© 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
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 |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 system13 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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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.
|
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).
|
|
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
).
|
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).
|
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).
|
|
|
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.
|
) or with vehicle
alone (
). PAF was detected associated with cells only. The numbers
are mean±SD of 3 different experiments. |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 1995;146:1029–1039.[Abstract]
2. Ferrara N. Vascular endothelial growth factor. Eur J Cancer. 1996;14:2413–2422.
3.
Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey
VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that
promotes accumulation of ascites fluid. Science. 1983;219:983–985.
4. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun. 1989;161:851–858.[Medline] [Order article via Infotrieve]
5.
Keck PJ, Hauser S, Krivi G, Sanzo K, Warren T, Feder
J, Connolly DT. Vascular permeability factor, an
endothelial cell mitogen related to platelet
derived growth factor. Science. 1989;246:1309–1311.
6.
Leung DW, Cachianes G, Kuang W-J, Goeddel DV, Ferrara
N. Vascular endothelial growth factor is a secreted
angiogenic mitogen. Science. 1989;246:1306–1309.
7. Plouet J, Schilling J, Gospodarowicz D. Isolation and characterization of a newly identified endothelial cell mitogen produced by At20 cells. EMBO J. 1989;8:3801–3807.[Medline] [Order article via Infotrieve]
8. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest. 1989;84:1470–1478.
9. Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. Am J Pathol. 1991;138:213–221.[Abstract]
10.
Senger DR, Ledbetter SR, Claffey KP,
Panadopoulos-Sergiou A, Perruzzi CA, Dednar M. Stimulation of
endothelial cell migration by vascular permeability
factor/vascular endothelial growth factor through
cooperative mechanisms involving the
vß3 integrin,
osteopontin, and thrombin. Am J Pathol. 1996;149:293–305.[Abstract]
11. Wilting J, Christ B, Bokeloh M, Weich HA. In vivo effects of vascular endothelial growth factor on the chicken chorioallantoic membrane. Cell Tissue Res. 1993;274:163–172.[Medline] [Order article via Infotrieve]
12. Folkman J. Angiogenesis and angiogenesis inhibition: an overview. EXS. 1997;79:1–8.[Medline] [Order article via Infotrieve]
13. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435–439.[Medline] [Order article via Infotrieve]
14. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–442.[Medline] [Order article via Infotrieve]
15. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999;103:1231–1236.[Medline] [Order article via Infotrieve]
16. Prescott SM, Zimmerman GA, McIntyre TM. Platelet-activating factor. J Biol Chem. 1996;268:17381–17384.
17. McManus LM, Woodard DS, Deavers SI, Pinckard RN. PAF molecular heterogeneity: pathobiological implications. Lab Invest. 1993;69:639–650.[Medline] [Order article via Infotrieve]
18.
Montrucchio G, Lupia E, Battaglia E, Passerini G,
Bussolino F, Emanuelli G, Camussi G. Tumor-necrosis factor
-induced angiogenesis depends on in situ platelet
activating factor biosynthesis. J Exp Med. 1994;180:377–382.
19. Camussi G, Montrucchio G, Lupia E, Soldi R, Comoglio PM, Bussolino F. Angiogenesis induced in vivo by hepatocyte growth factor is mediated by platelet activating factor synthesis from macrophages. J Immunol. 1997;158:1302–1309.[Abstract]
20. Camussi G, Montrucchio G, Lupia E, DeMartino A, Perona L, Arese M, Vercellone A, Toniolo A, Bussolino F. Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism. J Immunol. 1995;154:6492–6501.[Abstract]
21. Bussolino F, Arese M, Montrucchio G, Barra L, Primo L, Benelli R, Sanavio F, Aglietta M, Ghigo D, Rola-Pleszczynski M, Bosia A, Albini A, Camussi G. Platelet activating factor produced in vitro by Kaposi’s sarcoma cells induces and sustains in vivo angiogenesis. J Clin Invest. 1995;96:940–952.
22. Honda Z, Nakamura M, Miki I, Minami M, Watanabe T, Seyama Y, Okado H, Toh H, Ito K, Myamoto T, Shimizu T. Cloning by functional expression of platelet activating factor receptor from guinea pig lung. Nature. 1991;349:342–346.[Medline] [Order article via Infotrieve]
23. Sirois MG, Edelman ER. VEGF effect on vascular permeability is mediated by synthesis of platelet-activating factor. Am J Physiol. 1997;41:H2746–H2756.
24.
Heuer HO, Casals-Stenzel J, Muacevic G, Weber KH.
Pharmacologic activity of bepafant (WEB 2170), a new and selective
hetrazepinoic antagonist of platelet-activating factor.
J Pharmacol Exp Ther. 1990;255:962–970.
25. Terashita Z, Tsushima S, Yoshioka S, Namoto H, Inada Y, Nishikawa K. CV 3988: a specific antagonist of platelet-activating factor (PAF-acether). Life Sci. 1983;32:1975–1982.[Medline] [Order article via Infotrieve]
26. Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest. 1992;67:519–528.[Medline] [Order article via Infotrieve]
27. Nachlas MM, Seligman AM. Histochemical demonstration of esterase. J Natl Cancer Inst. 1949;9:415–421.[Medline] [Order article via Infotrieve]
28.
Kibbey MC, Grant DS, Klieinman HK. Role of the SIKVAV
site of laminin in promotion of angiogenesis and tumor growth: an in
vivo Matrigel model. J Natl Cancer Inst. 1992;84:1633–1638.
29. Miles AA, Miles EM. Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs. J Physiol. 1952;118:228–257.
30. Bussolino F, Camussi G, Aglietta M, Braquet P, Bosia A, Pescarmona GP, Sanavio F, D’Urso M, Marchisio PC. Human endothelial cells are target for platelet-activating factor: platelet-activating factor induces changes in cytoskeleton structures. J Immunol. 1987;139:2439–2446.[Abstract]
31.
Rotrosen D, Gallin JI. Histamine type I receptor
occupancy increases endothelial cytosolic calcium,
reduces F-actin and promotes albumin diffusion across cultured
endothelial monolayers. J Cell Biol. 1986;103:2379–2387.
32. Kueng W, Silfer E, Eppemberger U. Quantification of cell cultured on 96-well plates. Anal Biochem. 1989;182:16–19.[Medline] [Order article via Infotrieve]
33.
Thomas L, Randolph Byers H, Vink J, Stamenkovic I.
CD-44H regulates tumor cell migration on hyaluronate-coated substrate.
J Cell Biol. 1992;118:971–977.
34.
Romanov-Jessen L, Petersen OW. A function for
filamentous -smooth muscle actine: retardation of motility in
fibroblast. J Cell Biol. 1996;134:67–80.
35.
Camussi G, Bussolino F, Salvidio G, Baglioni C. Tumor
necrosis factor/cachectin stimulates peritoneal macrophages,
polymorphonuclear neutrophils, and vascular
endothelial cells to synthesize and release
platelet-activating factor. J Exp Med. 1987;166:1390–1404.
36. Camussi G, Aglietta M, Malavasi F, Tetta C, Piacibello W, Sanavio F, Bussolino F. The release of platelet-activating factor from human endothelial cells in culture. J Immunol. 1983;131:2397–2403.[Abstract]
37. Tufano MA, Biancone L, Rossano F, Capasso C, Baroni A, De Martino A, Iorio EL, Silvestro L, Camussi G. Outer-membrane porins from Gram-negative bacteria stimulate platelet-activating factor biosynthesis by cultured human endothelial cells. Eur J Biochem. 1993;214:685–693.[Medline] [Order article via Infotrieve]
38. Bligh EJ, Dyer WY. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917.
39. Triggiani M, Hubbard WC, Chilton FH. Synthesis of 1-O-acyl-2-acetyl sn -glycero 3-phosphocholine by an enriched preparation of the human lung mast cell. J Immunol. 1990;144:4773–4780.[Abstract]
40. Prescott SM, McEntyre TM, Zimmerman GA. The role of platelet activating factor in endothelial cells. Thromb Haemost. 1990;64:99–103.[Medline] [Order article via Infotrieve]
41. Ziche M, Morbidelli L, Choudhuri R, Zhang H-T, Donnini S, Granger HJ. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99:2625–2634.[Medline] [Order article via Infotrieve]
42. Collins PD, Connolly DT, Williams TJ. Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharmacol. 1993;109:195–199.[Medline] [Order article via Infotrieve]
43.
Murohara T, Horowitz JR, Silver M, Tsurumi Y, Chen D,
Sullivan A, Isner JM. Vascular endothelial growth
factor/vascular permeability factor enhances vascular permeability via
nitric oxide and prostacyclin. Circulation. 1998;97:99–107.
44. Ihida K, Predescu D, Czekay RP, Palade GE. Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells. J Cell Sci. 1999;112:285–295.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  S. Doublier, M. Ceretto, E. Lupia, S. Bravo, B. Bussolati, and G. Camussi The Proangiogenic Phenotype of Tumor-Derived Endothelial Cells is Reverted by the Overexpression of Platelet-Activating Factor Acetylhydrolase Clin. Cancer Res., October 1, 2007; 13(19): 5710 - 5718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Denizot, T. Chianea, F. Labrousse, V. Truffinet, M. Delage, and M. Mathonnet Platelet-activating factor and human thyroid cancer Eur. J. Endocrinol., July 1, 2005; 153(1): 31 - 40. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Russo, B. Bussolati, I. Deambrosis, F. Mariano, and G. Camussi Platelet-Activating Factor Mediates CD40-Dependent Angiogenesis and Endothelial-Smooth Muscle Cell Interaction J. Immunol., November 15, 2003; 171(10): 5489 - 5497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Biancone, V. Cantaluppi, L. Del Sorbo, S. Russo, L. W. Tjoelker, and G. Camussi Platelet-activating Factor Inactivation by Local Expression of Platelet-activating Factor Acetyl-Hydrolase Modifies Tumor Vascularization and Growth Clin. Cancer Res., September 15, 2003; 9(11): 4214 - 4220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bussolati, L. Biancone, P. Cassoni, S. Russo, M. Rola-Pleszczynski, G. Montrucchio, and G. Camussi PAF Produced by Human Breast Cancer Cells Promotes Migration and Proliferation of Tumor Cells and Neo-Angiogenesis Am. J. Pathol., November 1, 2000; 157(5): 1713 - 1725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Montrucchio, G. Alloatti, and G. Camussi Role of Platelet-Activating Factor in Cardiovascular Pathophysiology Physiol Rev, October 1, 2000; 80(4): 1669 - 1699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hardy, I. Dumont, M. Bhattacharya, X. Hou, P. Lachapelle, D. R. Varma, and S. Chemtob Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy Cardiovasc Res, August 18, 2000; 47(3): 489 - 509. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||