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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:80.)
© 2000 American Heart Association, Inc.

Vascular Biology

Platelet-Activating Factor Enhances Vascular Endothelial Growth Factor–Induced Endothelial Cell Motility and Neoangiogenesis in a Murine Matrigel Model

Giuseppe Montrucchio; Enrico Lupia; Edda Battaglia; Lorenzo Del Sorbo; Mariarosaria Boccellino; Luigi Biancone; Giorgio Emanuelli; Giovanni Camussi

From the Dipartimento di Fisiopatologia Clinica, Università di Torino (G.M., E.L., E.B., L.D.S., G.E.); the Ospedale Gradenigo (E.L.); and the Dipartimento di Medicina Interna, Università di Torino (M.B., L.B., G.C.), Torino, Italy.

Correspondence to Dr G. Camussi, Cattedra di Nefrologia, Dipartimento di Medicina Interna, Corso Dogliotti 14, 10126, Torino, Italy. E-mail giovanni.camussi{at}unito.it

	Abstract

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Abstract—We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of ¹²⁵I-albumin. The synthesis of PAF, cell motility, and the increased ¹²⁵I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein–derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.

Key Words: vascular endothelial growth factor • platelet-activating factor • angiogenesis

	Introduction

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Vascular endothelial growth factor (VEGF) is a potent, pleiotropic cytokine that possesses several biological and possibly independent actions on the vascular endothelium.^{1 2} VEGF was originally isolated as a tumor-released factor that enhances vascular permeability to circulating macromolecules.^{3 4} It was subsequently found that VEGF acts directly on endothelial cells by promoting proliferation,^{5 6 7 8} a transient increase in cytoplasmic calcium,⁹ shape change, and migration.¹⁰ Moreover, VEGF has emerged as a potent mediator of angiogenesis in vivo.^{6 8 11} Angiogenesis is a process involved in wound healing, tumor growth, and inflammation, which implicates a concerted sequence of events including directional migration and proliferation of endothelial cells and canalization of solid, endothelial cords penetrating the tissue.¹² A number of diffusible angiogenic factors have been recently characterized.¹² Among these angiogenic factors, VEGF has been implicated as a mediator in the development of the vascular system^{13 14} and in angiogenesis associated with wound healing, cancer, rheumatoid arthritis, psoriasis, and proliferative retinopathies.^{1 2 12} Recently, experimental and clinical studies have exploited the angiogenic properties of VEGF to favor the revascularization of ischemic tissue.¹⁵ VEGF, as well as other mediators, including platelet-activating factor (PAF),^{16 17} can be produced by endothelial cells and may possess an autocrine modulatory role on endothelial cell function. Recently, we found that PAF contributes to the angiogenic activity of certain cytokines, including tumor necrosis factor (TNF)¹⁸ and hepatocyte growth factor (HGF).¹⁹ PAF directly stimulates in vitro migration of endothelial cells,²⁰ enhances vascular permeability,^{16 17} and promotes in vivo angiogenesis,^{20 21} all biological activities that resemble that of VEGF. PAF belongs to the structurally related family of acetylated alkyl phosphoglycerides produced by a broad range of cells, including neutrophils, macrophages, and endothelial cells.^{16 17} It acts through a specific receptor belonging to the family of 7 spanning membrane domain receptors²² and elicits diverse and potent biological properties relevant to the development of the inflammatory reaction, embryogenesis, and cell differentiation.^{16 17} Recently, it has been shown that the enhanced permeability induced in vivo by VEGF is inhibited by selective PAF-receptor antagonists in some organs.²³

The aim of the present study was to evaluate whether PAF synthesis mediates some of the biological properties of VEGF. For this purpose, experiments were performed in vivo by evaluating the synthesis of PAF during angiogenesis induced by VEGF in a murine model of subcutaneous Matrigel implantation and the inhibitory effect of 2 chemically unrelated PAF-receptor antagonists on both VEGF-induced angiogenesis and enhanced vascular permeability. In vitro, we evaluated whether VEGF may trigger the synthesis of PAF by cultured human endothelial cells and whether PAF-receptor antagonists may inhibit endothelial cell motility, proliferation, and albumin transfer across the endothelial monolayer.

	Methods

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Reagents
Growth factor–reduced Matrigel basement membrane matrix was obtained from Becton Dickinson Labware. Recombinant human VEGF 165 was obtained from R&D Systems. Synthetic PAF (1-hexadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine; alkyl-PAF C16:O) was obtained from Bachem Feinchemikalien. WEB2170 was obtained from Boehringer Mannheim. CV3988 was from Takeda Chemical Industries. Anti-mouse T-cell serum, anti-L3/T4, anti-Ly2 monoclonal antibodies (mAbs), and anti–MAC-1 FITC-conjugated mAb were purchased from Cedarlane. FITC-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Cappel Laboratories. Rabbit anti-human von Willebrand factor, rabbit IgG, and anti-rabbit IgG mAb (RG-96) were obtained from Sigma Chemical Co. Rabbit polyclonal anti–basic fibroblast growth factor (bFGF) antibody was from Immunogenex. Silica gel 60F254 thin-layer chromatography (TLC) plates were obtained from Merck. µPorasil high-performance liquid chromatography columns were provided from the Millipore chromatographic division (Waters). Bovine calf serum (BCS) was from Hyclone Labs.

In Vivo Experimental Protocol
The angiogenic effect of various doses of VEGF (2, 20, 40, 80, or 160 ng/mL) in 0.5 mL of Matrigel was studied at different times and in the presence of 64 U/mL heparin. In selected experiments, the effect of WEB2170 and CV3988, 2 chemically unrelated PAF-receptor antagonists,^{24 25} or of anti-bFGF neutralizing antibody (10 µg/mL) on VEGF-induced angiogenesis was evaluated. WEB2170 was included in the Matrigel plug (final concentration 250 ng/mL) and injected intraperitoneally (10 mg/kg) 30 minutes before the subcutaneous injection and daily thereafter for 6 days. CV3988 was included in the Matrigel plug (final concentration 500 ng/mL) and injected intraperitoneally (20 mg/kg) 30 minutes before the subcutaneous injection and daily thereafter for 6 days. As controls, VEGF, WEB2170, or CV3988 boiled for 5 minutes was used at the same doses. In other experiments, the angiogenic effect of bFGF (10 ng/mL) or PAF (10 ng/mL) in the presence or absence of WEB2170 was evaluated. Moreover, experiments with VEGF (2 ng/mL) or bFGF (1 ng/mL) combined with PAF (4 ng/mL) were performed. The angiogenic effect of acyl-PAF (10 ng/mL) was also studied. To evaluate whether the angiogenic factors induced in vivo PAF synthesis, PAF was extracted and purified from Matrigel at different times after the injection and characterized as described below.

Murine Angiogenesis Assay
Female C57 mice were used at 6 to 8 weeks of age. Mice were cared for and handled according to accepted ethical practices. Angiogenesis was assayed as growth of blood vessels from subcutaneous tissue into a solid gel of basement membrane containing the test sample.²⁶ Matrigel (8.83 mg/mL), in liquid form at 4°C, was mixed with the experimental substances and injected (0.5 mL) into the abdominal subcutaneous tissue of mice along the peritoneal midline. Matrigel rapidly forms a solid gel at body temperature, thereby trapping the factors and allowing their slow release and prolonged exposure to surrounding tissues. At various times the mice were subsequently killed with sodium pentobarbital (100 mg/kg IP), and the gels were recovered and processed for histology. Typically, the overlying skin was removed, and gels were cut out by retaining the peritoneal lining for support. Part of the tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections cut at 3 µm and stained with hematoxylin and eosin were studied by light microscopy. Other sections, obtained from frozen tissue cut with a cryostat, were stained for nonspecific esterase activity²⁷ or processed for immunofluorescence microscopy, performed as previously described.^{18 19} Vessel area and the total Matrigel area were planimetrically assessed from stained sections, as described by Kibbey et al.²⁸ We considered as "vessels" only those structures possessing a patent lumen and containing erythrocytes. Results were expressed as the mean±SD (in percent) of the vessel area to the total Matrigel area.

Miles Vessel-Permeability Assay
The Miles assay was performed as described previously.²⁹ Depilated mice were injected intravenously with ¹²⁵I-albumin (2.5 µCi per animal) and Evans blue dye (0.5% in sterile saline). Test samples (10 and 20 ng/mL VEGF, 10 ng/mL PAF, a mixture of 20 ng/mL VEGF and 3 µmol/L WEB2170, a mixture of 10 ng/mL PAF and 3 µmol/L WEB2170, or a mixture of vehicle alone plus 3 µmol/L WEB2170) were injected intradermally into the dorsal skin in 4 replicate sites (100 µL per skin site) according to a balanced site injection plan. Test sites were excised and quantified for ¹²⁵I-albumin in a gamma counter.

In Vitro Permeability Studies
Human umbilical cord vein–derived endothelial cells (HUVECs) were grown to confluence on polycarbonate filters (pore size 0.4 µm) of Transwell chamber assemblies (Costar) coated with fibronectin. The permeability of HUVEC monolayers was measured by diffusion of ¹²⁵I-albumin.³⁰ The upper chamber was filled with 0.5 mL of Iscove’s medium containing 0.1 µCi of ¹²⁵I-albumin. Fluid volumes were selected to avoid hydrostatic pressure gradients across monolayers. The chambers were incubated at 37°C in 5% CO₂ with continuous agitation with a trypan blue–albumin complex, prepared as described by Rotrosen and Gallin.³¹ Monolayers with no leakage of the dye after 5 minutes of incubation were used for the experiments. The transport of albumin across HUVEC monolayers was determined by sampling aliquots and measuring the radioactivity in the outer and inner wells in triplicate. The passage of albumin across the HUVEC monolayer was studied, as induced by VEGF (20 ng/mL), PAF (10 ng/mL), vehicle alone as control, VEGF plus WEB2170 (3 µmol/L), PAF plus WEB2170 (3 µmol/L), or vehicle alone plus WEB2170 (3 µmol/L).

In Vitro Endothelial Cell Growth Assay
The effect of vehicle alone (saline containing 0.25% BSA) or VEGF (20 ng/mL) in the presence or absence of 3 µmol/L WEB2170 on endothelial cell proliferation was evaluated as described below. HUVECs (5x10³)were plated in 96-well plates (Costar) coated with gelatin (0.05% for 1 hour at 22°C) in medium 199 containing 20% BCS. After 24 hours, the medium was removed and replaced with medium 199 supplemented with 6 mg/L transferrin, 5 mg/L insulin, 100 mg of soybean lecithin, 6.73 µg/L sodium selenite, 400 mg/L BSA, and 0.25% Ultroser HY (IBF Biotechnics) with or without tested factors. The incubation media were changed every 2 days. After 8 days, endothelial cell number was estimated by a colorimetric assay after the cells were stained with crystal violet, according to the method of Kueng et al.³² In brief, the cells were fixed for 20 minutes at room temperature with 2.5% glutaraldehyde and then stained with 0.1% crystal violet in 20% methanol. After solubilization of stained endothelial cells with acetic acid (10%), the samples were read at 595 nm in a microplate reader (Bio-Rad model 3550). A calibration curve was constructed by using a known number of cells.

In Vitro Endothelial Cell Migration: Cell Motility Assay
Cell motility was measured as the speed at which cells migrated into a "wound" introduced in a confluent monolayer.^{33 34} Cells (2x10⁵ per well) were plated from essentially nondividing, confluent cultures. The cell monolayers were allowed to rest for 12 hours with medium 199 containing 1% BCS, wounded by rapid scraping, washed 3 times with PBS, and then incubated with RPMI 1640 and the agonist. Cell division did not start, to any significant degree, during the experiments. Cell migration was studied over a 4-hour period under a Nikon Diaphot inverted microscope with a 10x phase-contrast objective in an attached, hermetically sealed Plexiglas Nikon NP-2 incubator at 37°C. Cell migration was recorded by using a JVC 1CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging srl) and an IBM-compatible system equipped with a video card (Targa 2000, Truevision). Image analysis was performed by digital recording and storage of images at 30-minute intervals. Migration tracks were generated by marking the position of the nucleus of individual cells on each image. The net migratory speed (straight-line velocity) was calculated by the MicroImage software on the basis of the straight-line distance between the starting and end points divided by the time of observation. Migration of at least 30 cells was analyzed for each experimental condition. HUVECs were incubated with vehicle alone, PAF (10 ng/mL), or VEGF (20 ng/mL) in the presence or absence of 3 µmol/L WEB2170.

In Vitro PAF Synthesis by Endothelial Cells
HUVECs were prepared, grown, and characterized as described previously.³⁵ In standard PAF-synthesis assays, confluent HUVECs maintained for 24 hours in Dulbecco’s modified Eagle’s medium without BCS were stimulated with VEGF (0.1 to 40 ng/mL) in 1 mL of Iscove’s medium containing 0.25% BSA for various periods of time.

Assay and Quantification of PAF
PAF extracted and purified by the Matrigel plugs obtained from mice or by cultured HUVECs was quantified by bioassay on washed rabbit platelets.³⁶ PAF bioactivity, tested after extraction³⁶ and purification by TLC and high-performance liquid chromatography,³⁷ was characterized by comparison with synthetic PAF according to the following criteria: (1) induction of platelet aggregation by a pathway independent of both ADP- and arachidonic acid/thromboxane A₂-mediated pathways; (2) specificity of platelet aggregation as inferred from the inhibitory effect of the PAF-receptor antagonist WEB2170 (3 µmol/L); (3) TLC and high-performance liquid chromatographic behavior and physicochemical characteristics, such as inactivation by strong bases and heating for 5 minutes in boiling water. The methods used were previously described in detail.³⁶

To study the incorporation of radioactive precursors, 5x10⁵ HUVECs were incubated in 1 mL of RPMI 1640 for 30 minutes with 30 µCi of [³H]acetate (from NEN Life Sciences Products) before stimulation.³⁷ The cell pellets were extracted according to a modification of the Bligh and Dyer procedure,³⁸ with formic acid added to lower the pH of the aqueous phase to 3.0,³⁷ and lipids were fractionated by TLC on aluminum-sheet silica-gel plates (silica gel 60, F254, 0.2-mm thickness, Merck Darmstadt) by using a solvent of chloroform/methanol/acetic acid/water (50:25:8:4, vol/vol/vol/vol). The plates were cut into 1-cm sections and the radioactivity of each was measured. Radiolabeled PAF was used as a standard. To discriminate between the presence of an ester or ether group at the sn-1 position, the TLC-extracted radiolabeled lipid, comigrating with PAF were treated with phospholipase C from Bacillus cereus for 2 hours.³⁹ Control acyl-PAF C16:O was obtained by acetylation of 1-palmitoyl-sn-glyceryl-3-phosphorylcholine (Bachem Feinchemikalien) with acetic anhydride and dimethylaminopyridine as previously described.³⁷ The 1-radyl-2-[³H]acetylglycerols, obtained by phospholipase C treatment, were then acetylated at position 3 by incubation for 16 hours at 37°C with 0.5 mL of acetic anhydride and 0.1 mL of pyridine.^{37 39} The radiolabeled 1-radyl-2,3-diacetylglycerols obtained by reaction were extracted with hexane/diethyl ether (1:1, vol/vol) and analyzed by TLC on silica-gel plates that were developed with hexane/diethyl ether/formic acid (90:60:6, vol/vol/vol) that allows the separation of 1-alkyl-2,3-diacetylglycerol from 1-acyl-2,3-diacetylglycerol.^{37 39}

Statistical Analysis
All data are expressed as mean±SD. Statistical analysis was performed by ANOVA with Newman-Keuls multiple comparison test.

	Results

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In Vivo Angiogenic Effect of VEGF
Figures 1 and 2 show the results of experiments performed to evaluate the in vivo angiogenic effect of VEGF. Matrigel containing 64 U/mL heparin and various concentrations of VEGF, VEGF plus WEB2170, VEGF plus CV3988, VEGF plus anti-bFGF, VEGF boiled for 5 minutes, WEB2170, CV3988, bFGF, bFGF plus WEB2170, PAF, PAF plus WEB2170, acyl-PAF, or sterile saline used as vehicle for all different agents was injected subcutaneously into mice. The histological and morphometric analyses of Matrigel plugs were performed when the mice were humanely killed 6 days later. VEGF induced a dose-dependent angiogenic response that was absent in controls containing heparin plus saline (Figure 1). As shown in Figure 2, canalized vessels (Figure 2A) and microaneurysmatic structures (Figure 2B) containing erythrocytes and leukocytes were seen. Sections of the gel were stained with anti–von Willebrand factor antibodies, which confirmed the presence of endothelial cells in association with the vessels (data not shown). Infiltration into the Matrigel of cells positive for pan-T lymphocyte markers, L3/T4 or Ly2 antigens, or polymorphonuclear cells was never observed. In contrast, a few MAC-1– and nonspecific esterase–positive monocytes/macrophages were observed infiltrating the Matrigel at day 6. VEGF-induced angiogenesis was significantly reduced in mice treated with VEGF plus WEB2170 (Figures 1A, 1B, and 2C) or VEGF plus CV3988 (Figure 1B). As previously shown,²⁰ WEB2170 prevented angiogenesis induced by synthetic PAF but not that induced by bFGF (Figure 1B). Neutralizing antibodies against bFGF did not significantly reduce VEGF-induced angiogenesis (Figure 1B). At day 6, no angiogenesis was observed in controls injected with 64 U/mL heparin and vehicle alone (Figure 1A). In Matrigel containing 64 U/mL heparin alone or heparin plus VEGF boiled for 5 minutes (as controls), significant angiogenesis was never observed (Figure 1B). Control mice injected with vehicle alone and treated as above with WEB2170 or CV3988 alone did not exhibit any cell infiltration within Matrigel (Figure 1B). As shown in Figure 3, a combination of PAF and VEGF did not significantly enhance neoangiogenesis. In contrast, bFGF in combination with PAF was synergistic.

View larger version (26K): [in this window] [in a new window]   Figure 1. Angiogenic effect of VEGF in vivo. Matrigel (0.5 mL) containing 64 U/mL heparin was mixed with 2 (n=11 mice), 20 (n=5 mice), 40 (n=9 mice), 80 (n=5 mice), or 160 (n=5 mice) ng/mL VEGF or with vehicle alone (control, n=14 mice) and was injected subcutaneously into mice (A). After 6 days, the mice were killed and the Matrigel plugs were excised and processed for light microscopy. Quantitation of neovascularization was performed on hematoxylin-eosin–stained histological sections as described in Methods, and results were expressed as the percent mean±SD of the vessel area to the total Matrigel area. For each dose of VEGF (2, 20, 40, 80, or 160 ng/mL), 5, 7, 6, 3, and 3 mice, respectively, were treated with WEB2170. WEB2170 was included in the Matrigel plug (final concentration 250 ng/mL) and was injected (10 mg/kg IP) 30 minutes before the subcutaneous injection and daily thereafter for 6 days. ANOVA with Newman-Keuls multicomparison test was performed: Control vs VEGF 2, 20, 40, 80, or 160 ng/mL (*P<0.05); VEGF vs VEGF+WEB2170 (P<0.05). B, Effect of VEGF (40 ng/mL) in the absence (n=9 mice) or presence of 2 chemically unrelated PAF-receptor antagonists, WEB2170 (n=6 mice) and CV3988 (n=6 mice) or of anti-bFGF neutralizing antibody (10 µg/mL; n=6 mice). Treatment with WEB2170 was performed as described above. CV3988 was included in the Matrigel plug (final concentration 500 ng/mL and injected (20 mg/kg IP) 30 minutes before the subcutaneous injection and daily thereafter for 6 days. As controls, VEGF boiled for 5 minutes, WEB2170, and CV3988 were used at the same doses. In other experiments, the angiogenic effect of bFGF (10 ng/mL; n=10 mice) and PAF (10 ng/mL; n=10 mice) in the presence or absence of WEB2170 (n=10 mice) was evaluated. The angiogenic effect of acyl-PAF (10 ng/mL) was also studied. Results are expressed as percent mean±SD of the vessel area to the total Matrigel area. ANOVA with Newman-Keuls multicomparison test was performed: Control vs VEGF, VEGF+WEB2170, VEGF+CV3988, VEGF+anti-bFGF, heat-inactivated VEGF, WEB2170 alone, CV3988 alone, bFGF, bFGF+WEB2170, PAF, PAF+WEB2170, and acyl-PAF (*P<0.05); VEGF vs VEGF+WEB2170 and VEGF+CV3988 (P<0.05); bFGF vs bFGF+WEB2170 (not significant); PAF vs PAF+WEB2170 and acyl-PAF (P<0.05).

View larger version (76K): [in this window] [in a new window]   Figure 2. Histological analysis of Matrigel plugs. Hematoxylin-eosin staining of Matrigel containing 64 U/mL heparin and 20 ng/mL VEGF excised 6 days after injection. Canalized vessels (A) and microaneurysmatic structures (B) containing erythrocytes and leukocytes are shown. C, Inhibitory effect of WEB2170 on neovascularization of Matrigel containing 64 U/mL heparin and 20 ng/mL VEGF excised 6 days after injection (x250).

View larger version (22K): [in this window] [in a new window]   Figure 3. Comparison of angiogenic activity of individual mediators with that of a combination of PAF (4 ng/mL) and bFGF (1 ng/mL) or VEGF (2 ng/mL). Each group included 4 mice. Results are expressed as percent mean±SD of the vessel area to the total Matrigel area. ANOVA with Newman-Keuls multicomparison test was performed: bFGF+PAF vs bFGF or PAF alone (*P<0.05); VEGF+PAF vs VEGF or PAF alone (not significant).

In some experiments, Matrigel was subjected to PAF extraction at 6, 12, 24, and 48 hours and 6 days after the beginning of the experiment. PAF was detected at 24 and 48 hours in mice injected with Matrigel containing VEGF but not in controls (Figure 4). When the animals were injected with Matrigel containing an amount of synthetic PAF comparable with that extracted from VEGF-treated mice, an angiogenic response was observed, whereas when animals were injected with acyl-PAF, only a slight angiogenic response was observed (Figure 1B).

View larger version (16K): [in this window] [in a new window]   Figure 4. PAF synthesis within VEGF-containing Matrigel implanted subcutaneously in mice. PAF extracted and purified, at various times, by the Matrigel plugs obtained from mice containing 64 U/mL heparin and 20 ng/mL VEGF (dashed columns) or heparin and vehicle alone (black columns) was used as controls. The protein content of Matrigel plugs extracted for PAF determinations was 12.8±1.3 mg. Results are expressed as mean±SD of 4 experiments. ANOVA with Newman-Keuls multicomparison test was performed. VEGF vs controls (*P<0.05).

In Vivo and In Vitro Endothelial Permeability Studies
As shown in Figure 5A, WEB2170 inhibited in vivo dermal permeability induced by PAF but was ineffective on that induced by VEGF. Similar results were obtained in vitro, in which the VEGF-induced increased passage of ¹²⁵I-albumin across the HUVEC monolayer was not inhibited by WEB2170 (Figure 5B).

View larger version (28K): [in this window] [in a new window]   Figure 5. Endothelial permeability in vivo and in vitro. A, Dermal permeability induced by PAF (10 ng/mL), VEGF (10 and 20 ng/mL), or vehicle alone (dashed columns) and PAF+WEB2170 (3 µmol/L), VEGF+WEB2170 (3 µmol/L), or vehicle alone+WEB 2170 (3 µmol/L; open columns). Results are expressed as cpm/mg skin tissue, mean±SD of 3 experiments. ANOVA with Newman-Keuls multicomparison test was performed: Control vs VEGF 10 ng/mL, VEGF 20 ng/mL, or PAF (*P<0.05); VEGF vs VEGF+WEB2170 or PAF versus PAF+WEB2170 (P<0.05). B, Passage of albumin across the HUVEC monolayer, induced by VEGF (20 ng/mL), PAF (10 ng/mL), or vehicle alone as control (dashed columns) and VEGF+WEB2170 (3 µmol/L), PAF+WEB2170 (3 µmol/L), or vehicle alone+WEB2170 (3 µmol/L; open columns). Results are expressed as percent of 125I albumin transfer, mean±SD of 6 experiments. ANOVA with Newman-Keuls multicomparison test was performed; Control vs VEGF and PAF (*P<0.05); VEGF vs VEGF+WEB2170 or PAF vs PAF+WEB2170 (P<0.05).

In Vitro Growth and Migration of HUVECs
To evaluate the role of PAF on cell proliferation induced by VEGF and bFGF, HUVECs were stimulated every 2 days with 20 ng/mL VEGF or 10 ng/mL bFGF in the presence or absence of 3 µmol/L WEB2170. As shown in Figure 6, both VEGF and bFGF stimulated cell growth, which was not inhibited by WEB2170. Cell motility was measured as the speed at which cells migrated into a "wound" introduced in a confluent monolayer over a 4-hour period. No significant motility of unstimulated HUVECs was recorded. Incubation with VEGF (20 ng/mL) resulted in the activation of cell motility (Figures 7 and 8A), which was detectable as early as 30 minutes after the beginning of the experiment. Similar enhancement of cell motility was observed after stimulation of HUVECs with PAF (10 ng/mL; Figure 7). WEB2170 and CV3988, which completely abrogated PAF-induced motility, also significantly inhibited motility stimulated by VEGF (Figures 7 and 8B).

View larger version (40K): [in this window] [in a new window]   Figure 6. Effect of WEB2170 on HUVEC proliferation induced by VEGF. HUVECs (5x103) were cultured in 6-well microtiter plates in 0.2 mL of medium 199 supplemented with 6 mg/L transferrin, 5 mg/L insulin, 100 mg of soybean lecithin, 6.73 µg/L sodium selenite, 400 mg/L BSA, and 0.25% Ultroser HY. Stimuli (20 ng/mL VEGF; 20 ng/mL VEGF+3 µmol/L WEB2170; 10 ng/mL bFGF; 10 ng/mL bFGF+3 µmol/L WEB2170; 10 ng/mL PAF; or vehicle alone as control) were added every 2 days, and cell number was determined on day 8. Data are cell numbers determined as described in Methods (mean±SD of 3 individual experiments). ANOVA with Newman-Keuls multicomparison test was performed: Control vs experimental sets (*P<0.05); VEGF vs VEGF+WEB2170 and bFGF vs bFGF+WEB2170 (not significant).

View larger version (27K): [in this window] [in a new window]   Figure 7. Motility of HUVECs measured as the speed at which cells migrated into a wound introduced in a confluent monolayer 4 hours after stimulation (see Methods). HUVECs were incubated with vehicle alone, PAF (10 ng/mL), or VEGF (20 ng/mL) in the presence or absence of 3 µmol/L WEB2170 or CV3988. Each experimental group included 3 different experiments. Results are expressed as mean±SD. ANOVA with Newman-Keuls multicomparison test was performed: Cells incubated with vehicle alone (Control) vs cells incubated with PAF or VEGF (*P<0.05); VEGF vs VEGF+WEB2170, VEGF+CV3988 or PAF vs PAF+WEB2170 or PAF+CV3988 (P<0.05).

View larger version (200K): [in this window] [in a new window]   Figure 8. Photomicrographs representative of time-lapse analysis of HUVEC motility performed by digital image storage at 30-minute intervals. Migration tracks were generated by marking the positions of nuclei of individual cells in each image (see Methods). A, HUVECs incubated with VEGF (20 ng/mL) for 4 hours. B, HUVECs incubated with VEGF (20 ng/mL) for 4 hours in the presence of 3 µmol/L WEB2170.

Synthesis of PAF by VEGF-Stimulated HUVECs
As shown in Figure 9, HUVECs synthesized PAF after these cells were stimulated with VEGF. The PAF synthesized by HUVECs after their stimulation with VEGF remained cell associated. Using radioactive acetate as a substrate for PAF synthesis, we found that the PAF detected after stimulation with VEGF was newly synthesized. The TLC analysis of lipid fractions extracted 30 minutes after addition of VEGF to HUVECs preincubated with [³H]acetate demonstrated the presence of 1 main peak of radioactivity that comigrated with synthetic [³H]C16-PAF (data not shown). This peak was absent in the lipid fractions extracted from unstimulated HUVECs. Several studies have shown the heterogeneity of acetylated glycerophospholipids produced by HUVECs concerning the type of group present at the sn-1 position.¹⁷ Therefore, to evaluate the molecular species of PAF produced by VEGF-stimulated HUVECs, the ³H-labeled lipids extracted and purified by TLC were modified into 1-radyl-2,3-diacetylglycerol as described in Methods. When the labeled 1-radyl-2,3-diacetylglycerols were separated by TLC, the radioactive products appeared in 2 distinct regions of the chromatogram, corresponding to the Rf’s of radiolabeled 1-palmitoyl-2,3-diacetylglycerol (peak 1) and 1-O-hexadecyl-2,3-diacetylglycerol (peak 2). The major radiolabeled compound produced by HUVECs was the 1-acyl derivative of PAF (30.74%), which is 1000-fold less active then the alkyl derivative in terms of platelet activation.¹⁷ The compound migrating with the 1-alkyl derivative of PAF accounted for 25.48% of the radiolabeled 1-radyl-2,3-diacetylglycerol subjected to TLC.

View larger version (26K): [in this window] [in a new window]   Figure 9. PAF synthesis by HUVECs. A, Effect of different doses of VEGF on PAF synthesis by HUVECs. B, Time course of PAF synthesis by HUVECs stimulated with 20 ng/mL VEGF () or with vehicle alone (). PAF was detected associated with cells only. The numbers are mean±SD of 3 different experiments.

	Discussion

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The results of the present study indicate that PAF mediates endothelial cell motility induced by VEGF and contributes to its angiogenic effect in the in vivo Matrigel model. PAF is a phospholipid mediator with multiple biological activities relevant for the development of several pathological and physiological processes, such as embryogenesis, cell differentiation, shock, and inflammation.^{16 17 40} It has recently been shown that PAF directly stimulates the in vitro migration of endothelial cells and promotes in vivo angiogenesis.^{20 21} Several lines of evidence suggest that PAF may act as a secondary mediator of angiogenesis induced by certain polypeptidic mediators such as TNF¹⁸ and HGF.¹⁹ In contrast, the angiogenic effect of bFGF is completely independent of PAF synthesis, suggesting that the involvement of PAF as a secondary mediator occurs only for certain agonists.²⁰

The present observation that PAF is synthesized within Matrigel during vascularization induced by VEGF and that 2 chemically unrelated, specific PAF-receptor antagonists inhibit the angiogenic process suggests that this phospholipid may act as a secondary mediator for VEGF also. The combination of PAF and VEGF did not significantly enhance angiogenesis, whereas bFGF together with PAF was synergistic, suggesting that the effect of VEGF but not of bFGF is mediated by the synthesis of PAF. Taken together, these results indicate that PAF synthesis may contribute in vivo to the angiogenesis due to agents, such as TNF¹⁸ and VEGF, that induce different types of angiogenic response. A possible explanation for these observations is provided by the nature of the angiogenic process itself, which is a complex, multistep process involving recruitment of endothelial cells, matrix degradation, proliferation of endothelial cells, and canalization of endothelial cords. Therefore, it is possible that different agents may, in some of these steps, overlap on signals or on secondary mediators. This is not only the case of PAF but also of nitric oxide (NO), which is involved in angiogenesis induced by both TNF and VEGF but not in that induced by bFGF.⁴¹

The present study does not address the cellular origin of the PAF detected in Matrigel. However, we found that the infiltration of endothelial cells preceded the formation of new vessels within the Matrigel in the absence of an accumulation of inflammatory cells. Therefore, endothelial cells are potential candidate for VEGF-induced synthesis of PAF within Matrigel. This hypothesis is strengthened by the present observation that HUVECs synthesized PAF after their stimulation with VEGF.

Because previous studies had shown that VEGF stimulates endothelial cell proliferation and microvascular hyperpermeability,^{1 2 5 6 7 8} we evaluated whether the synthesis of PAF might affect these biological activities of VEGF. The results obtained indicate that VEGF-induced proliferation of HUVECs does not depend on the synthesis of PAF. Indeed, the PAF receptor antagonist WEB2170 was found to be ineffective with respect to HUVEC proliferation induced by VEGF. This result is consistent with the previously reported observation that PAF does not stimulate the proliferation of HUVECs.²⁰ Moreover, the blockade of PAF receptors did not prevent the increased dermal permeability in mice and the in vitro HUVEC permeability induced by VEGF. These results are consistent with the recent report that VEGF cutaneous permeability is PAF independent in rats²³ and rabbits.⁴² In contrast, the effect of VEGF on vascular cutaneous permeability is significantly reduced by inhibitors of NO and prostacyclin synthesis.⁴³ Because newly formed vessels in Matrigel implants are derived from subcutaneous tissue, the VEGF-induced angiogenesis seems, at least in this model, independent of a PAF-enhanced vascular permeability. In contrast, the VEGF-induced vascular permeability is PAF dependent in the airways, pancreas, and duodenum, suggesting a differential role for PAF as a secondary mediator of VEGF in different tissues.²³ Taken together, these results suggest that VEGF-induced vascular permeability depends on the production of secondary mediators that may contribute differently, depending on the vascular districts. In contrast, we found that in vitro VEGF-induced endothelial cell motility was PAF dependent, because it was inhibited by PAF-receptor antagonists. This observation suggests that the role of PAF in the in vivo neoangiogenesis induced by VEGF is mainly related to its ability to enhance the motility of endothelial cells.

In conclusion, in the Matrigel model of angiogenesis, PAF may have a dual role: (1) Since endothelial cells express intracellular PAF receptors,⁴⁴ the synthesis of PAF induced by VEGF may mediate, in an autocrine manner, VEGF-triggered endothelial cell motility, which is required for the recruitment and structural organization of endothelial cells within Matrigel. (2) PAF may be involved in the amplification of some of the signals triggered by VEGF. Indeed, it has been shown that PAF induces the expression within Matrigel of several angiogenic factors and chemokines, such as bFGF, placental growth factor, KC, macrophage inflammatory protein-2, HGF, VEGF itself, and its specific receptor flk-1.²¹ These results suggest a cooperative circuit among various angiogenic growth factors of endothelial origin and PAF. The inhibitory effect of a PAF-receptor antagonist emphasizes the critical role of PAF in this context.

	Acknowledgments

 
This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC); by the National Research Council (CNR), Targeted Project "Biotechnology"; and Istituto Superiore di Sanità, Targeted Project "AIDS" and "Artificial organs and organ transplantation" to G.C.

Received June 23, 1999; accepted June 24, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

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