| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 47, 46681-46691, November 21, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
**
From the
Supratek Pharma Inc., Dorval, Quebec H9S 1A9, Canada, Wyeth Research, Cambridge, Massachusetts 02140, the ¶Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada, and the ||Institut National de Recherche Scientifique, Laval, Quebec H7V 1B7, Canada
Received for publication, August 6, 2003 , and in revised form, August 29, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2--gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
VEGF activities are mediated through binding to two high affinity receptors, human kinase domain receptor (KDR) and Fms-like tyrosine kinase receptor (Flt-1) (5), both of which are selectively expressed on endothelial cells during embryogenesis and VEGF-related pathologies (6). Both of these receptors are class III tyrosine kinases (7, 8) that undergo ligand-induced dimerization that triggers signal transduction. Studies in mice have shown that the expression of KDR reaches the highest levels during embryonic vasculogenesis and angiogenesis (6). In contrast, low Flt-1 mRNA levels were found during fetal growth, moderate during organogenesis, and high in newborn mice (9). The third VEGF receptor from the tyrosine kinase family, Flt-4, is largely confined to the lymphatic vasculature and has a role in lymph angiogenesis (10). Flt-4 binds VEGF-C and -D, but not VEGF (11). Neuropillin-1 (NRP-1) was recently identified as a new receptor for VEGF (12). It is expressed in the enocardium, coronary vessels, myocardial capillaries, and epicardial blood vessels of human fetal heart (12). Experiments with knockout mice deficient in Flt-1 or KDR receptor revealed that KDR is essential for the development of endothelial cells, whereas Flt-1 is necessary for the organization of embryonic vasculature (13, 14). The VEGF-Flt-1 receptor system also plays an important role in the simulation of tumor angiogenesis, which makes Flt-1 an interesting target for antiangiogenic drugs (15).
Human Flt-1 receptor is composed of seven extracellular Ig-like domains containing the ligand binding region, a single short membrane-spanning sequence, and an intracellular region containing the tyrosine kinase domain. The amino acid sequences of Flt-1 and KDR show 
45% identity; however, Flt-1 has a higher affinity for VEGF (KD = 10–20 pM) compared with KDR (KD = 75–125 pM). The activation of Flt-1 receptor by VEGF regulates interactions of endothelial cells with each other or the basement membrane on which they reside (16, 17). Alternative splicing of the Flt-1 receptor results in two forms, the full-length membrane-spanning receptor and a soluble form, denoted sFlt-1. Isolated sFlt-1 retains specific high affinity binding for VEGF and fully inhibits VEGF-stimulated endothelial cell mitogenesis by dominant negative mechanism (18). Furthermore, it was suggested that sFlt-1 might form heterodimeric complexes with KDR with potentially negative effect on KDR signal transduction (19).
Because VEGF receptors are implicated in several pathologies, pharmacological interference with the VEGF/VEGF receptor system antagonists is clinically attractive. Humanized neutralizing antibodies that interact with VEGF near the KDR and Flt-1 binding sites (20–22) and systemic evolution of ligands by exponential enrichment (SELEX)-derived RNA molecules (23) that selectively bind to Flt-1 have been shown to block tumor growth dependent on vascularization of adjacent normal tissue (24). Similarly, anti-KDR monoclonal antibodies inhibited VEGF-induced signaling and demonstrated a high antitumor activity (25). Soluble Flt receptor (26), fragments of VEGF, as well as small molecule inhibitors of the VEGF receptors tyrosine kinase activity, such as PTK787/ZK222584 (27) and ZD4190 (28), have been shown to inhibit angiogenesis in vivo. Anti-VEGF antisense oligonucleotide has been shown to inhibit VEGF expression, VEGF-induced neovascularization, and tumor implantation and growth (29–31). However, no truly antagonistic compounds that would selectively discriminate between Flt-1 and KDR receptors are available yet.
In the present study, a new peptide motif that inhibits VEGF binding to Flt-1 has been identified using a phage-displayed peptide library. A random 16-mer peptide library displayed on the surface of the filamentous phage M13 was screened against the extracellular domain of Flt-1. This screening resulted in a peptide (SP5.2) that competed with VEGF for the Flt-1 binding and inhibited a broad range of VEGF-induced events in cultured endothelial cells. Potential use of SP5.2 as a targeting agent was evaluated using both synthetic and genetically constructed conjugates of the peptide and reporter proteins.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-galactosidase clone Gal-13 and anti-mouse IgG peroxidase conjugate developed in goat and absorbed with rat serum proteins were obtained from Sigma (Oakville, Ontario, Canada). The anti-histidine HRP conjugate antibody was purchased from Invitrogen (Burlington, Ontario, Canada). Scrambled peptide was synthesized at Supratek (Laval, Quebec, Canada). Cell Culture—Human umbilical vein endothelial cells (HUVECs) were purchased from BioWhittaker Inc. (Walkersville, MD). The cells were cultured in the endothelial growth medium, Clonetics media EGM & EGM BulletKitTM (BioWhittaker Inc.). The cells were used for the experiments at their 3rd or 4th growth passage.
HCECs were isolated using previously described protocols (32). Purity of HCEC cultures generated by these procedures was routinely assessed by the immunocytochemical staining for Factor VIII-related antigen and the lack of staining for smooth muscle -actin and was estimated to be >95%. The morphological, phenotypic, biochemical, and functional characteristics of these HCEC cultures have been described in detail previously (32, 33). For endothelial permeability studies, HCECs were used as an in vitro blood-brain barrier model described previously (33).
Bacterial Strains and Bacteriophages—K91Kan cells obtained from G. Smith were grown on LB agar supplemented with kanamycin (100 µg/ml). Cells were made competent using the protocol described previously by Smith (34). M15 was purchased from Qiagen (Mississauga, Ontario, Canada) cells made competent following the protocol from Qiagen. pQE16 vector purchased from Qiagen and pCMVb vector from Clontech (Palo Alto, CA).
The phage library containing the 16-amino acid peptide was constructed essentially as previously described, using fUSE5 as the phage vector (35). This linear library consisted of 109 independent recombinant phages recovered as tetracycline-resistant colonies. Sequencing of randomly selected clones indicated that the majority of these phages (>90%) contain inserts.
Screening the Phage Display Library with Flt-1-coated Magnetic Particles—The receptor, rhFlt-1 chimera (10 µg in 50 µl of 0.1% BSA/PBS), was immobilized on streptavidin-coated magnetic particles (Roche Applied Science, Laval, Canada) using biotinylated anti-hIgG(Fc) antibody. The Flt-1-coated magnetic particles (Flt-MPs) were blocked with 3% BSA/PBS for 2 h at room temperature. For selection, phage (1 x 1011 cfu) from the linear 16-amino acid random peptide phage display library diluted in 0.1% BSA/PBS were added to Flt-MP and incubated overnight at 4 °C. After extensive washing with 0.1% BSA/PBS, the bound phage were either eluted with a low pH buffer (0.2 M glycine-HCl, pH 2.2) or displaced from Flt-MP with 10 µg/ml rhVEGF165 for 1 h at 22 °C. Recovered phages were amplified using competent K91Kan Escherichia coli cells and then subjected to four subsequent rounds of selection on Flt-MP. Phage binding was quantified by counting the phage titer in eluted aliquots from Flt-MP as described earlier (34). Phages from selected clones were sequenced at Sheldon Biotechnology center (McGill, Montreal).
ELISA for the Displacement of Phage Binding—The rhFlt-1 receptor chimera were immobilized on Streptavidin-coated microtiter plates (total binding capacity for biotin-labeled AB = 1.5 µg/well, Roche Applied Science). Biotin-labeled anti-human FC antibody (ICN), at a concentration of 10 µg/ml in 0.1% BSA/PBS, were added into each well of the plates. The plates with assay mixtures were incubated at 4 °C for 8 h in a humidified container and then washed four times with 0.1% BSA/PBS. Recombinant human Flt-1/Fc chimera (1 µg/well in 0.1% BSA/PBS, R&D Systems, Minneapolis, MN) were added into each well, and the plates were incubated overnight at 4 °C in a sealed container to allow the receptor to attach to the ligand. Unbound receptor was washed away with 2 ml of 0.1% BSA/PBS. Each well was then filled with 250 µl of blocking buffer (2% nonfat dry milk in PBS) and incubated at room temperature for 2–3 h. As a negative control, three of the wells were blocked with the blocking buffer (2% nonfat milk in PBS). Phage particles (5 x 109 to 1 x 1010 cfu/well) were added to each well and incubated for 2 h at room temperature. In competition experiments, phage suspension was premixed with various concentrations of competing agent (SP5.2 peptide, rhVEGF165) and then added to immobilized receptor. Wells were washed with 0.1% BSA/PBS, and the amount of bound phages was detected with peroxidase-conjugated anti-M13 antibody (Amersham Biosciences). After the addition of the substrate, ABTS, and H2O2, antibody reaction was analyzed in a microtiter plate reader at 405 nm.
Production of Mutant Variants of SP5.2 Phage—Several mutations in SP5.2 coding oligonucleotide inserts were carried out to identify the amino acids involved in the receptor binding site domain. The single point mutations in V5.2 phage insert coding sequence were produced as described previously (36). Briefly, a series of SP5.2-coding oligonucleotides, with a particular amino acid coding triplet replaced with GCT (alanine-coding triplet), was synthesized by Invitrogen (Ontario, Canada). The mutant oligonucleotides were cloned into fUSE5 phage vector as described (35). All mutants were purified and verified by DNA sequencing.
Peptide Synthesis—Peptide amides were synthesized manually on solid Rink amide resin (Nova Biochem) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocol and (benzotriazol-1-yloxy)tripyrro-lidinophosphonium hexafluorophosphate (PyBOP) activation. The fluorescein residue was introduced into the peptide by standard coupling of N-terminal-deprotected peptide with fluorescein-5-carboxylic acid. Peptide amides were cleaved from the resin with trifluoroacetic acid:water: ethanedithiol:triisopropylsilane (95:5:2.5:2.5, v/v) and were recovered by precipitation with ice-cold diethyl ether. Crude products were purified by high performance liquid chromatography on a Vydac C18 column using a linear gradient of 30% to 70% acetonitrile/water (0.1% trifluoroacetic acid), for 60 min at a 5-ml/minute flow rate. The identity of peptides was verified by electrospray mass spectrometry (PE Sciex API III Biomolecular Mass Analyzer, Applied Biosystems, CA).
Binding Competition for Flt-1 Receptor—Fluorescein-labeled peptide in 0.1% BSA/PBS buffer, pH 8.5, was added at various concentrations (0, 0.1, 1, 10, 50, and 100 µM) into wells of an ELISA plate coated with immobilized Flt-1 receptor. In competition experiments, the various concentrations of phage or VEGF165 were mixed and added into wells. After 2-h incubation, microtiter plates were washed 10 times with 0.1% BSA/PBS buffer, pH 8.5, and the bound peptide was measured using a microplate fluorescence reader (FL600, BioTek) (
ex = 485 nm; em = 530 nm).
Flt-1 Receptor Immunochemistry—HCECs were grown on glass coverslips in a 24-well dish for 3 days until sub-confluent. Media was aspirated out; cells were washed two times in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were subsequently washed three times in PBS, incubated in blocking solution containing 5% goat serum in PBS for 1 h at room temperature, and exposed to the primary antibody (5 µg/ml goat anti-human Flt-1 in blocking solution; R&D Systems Inc) for 1 h at room temperature. After washing three times in blocking solution, cells were exposed to the secondary antibody (1:20 dilution of donkey anti-goat IgG-immunogold in blocking solution; Accurate Chemical and Scientific Corp.) for 30 min at room temperature. Cells were then extensively washed (three times with blocking solution and three times with distilled water) and incubated in silver enhancing solution for 30 min (1:1 dilution of reagents A and B). After washing with distilled water (two times for 5 min), slides were viewed under a microscope.
SP5.2 Binding to Cells—Fluorescein-labeled SP5.2 peptide was added to HCECs cells at different concentrations, (1, 10, and 50 µM) and incubated for 30 min. The neuroblastoma ShyY cell line was used as a negative control. Media were aspirated, and cells were washed in phenol red-free M199 and observed under a fluorescence microscope.
Endothelial Cell Proliferation Assay—HUVECs were placed in 96-well plates (Costar) at 104 cells per well in 200 µl of EGM-2 medium (Clonetics) supplemented with 0.5% heat-inactivated fetal bovine serum. Cells were incubated for 24 h at 37 °C in 5% CO2. HCECs were placed in 12-well plates and allowed to grow for 3 days until sub-confluent. Cells were then washed once by PBS and incubated in a serum- and glucose-free DMEM for 24 h to suppress cell growth. Both HCECs and HUVECs were then exposed to various treatments (i.e. 10% fetal bovine serum, VEGF165 (20 ng/ml), PLGF (100 ng/ml), bFGF (10 ng/ml) in the absence or presence of SP5.2 peptide). Scrambled peptide NGSAIAASSAVTHGMS at the same concentration was used as control. After 48 h of incubation, 1 µCi of [methyl-3H]thymidine (20 Ci/mmol; ICN) was added in each well, and plates were incubated for an additional 24 h. The cells were then placed on ice, washed twice with EGM-2 medium containing 10% fetal bovine serum, and fixed for 10 min in ice-cold 10% trichloroacetic acid. After washing with ice-cold water, cells were lysed and DNA was solubilized in 50 µl of 2% SDS. [3H]Thymidine incorporation was determined by scintillation counting.
Endothelial Cell Migration—Migration of calcein-AM labeled (Molecular Probes, Eugene, OR, 10 µM for 20 min at 37 °C) HCECs stimulated by VEGF165 were tested in the absence or presence of SP5.2 peptide or scrambled peptide using a ChemoTx #101-5 assembly (Neuro Probe, Inc., Gaithersburg, MD) consisting of a 96-well plate and a polycarbonate filter membrane, as described in a previous study (37). Briefly, the wells of the plates were loaded with buffer containing various chemoattractants or phenol red- and serum-free media conditioned by cancer cells. The framed filter membrane was positioned on top, and 50,000 calcein-AM-labeled HCECs were suspended in 20 µl of matching buffer, or media was applied on the top of each membrane/well. The assembly was incubated for 120 min at 37 °C, and the number of endothelial cells transmigrated into the wells was quantified by measuring the intensity of fluorescence (excitation/emission: 485/530 nm) in a CytoFluor 2350 reader (Millipore, Bedford, MA).
Capillary Tube Formation in MatrigelTM—HCECs were seeded (8 x 104 cells) on a semi-solid basement membrane matrix, MatrigelTM (Collaborative Biomedical Products, Bedford, MA)-coated 24-well plates and exposed to serum-free media containing various treatments. Cells were treated with 10 nM VEGF165 or glioblastoma cell (U-87MG, ATCC, Rockville, MD)-conditioned media (serum-free Dulbecco's modified Eagle's medium conditioned for 48–72 h) in the absence or presence of SP5.2 or control peptide for 24 h. HCECs were then labeled with a vital dye, calcein-AM (2 µM) for 30 min. Cells were then washed in PBS and capillary-like tube formation, a measure of in vitro angiogenesis, was observed using a Zeiss LSM 410 inverted laser scanning microscope (Thornwood, NY) with an argon/krypton ion laser and a Zeiss LD achroplan 5x, 0.6-numerical aperture objective. Confocal apertures for each recorded wavelength were adjusted to a full-width half maximum of 20 µm. The emitted light was collected through a 515- to 540-nm band-pass filter using an excitation of 488 nm. 8–32 individual optical sections were collected, and the average was calculated. Standard image processing was performed to enhance brightness and contrast using ImageSpace software (Molecular Dynamics Inc., Sunnyvale, CA).
SP5.2--gal Fusion Construction—The prokaryotic expression vector, pQE-16, purchased from Qiagen, was used to construct the fusion protein SP5.2--gal, containing SP5.2 peptide on the N terminus and His6 on the C terminus of -galactosidase. For this the vector pCMV- from Clontech (Palo Alto, CA) was digested with BamH1 (New England Biolabs), the resulting -galactosidase gene was purified from agarose gel and modified by PCR as so to introduce BglII at the 3'-end for cloning into pQE16 instead of dihydrofolate reductase, which has been removed by digestion of plasmid with BamHI and BglII. The SP5.2 insert, containing the EcoRI site at the 5'-end and the BamHI site at the 3'-end, was prepared by annealing the two single-stranded oligonucleotides synthesized by INRS-IAF (Laval, Quebec, Canada) and inserting them into the pQE16--gal vector. pQE16-SP5.2--gal was used to transform competent E. coli M15 cells. The resulting clones were sequenced to verify the fusion gene.
Fusion Protein Expression and Purification—An overnight culture of a single colony was grown in 400 ml of LB medium containing 100 µg/ml ampicillin and 25 µg/ml kanamycin to an optical density of 0.6 at 600 nm. The culture was induced with 1 mM isopropyl-1-thio--D-galacto-pyranoside for 4 h at 37 °C, centrifuged, and the resulting pellet was stored at –80 °C. The expression of -galactosidase and the presence of His tag were verified by Western blot using anti--galactosidase (Sigma, Ontario, Canada) and anti-His-HRP antibody (Invitrogen, Ontario, Canada). The pellet was thawed and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and lysozyme was added to a concentration of 1 mg/ml. After incubation for 30 min at 4 °C, 400 µl of 50% nickel-nitrilotriacetic acid resin (Qiagen, Ontario, Canada) was added to the supernatant and incubated 1 h shaking at 4 °C. The lysate/nickel-nitrilotriacetic acid mixture was loaded into a column (Qiagen), washed with washing buffer (50 mM NaH2PO4, 10 mM NaCl, 20 mM imidazole, pH 8.0), and the protein SP5.2--gal was eluted with the elution buffer (50 mM NaH2PO4,10mM NaCl, 250 mM imidazole, pH 8.0). The eluted protein was washed by PBS to decrease imidazole concentration and concentrated by Centricons (Fisher) to get a final concentration of 1 µg/ml.
SP5.2--gal Fusion Protein Binding Assay—Streptavidin-coated microtiter plates (total binding capacity for biotin-labeled AB = 1.5 µg/well, Roche Applied Science) were used to immobilize the rhFlt-1 receptor chimera. Biotin-labeled anti-human Fc antibody (ICN) 10 µg/ml in 0.1% BSA/PBS were added into each well of streptavidin-coated plate. The microtiter plates were incubated at 4 °C for 16 h in a humidified container and then washed for four times with 0.1% BSA/PBS. Recombinant human Flt-1/Fc chimera (1 µg/well in 0.1% BSA/PBS, R&D Systems) was added into each well. Plates were incubated at 4 °C 8 h in sealed container to let receptor to immobilize. Unbound receptor was washed away with 2 ml of 0.1% BSA/PBS. Then each well was filled with 350 µl of blocking buffer (2% nonfat dry milk in PBS) and blocked for 16 h at 4 °C. As a negative control, three wells with immobilized anti-Fc antibody were blocked with 2% nonfat milk in PBS. SP5.2--gal fusion protein and recombinant -galactosidase from Sigma (Oakville, Ontario, Canada), used as control, were added to each well and incubated for 2 h at room temperature. Wells were washed with 0.1% BSA/PBS, and the amount of bound protein expressing SP5.2--gal was detected with anti--gal (Sigma) and IgG anti-mouse HRP conjugate (Sigma). After substrate addition (ABTS containing H2O2), wells were analyzed in microtiter plate reader at 405 nm.
Synthesis of SP5.2-Peroxidase-conjugated Peptide—3 mg of peroxidase (ICN) was dissolved in 1 ml of phosphate buffer (0.1 M Na2HPO4, 0.1 M NaCl, 1 mM EDTA, pH 8.5). N-Succinimidy 1,3-(2-pyridylthio)propionate (SPDP; Sigma) was dissolved in dimethylformamide in a proportion of 0.234 mg of SPDP/39 µl of dimethylformamide. The solution of SPDP was added to the solution of peroxidase and stirred 30 min at room temperature. The solution of activated peroxidase was applied to Sephadex G-25 column (Fisher) and eluted with 50 ml of phosphate buffer, fractions of 1 ml were collected and detected at 280 nm at a sensitivity level of 50, lamp intensity 0.005, and recorder prints at 5 mm/min. The fractions containing modified peroxidase were selected and combined. 1 mg of SP5.2 peptide synthesized at Supratek was dissolved in phosphate buffer, added to active peroxidase, and incubated stirring for 24 h at room temperature. The reaction was controlled by UV detection at 343 nm. The conjugate was applied on G-25 Sephadex column for purification, and the conjugate fractions were collected and combined. Activity of conjugate was determined by checking peroxidase activity itself using ABTS substrate containing H2O2. Cysteine conjugated to peroxidase was synthesized at Supratek to be used as a negative control.
SP5.2-Peroxidase Conjugate Binding Assay—Streptavidin-coated microtiter plates (total binding capacity for biotin-labeled AB = 1.5 µg/well, Roche Applied Science) were used to immobilize the rhFlt-1 receptor chimera. Biotin-labeled anti-human FC antibody (ICN, 10 µg/ml in 0.1% BSA/PBS) was added into each well of streptavidin-coated plate. The microtiter plates were incubated at 4 °C for 16 h in a humidified container and then washed for four times with 0.1% BSA/PBS. Recombinant human Flt-1/Fc chimera (1 µg/well in 0.1% BSA/PBS, R&D Systems) was added into each well. Plates were incubated at 4 °C 8 h in a sealed container to let receptor to immobilize. Unbound receptor was washed away with 2 ml of 0.1% BSA/PBS. Then each well was filled with 350 µl of blocking buffer (2% nonfat dry milk in PBS) and blocked for 16 h at 4 °C. As a negative control, three wells with immobilized anti-Fc antibody were blocked with 2% nonfat milk in PBS. SP5.2-peroxidase conjugate and Cys-peroxidase, used as control, were added to each well and incubated for 2 h at room temperature. Wells were washed with 0.1% BSA/PBS, and the amount of bound conjugate was detected with peroxidase activity. After substrate addition (ABTS containing H2O2), wells were analyzed in a microtiter plate reader at 405 nm.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
1 x 109 independent peptide sequences was screened for binding to immobilized human Flt-1 receptor fused with Fc fragment of human IgG antibody (Flt-1/Fc chimera). In one setting, the Flt-1-bound phages were recovered by elution with acidic buffer (0.2 M glycine-HCl, pH 2.2). In another protocol, used to identify phages that interact with the VEGF binding site of Flt-1, 10 ng/ml rhVEGF165 was used to displace the bound phages. A marked enrichment in the Flt-1-binding phage relative to bovine serum albumin (BSA), used as a control target protein, was detected with each consecutive panning round, reaching the maximum (2500-fold in the case of acidic elution and 400-fold in the case of elution with VEGF) (Fig. 1) after the fourth panning round. At the end of the selection protocols, a total of 20 clones were isolated and analyzed. DNA sequencing of these clones showed that two independent peptide sequences (A1 and A2) were selected with acidic elution, and four homologous peptide sequences (V5.2, V5.10, V40, and V5.8) were selected with VEGF displacement. A2 and V5.2 sequences, isolated in both protocols, were identical (Table I).
|
|
|
|
|
|
|
|
Inhibition of VEGF-mediated Proliferation of Endothelial Cells—The effects of SP5.2 peptide on endothelial proliferation were assessed in HUVECs and HCECs cells stimulated with VEGF using the DNA synthesis rate as a measure of cell proliferation.
HUVECs were stimulated with 10 ng/ml rhVEGF165 and then treated with various concentrations of SP5.2 peptide for 48 h. The peptide inhibited the proliferation of HUVECs in a dose-dependent manner with an IC50 of 5 µM (Fig. 8A).
|
Inhibition of VEGF-mediated Migration of HCECs—Migration (chemotaxis) of calcein-AM-labeled HCECs induced by several stimuli, including VEGF165 and glioblastoma-conditioned media, was tested in the absence or presence of SP5.2 peptide or scrambled peptide as described previously (37). VEGF stimulated migration of HCECs across the filter by 25% (Fig. 9), and this effect was counteracted by SP5.2 but not by the control peptide (Fig. 9). SP5.2 also inhibited migration of HCECs induced by glioblastoma cell-conditioned media, known to contain high amount of secreted angiogenic factors.
|
|
|
-gal Fusion Protein—To explore the possibility that the SP5.2 sequence can be used as a targeting moiety for delivery of biologically active agents such as genes, polypeptides and particulate delivery vehicles to Flt-1-expressing cells/ tissues, a fusion protein, SP5.2--gal, expressing the peptide sequence at the N terminus and the His6 tag at the C terminus of -galactosidase, was produced and purified by His tag affinity chromatography. The SP5.2--gal affinity and selectivity for the Flt-1 receptor was determined by ELISA assay using a recombinant -galactosidase as control. Fig. 12A shows that SP5.2--gal construct binds to Flt-1 receptor with an apparent KD of 200 nM, suggesting that the fusion protein has about 200-fold binding affinity compared with the synthetic SP5.2 peptide. At the same time, SP5.2--gal showed no significant interaction to KDR receptor (Fig. 12B). This result suggests that SP5.2 can potentially be used as a targeting element for genetically constructed therapeutic proteins to be delivered into specific Flt-1-rich compartments such as tumor endothelium, inflammation sites, or a damaged blood-brain barrier.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In this study, five rounds of biopanning of a 16-mer peptide phage display library against Flt-1 immobilized on magnetic particles resulted in identification of five different phage clones capable of binding to immobilized Flt-1. The most efficient of these, a phage V5.2, expressing the SP5.2 peptide (NGYEIEWYSWVTHGMY) was chosen for further analysis. Chemically synthesized SP5.2 peptide was tested for its ability to bind other endothelial receptors such as ICAM-1, Flk-1, and KDR, none showed any significant binding compared with Flt-1. However, there was a significant and selective cross interaction with the murine analog of Flt-1, also to murine NRP-1. An alanine scan was used to identify functionally important amino acids in the SP5.2 sequence, and the binding properties of the obtained mutated phages were analyzed by ELISA. Based on these results a critical structural motif of the SP5.2 peptide important for the binding to Flt-1 receptor was identified as: NXXEIEXYXWXXXXXY, were X represents amino acid residues not involved in the Flt-1 binding.
Cell-based experiments have demonstrated that SP5.2 produces inhibition of VEGF-mediated functional events in Flt-1-positive cells such as HUVECs and HCECs. SP5.2 was found to inhibit several components of angiogenic response in these cells induced by VEGF, including proliferation, migration, early angiogenesis, and vascular permeability. All these inhibitory effects were sequence-specific and selective with respect to VEGF compared with other growth factors. SP5.2 was also shown to antagonize some effects of PLGF, known to share more than 50% homology with VEGF and to act through Flt-1 and NRP-1 receptors (48, 49).
Most previous studies, aiming to establish functional roles of Flt-1 and KDR receptors, have produced a general agreement that events mediated through Flt-1 are mainly responsible for early stages of VEGF-induced angiogenesis, such as modulation of cell motility and adhesion of endothelial cells, whereas KDR is more important in regulating endothelial proliferative response (50–52). However, it has recently been demonstrated that Flt-1 and KDR can form heterodimers in which Flt-1 may participate in the regulation of proliferative response to VEGF (43). Also, NRP-1 when coexpressed in cells with KDR may present VEGF165 to the KDR receptor in a manner that enhances the effectiveness of KDR-mediated signal transduction (53). SP5.2, although selectively binding to Flt-1 receptor in in vitro studies, was capable of inhibiting both Flt-1-mediated cellular events such as capillary-like tube formation, vascular permeability, endothelial migration, and predominantly KDR-mediated endothelial proliferation. This result is in agreement with recent studies suggesting that Flt-1 is involved in the proliferative control of endothelium most likely through its heterodimerization with KDR (43). KDR-mediated signal transduction and its mitogenic activity for endothelial cells can also be inhibited while SP5.2 binding to the NRP-1 receptor occurs. Further studies are required for more detailed mechanistic understanding of the antiproliferative effects of SP5.2.
It is well known that peptides identified during phage display library screening often show much lower potencies when synthesized as individual compounds (54, 55). The two main reasons for this are: (i) small synthetic peptides most often do not have sufficient conformational rigidity that, in the case of phage, is provided by phage filament protein in which the peptide is inserted and (ii) phage particles display multiple copies of inserted peptides (from three to five peptides in the case of P3 protein-based libraries), and this multivalency results in higher avidity compared with the synthetic peptide. The synthetic form of SP5.2 when presented as a single molecule demonstrated a micromolar affinity in a variety of binding assays. A possible strategy to increase binding of the synthetic peptide is to achieve its multivalency by attaching multiple copies to a structurally rigid moiety such as a protein, liposome, polymer, etc. (56–58). To explore a possibility of increasing potency of SP5.2, two derivatives of this molecule were produced. The first was a recombinant fusion of SP5.2 with -galactosidase in which SP5.2 was present as a single copy at the N terminus of the enzyme that was used as a carrier protein. The second derivative was a chemical conjugate of SP5.2 and peroxidase in which three to five copies of the peptide were attached to the protein. The resulting products showed considerably higher binding potencies to Flt-1 compared with the original synthetic peptide. The apparent dissociation constants established in the Flt-1 binding assays were 200 nM (200-fold lower compared with SP5.2) for the fusion with a single copy of the peptide, and 100 nM (400-fold lower compared with SP5.2) in the case of the chemically linked, polyvalent product. These results suggest that SP5.2 can potentially be used as an efficient targeting component that, as an added benefit, displays inherent antiangiogenic activity. Several possible therapeutic applications of SP5.2 are presently being explored in ongoing studies. These include combining SP5.2 with therapeutic proteins such as cytokines in targeted genetic constructs, as well as combining this peptide with polymeric micelles loaded with cytotoxic drugs.
In summary, this study describes discovery of the new peptide, SP5.2, which is an effective and selective Flt-1 antagonist. This molecule and its derivatives may have future utility in clinical applications for treating cancer, diabetic retinopathy, and other angiogenic or proliferative disorders associated with Flt-1 up-regulation in endothelial cells.
|
FOOTNOTES |
|---|
** To whom correspondence should be addressed: Supratek Pharma Inc., 215 Bouchard St., Dorval, Quebec H9S 1A9, Canada. Tel.: 514-422-9191 (ext. 207); Fax: 514-422-9410; E-mail: valery.alakhov{at}supratek.com.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; Flt-1, tyrosine kinase receptor VEGFRI; KDR, tyrosine kinase receptor VEGFRII; NRP-1, neuropillin-1; HRP, horseradish peroxidase; HCEC, human cerebral endothelial cell; HUVEC, human umbilical vein endothelial cell; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Flt-MP, Flt-1-coated magnetic particle; ELISA, enzyme-linked immunosorbent assay; cfu, colony-forming unit(s); bFGF, basic fibroblast growth factor; CMV, cytomegalovirus; SPDP, N-succinimidy 1,3-(2-pyridylthio)propionate; EC, endothelial cell; AB, antibody; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); PLGF, placenta growth factor.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
