor cells.32
This receptor has been cloned34 and revealed to be a
glycophosphatidylinositol (GPI)-anchored protein,35
consisting of 3 approximately 90-amino acid repeats of domains 1, 2, and 3. The urokinase binding site of the receptor is
contained within uPAR domain 1 residues 1-135.5 The uPAR
plays a critical part in initiating cell migration and enhancing
adhesion and regulatory proteolysis on the cell surface and by
increasing the activation of cell-associated plasminogen. Cell-bound
plasmin is relatively protected from inhibition by its natural
inhibitors36 and therefore may effectively enhance degradation of the extracellular matrix components, such as
fibronectin, laminin, and, indirectly, collagen by its capacity to
activate matrix collagenases.
Over the past several years, uPAR has been regarded as playing a
critical role in the mediation of angiogenesis. Bovine endothelial cell
migration induced by basic fibroblast growth factor
(FGF2) was shown to be mediated by uPA.37
Physiologic angiogenesis is dependent in vivo on the expression of uPA
and PAI-1.38 Human endothelial cells in vitro depend on
plasminogen activators and fibrin for repair-associated
angiogenesis.39 Both the proteolytic and angiogenic
properties of endothelial cells are stimulated concordantly by
FGF2.40 Invasion of a CAM is enhanced in the presence of
cell-associated urokinase. Moreover, metastasis of human prostate
carcinoma cells in mice is prevented by transfection of an inactive
urokinase mutant. Finally, the expression of uPAR is increased in
migrating endothelial cells.41 These observations support
the role of urokinase and uPAR in angiogenesis.
The initiating mechanism by which uPAR-bound proUK becomes activated to
an active enzyme is central to the expression of cell surface
proteolytic activity. The binding of proUK to uPA induces a
conformational change in the zymogen, which endows it with proteolytic activity.42 Alternatively, plasmin can cleave pro-UK to
UK,43 but this reaction is a positive feedback and does not
explain the mechanism of initiation. Recent studies from our
laboratory20 and others44 have defined a third
mechanism of proUK activation, which could be an initiating reaction.
Expression of plasmin activity on the endothelial cell surface is
significantly increased following the binding of HK and PK (Figure
8). Our studies have demonstrated that
following its HK-dependent binding to endothelial cells, PK is
activated to kallikrein by cell-associated cysteine protease and
subsequently promotes the activation of cell-bound proUK to uPA.20 However, a negative control mechanism may be
enhanced by cleavage of proUK to uPA, because the single chain proUK
bound to uPAR is more resistant to inhibition by PAI-1 than
uPA.45 In vivo, the relative importance of these mechanisms
of cell-associated plasmin formation is not clear at present.
View larger version (54K):
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| Fig 8.
Mechanisms underlying antiangiogenic activity of HKa.
Vitronectin (VN) is pictured bound to both the integrin ( v3) and
the urokinase receptor (uPAR) by domains 2 and 3 (D2, D3). In this
tentative hypothesis, HKa is pictured as binding by D5 to domains 2/3
of uPAR. By doing so, HKa displaces VN from the same domains of uPAR,
thus acting as an antiadhesive ligand. In addition, prekallikrein (PK),
which circulates in complex with HK or HKa bound to D6, is recruited to
the endothelial surface. PK is converted to kallikrein (K) by an
endothelial cell cysteine protease (CP). K then cleaves pro-uPA to uPA.
Plasminogen (PG) bound to 1 of its receptors, such as annexin 2, is
converted by uPA to plasmin (PN). Thus, HKa can augment cell-bound
plasmin-dependent proteolysis to facilitate endothelial cell migration.
Finally, pro-uPA is known to stimulate endothelial cell signal
transduction, probably by the association of uPAR with integrins such
as v3. It remains to be shown that the action of HKa to inhibit
cell proliferation is by a similar mechanism.
|
|
HK binds to uPAR domains 2 + 3, leading to the assembly of a complex
consisting of uPAR, proUK, HK, and PK on the endothelial cell surface,
which could be one of the initiating mechanisms for the activation of
proUK and, subsequently, plasminogen to plasma (Figure 8). We
hypothesize that this complex may facilitate the invasion of
endothelial cells through the basement membrane and extracellular
matrix, a critical step in angiogenesis. Our observation that HKa binds
to a site within domains 2 + 3 of uPAR, and the binding is competed
for by vitronectin, indicates that HKa may possess antiadhesive
properties, because uPAR is recognized as an adhesion receptor for
vitronectin. The inhibition of adhesion of tumor cells to vitronectin
by HKa was noted by Asakura et al.17 By displacing
vitronectin from uPAR at focal contact sites, HKa may facilitate
cellular migration, which could facilitate angiogenesis.
The similar activity of HKa and domain 5 is consistent with our
previous observations that the cleaving of HK to HKa enhances its
coagulant activity10 and causes major conformational
changes as judged by circular dichroism.46 Electron
microscopy reveals that domain 5 is more exposed after cleaving of HK
to HKa.47 The effects of D5 may not be due entirely to
binding to uPAR, because HK or HKa binds to other receptors on
endothelial cells such as cytokeratin 148 or the receptor
for the globular head of Clq.49 Indeed, we have postulated
that these receptors may exist in a complex on the endothelial cell
surface.50
The major unexpected finding of these studies was the potent inhibition
by GST-D5 and the HK peptide 440-455 of endothelial cell proliferation.
It should be noted that the latter peptide is more potent by an order
of magnitude than the 2 peptides, 475-485 and 483-497, that subsume the
endothelial binding site mapped by Hasan et al.15 Although
uPAR has no cytoplasmic domains, it is physically associated with
integrins51 and facilitates 2-integrin adhesion of human
monocytes.52 Thus, uPAR signaling through an endothelial
integrin such as v3 can be initiated by
uPAR occupancy by HKa (Figure 8), and inhibition of proliferation could
well be a consequence. Whether either cytokeratin 1 or the receptor for
the globular head of C1q is involved in cell signaling is not known.
Further studies, which are ongoing, will determine whether MAP kinase
or other components in the signaling cascade are affected by binding of
HKa to endothelial cells or to uPAR.
| |
Acknowledgments |
We thank Seema Mohamed for conducting the angiogenesis assays. We
appreciate the skillful preparation of the manuscript and some of the
figures by Rita Stewart and the preparation of additional figures by
Robin Pixley, PhD. This work was supported by grants from NIH P01
HL56914 and R01 CA63938 to R.W.C.
| |
Footnotes |
Submitted June 8, 1999; accepted August 27, 1999.
Reprints: Robert W. Colman, Sol Sherry Thrombosis Research
Center, Temple University School of Medicine, 3400 North Broad St,
Philadelphia, PA 19140; e-mail: colmanr{at}astro.temple.edu.
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.
| |
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[Abstract]
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V. Chhokar and A. L. Tucker
Angiogenesis: Basic Mechanisms and Clinical Applications
Seminars in Cardiothoracic and Vascular Anesthesia,
September 1, 2003;
7(3):
253 - 280.
[Abstract]
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A. H. Schmaier
The kallikrein-kinin and the renin-angiotensin systems have a multilayered interaction
Am J Physiol Regulatory Integrative Comp Physiol,
July 1, 2003;
285(1):
R1 - R13.
[Abstract]
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R. A. Skidgel, F. Alhenc-Gelas, and W. B. Campbell
Regulation of Cardiovascular Signaling by Kinins and Products of Similar Converting Enzyme Systems: Prologue: Kinins and related systems. New life for old discoveries
Am J Physiol Heart Circ Physiol,
June 1, 2003;
284(6):
H1886 - H1891.
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S. Wang, M. G. Hasham, I. Isordia-Salas, A. Y. Tsygankov, R. W. Colman, and Y.-L. Guo
Regulation of Cardiovascular Signaling by Kinins and Products of Similar Converting Enzyme Systems: Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen
Am J Physiol Heart Circ Physiol,
June 1, 2003;
284(6):
H1917 - H1923.
[Abstract]
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Y. Krijanovski, V. Proulle, F. Mahdi, M. Dreyfus, W. Muller-Esterl, and A. H. Schmaier
Characterization of molecular defects of Fitzgerald trait and another novel high-molecular-weight kininogen-deficient patient: insights into structural requirements for kininogen expression
Blood,
June 1, 2003;
101(11):
4430 - 4436.
[Abstract]
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C. Marcinkiewicz, P. H. Weinreb, J. J. Calvete, D. G. Kisiel, S. A. Mousa, G. P. Tuszynski, and R. R. Lobb
Obtustatin: A Potent Selective Inhibitor of {alpha}1{beta}1 Integrin in Vitro and Angiogenesis in Vivo
Cancer Res.,
May 1, 2003;
63(9):
2020 - 2023.
[Abstract]
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M. Peek, P. Moran, N. Mendoza, D. Wickramasinghe, and D. Kirchhofer
Unusual Proteolytic Activation of Pro-hepatocyte Growth Factor by Plasma Kallikrein and Coagulation Factor XIa
J. Biol. Chem.,
November 27, 2002;
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J.-C. Zhang, F. Donate, X. Qi, N. P. Ziats, J. C. Juarez, A. P. Mazar, Y.-P. Pang, and K. R. McCrae
The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin
PNAS,
September 17, 2002;
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12224 - 12229.
[Abstract]
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F. A. Scappaticci
Mechanisms and Future Directions for Angiogenesis-Based Cancer Therapies
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September 15, 2002;
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T. Chavakis, R. A. Pixley, I. Isordia-Salas, R. W. Colman, and K. T. Preissner
A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function
J. Biol. Chem.,
August 30, 2002;
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T. Chavakis, N. Boeckel, S. Santoso, R. Voss, I. Isordia-Salas, R. A. Pixley, E. Morgenstern, R. W. Colman, and K. T. Preissner
Inhibition of Platelet Adhesion and Aggregation by a Defined Region (Gly-486-Lys-502) of High Molecular Weight Kininogen
J. Biol. Chem.,
June 21, 2002;
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C. Emanueli and P. Madeddu
Letter to the Editor: Renin-Angiotensin and Kallikrein-Kinin Systems Coordinately Modulate Angiogenesis
Hypertension,
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T. CHAVAKIS, S. M. KANSE, R. A. PIXLEY, A. E. MAY, I. ISORDIA-SALAS, R. W. COLMAN, and K. T. PREISSNER
Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen
FASEB J,
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Y.-L. Guo, S. Wang, and R. W. Colman
Kininostatin, an Angiogenic Inhibitor, Inhibits Proliferation and Induces Apoptosis of Human Endothelial Cells
Arterioscler. Thromb. Vasc. Biol.,
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M. S. Pepper
Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis
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F. Mahdi, Z. Shariat-Madar, R. F. Todd III, C. D. Figueroa, and A. H. Schmaier
Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells
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O. Musso, M. Rehn, N. Théret, B. Turlin, P. Bioulac-Sage, D. Lotrian, J.-P. Campion, T. Pihlajaniemi, and B. Clément
Tumor Progression Is Associated with a Significant Decrease in the Expression of the Endostatin Precursor Collagen XVIII in Human Hepatocellular Carcinomas
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J.-C. ZHANG, K. CLAFFEY, R. SAKTHIVEL, Z. DARZYNKIEWICZ, D. E. SHAW, J. LEAL, Y.-C. WANG, F.-M. LU, and K. R. MCCRAE
Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5
FASEB J,
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T. Chavakis, S. M. Kanse, F. Lupu, H.-P. Hammes, W. Muller-Esterl, R. A. Pixley, R. W. Colman, and K. T. Preissner
Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions
Blood,
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Top
Abstract
Introduction