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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 543-550
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
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.
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)
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 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.
Materials
Recombinant proteins
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.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.
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.
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.
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.
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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Top
Abstract
Introduction
a
histidine-glycine-lysine-rich region (residues 475-502) that also
supports binding to an anionic surface.
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 
v