Human recombinant VEGF165, recombinant human Fc-VEGFR2 chimera, Fc-VEGFR1 chimera and Fc-Neuropilin 1 chimera were purchased from R&D systems (Minneapolis, MN). 125I-Bolton Hunter reagent was obtained from PerkinElmer (Boston, MA). Heparin was from Neoparin (San Leonardo, CA). Bovine serum albumin (BSA) was obtained from American Bioanalytical (Natick, MA). Purified human plasma fibronectin was from Chemicon International (Temecula, CA). Dulbecco's Modified Eagle's media (DMEM), phosphate buffered saline (PBS) containing no Ca2+ and Mg2+, penicillin/streptomycin, L-glutamine, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and trypsin-EDTA were obtained from Invitrogen (Carlsbad, CA). Calf serum (CS) was purchased from Hyclone (Logan, UT). NE (human) was obtained from Elastin Products (Owensville, MO).
Bovine aortic endothelial cells (BAEC passage 5-15) were maintained in low-glucose DMEM, supplemented with 10% CS, 5 mM L-glutamine, 100 U/ml penicillin G, and 100 (μg/ml streptomycin sulfate. Neonatal rat aortic smooth muscle cells (SMCs) were isolated from Sprague-Dawley rats, ages 1-3 days as described, and maintained in low-glucose DMEM, with 10% FBS, 5 mM L-glutamine, 100 U/ml penicillin G, and 100 (μg/ml streptomycin sulfate and 1% nonessential amino acids.
VEGF Fragment (VEGFf) Generation
125I-labeled VEGF165, prepared using a modified Bolton-Hunter procedure, was treated with varying NE concentrations in 44 mM sodium bicarbonate, pH 7.4, for various times at 37°C. Mock-treated 125I-VEGF was incubated in 44 mM sodium bicarbonate, pH 7.4 at 37°C. The reaction was stopped by adding 1 μM di-isopropyl fluorophosphate (DFP). For large VEGFf preparations, the samples (NE and mock treated) were dialyzed (10-kDa molecular weight cut off, Slide-A-Lyzer; Pierce, Rockford, IL) against PBS at 4°C to remove DFP. The concentration of 125I-VEGF165 and 125I-VEGFf in the samples was determined by trichloracetic acid precipitation.
VEGF/VEGFf Binding to VEGFR1 and Neuropillin 1/Fc chimera
Binding assays were performed with VEGF binding chimeras by incubating a range of 125I-VEGF and 125I-VEGFf concentrations with Fc-VEGFR1, Fc-NP-1, or both in binding buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mg/ml BSA) for 2 h at 4°C. The bound complexes were pulled down with magnetic protein-A beads (New England Biolabs, Beverly, MA). The beads were washed three times with binding buffer, and 125I-VEGF/125I-VEGFf associated with the beads was measured using a Cobra Auto-Gamma 5005 counter (Packard Instruments, Meridian,CT).
VEGF/VEGFf Binding to Fibronectin
Fibronectin (10 μg/ml) in the presence of heparin (10 μg/ml) was adsorbed overnight onto 96-well hydrophobic polystyrene plates in PBS at 4°C (100 μl/well). Following protein adsorption, the surface was washed three times with PBS and once with binding buffer. VEGF/VEGFf binding assays were then conducted with various concentrations of 125I-VEGF and 125I-VEGFf in 0.15 M NaCl, 25 mM HEPES, 1 mg/ml BSA, pH 7.5 for 2.5 h at 4°C (50 μl/well). In some cases, plates were treated with various NE concentrations for 30 min at 37°C, the NE solution removed and plates washed three times with PBS then incubated for 10 min with PBS containing 1 μM DFP to inhibit any residual NE. VEGF binding to treated plates was then measured by incubating the wells with the indicated concentration of 125I-VEGF/VEGFf. Unbound VEGF/VEGFf was removed by washing three times with binding buffer and bound VEGF/VEGFf was extracted by 1 h incubation with 5M NaCl, 25 mM HEPES, pH 7.5. The incubation was performed at room temperature with 50 μl of extraction solution per well. After each incubation period the wells were washed once more with the same extraction solution (50 μl/well) to ensure complete recovery of the respective fraction of bound VEGF/VEGFf. The radioactivity released was measured by a Cobra Auto-Gamma 5005 counter (Packard Instruments, Meridian, CT).
VEGF Binding to Cell Culture Extracellular Matrices
Primary rat aortic smooth muscle cells and pulmonary fibroblasts were plated onto 24-well plates (2 cm2/well) at an initial density of 5 × 104 per well. When the cells reached confluence, the culture media was changed to 1% serum. Five days past-confluence 125I-VEGF/VEGFf binding was conducted with isolated ECM. Isolated ECM was used to evaluate the effects of elastase on VEGF binding in order to avoid interference caused by the effects of high elastase concentrations on the living cells. To prepare cell-free ECM the cell layer was dissolved with 0.5% Triton, 20 mM NH4OH in PBS at 23°C for 3 min, followed by three washes with PBS. The isolated ECM was incubated with various concentrations of NE for 30 min at 37°C, the elastase was inactivated with 1 μM DFP and the ECM plates washed with PBS. Isolated ECM were incubated with 125I-VEGF or 125I-VEGFf in binding buffer (0.15 M NaCl, 25 mM HEPES, pH 7.5, 1 mg/ml BSA) at 4°C for 2 h. Unbound ligand was removed by washing the ECM layers three times with binding buffer and VEGF/VEGFf bound to ECM sites was extracted with 2 M NaCl, 25 mM HEPES, pH 7.5 and samples were counted in a gamma counter.
Cell Migration Assay
Migration assays were performed using a modified Boyden chamber technique using 24-well Transwell® permeable supports (Corning, NY) with migration inserts (5 μm pore size, 6.5 mm diameter). Serum starved BAEC were plated onto the transwell inserts at a density of 100,000 cells/insert in serum-free medium, 25 mM HEPES, pH 7.5 and 0.05% gelatin (v/v) and placed in 24-well plates containing binding buffer +/- chemoattractant (VEGF, VEGFf) at 0.45 nM. The assembled plate was placed at 37°C (5% CO2, humidified) for the duration of the migration time (2h). Once the migration time had finished, media from the lower and upper chambers were aspirated; migrated cells were washed once in PBS without Ca2+ and Mg2+ ions and the cells that had migrated to the other side of the membrane were fixed with 100% pre chilled methanol for 10 min. Cells were subsequently washed two times in PBS and the non migrated cells on the top side of the transwell membrane were swabbed using Q-tips. The migrated cells were then stained by incubating in 5 μg/ml propidium iodide in PBS (600 μl/well) for 10 min. Cells were subsequently washed two times in PBS and a microsurgery knife was used to cut out the transwell membrane. The membranes were placed on labeled glass slides with migrated cells facing up. A drop of Antifade Component A (Molecular Probes, Invitrogen, Carlbad, CA) was placed on top of each membrane prior to covering with a glass cover slip. Images of migrated cells were captured by fluorescent microscopy at six different fields/membrane at 100× magnification. The migrated cells were counted using Image J NIH software.
Experimental data was subjected to statistical analysis using the Analysis ToolPak in Microsoft Excel X for Mac. Dose response data were subject to regression analysis and analysis of variance (ANOVA). Data from multiple treatments/conditions was subjected to ANOVA followed by the Newman-Keuls multiple comparison t-test routine. Differences between groups were considered statistically significant when p was less than 0.05.
Computational Model Development
In this paper we develop a model of VEGF binding and regulation of cell surface binding by VEGFf (Figure , Table ). The model is based on mass-action kinetics describing binding and cell surface trafficking interactions for VEGF with VEGFR1, VEGFR2, NP-1, and ECM sites and builds on previous work from Popel and co-workers[15
]. In the model, VEGF can form a triad with VEGFR2 and NP-1 but not with VEGFR1. NP-1 can bind VEGFR1 in the absence of VEGF but not VEGFR2. Coupling and uncoupling rates were considered equivalent for all species and independent of VEGF binding. A 1:1 stoichiometry of VEGF to receptor or NP-1 was maintained. Heparan sulfate proteoglycans (HSPGs) were only explicitly included as ECM sites. Synthesis was based on steady-state receptor levels and internalization rates for all species were considered equivalent except where noted. The fluid phase is considered well-stirred.
Regulation of VEGF binding by VEGFf was investigated in two ways. VEGFf has been shown previously to bind to VEGFR1 with a similar affinity as VEGF, but does not bind VEGFR2[14
]. In this paper, we provide evidence that VEGFf does not bind to NP-1 or ECM sites. The model reflected these events but there is no direct evidence supporting or excluding VEGFf bound to VEGFR1 from interacting with NP-1 as shown for VEGF121
] but not VEGF. In model 1, VEGFf was therefore able to bind VEGFR1 and then couple with NP-1 or bind to VEGFR1-NP-1 complexes. In model 2, binding of VEGFf to VEGFR1 excluded further interactions with NP-1 and VEGFf was not able to bind to VEGFR-NP-1 complexes. In all other ways the models are identical.
The models are composed of a set of nonlinear ordinary differential equations. The equations listed below describe Model 2, where VEGFf bound to VEGFR1 excludes any interactions between VEGFR1 and NP-1. Symbols are defined in Tables and .
With Model 1 where VEGFf bound VEGFR1 can interact with NP-1 the following equations are altered:
Simulations were run in Matlab R2006b (The Mathworks, Inc., Natick, MA) using the stiff ordinary differential equation solver ode15s with the backwards differentiation formulas option and an absolute tolerance criteria of 1 × 10-20. Parameter values are listed in Table . Simulations were generally run for 3h. Values were used as reported in the literature with no attempt to adjust based on temperature.