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* Section of General Pathology, Department of Pathology, University of Verona, Verona, Italy;
Molecular Biology Laboratory and Tumor Progression Section, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; and
Section of General Pathology, Department of Biotechnology, University of Brescia, Brescia, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polymorphonuclear neutrophils (PMN)3 are the most abundant circulating blood leukocytes that, typically provide the first-line defense against infections. However, in recent years it has become clear that the contribution of neutrophils to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. Indeed, neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses, engaging them in a complex cross-talk with immune cells that bridges innate and adaptive immunity (15). Interestingly, activated neutrophils are also able to produce a number of molecules that appear to either positively or negatively modulate the angiogenic process (15). In addition, PMN store a number of proteinases in their granules, including gelatinase B (matrix metalloproteinase (MMP)-9) and urokinase (16), which, at least in vitro, are known to generate angiostatin K1–4 (5, 7). However, despite of this body of information, the role of PMN in angiogenesis has remained poorly investigated.
In the present study, we addressed the question of whether neutrophils could directly generate biologically active angiostatin from plasminogen. We show that activated neutrophils are indeed capable of generating anti-angiogenic peptides comprising kringle domains 1–3 of plasminogen. We also provide evidence that neutrophil elastase (NE), directly secreted by stimulated PMN, is the enzyme responsible for the generation of angiostatin K1–3. Finally, we show that the affinity-purified angiostatin peptides generated by neutrophils can inhibit basic fibroblast growth factor (FGF2) plus vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation in vitro, and both VEGF-induced angiogenesis in the matrigel assay as well as FGF2-induced angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified granulocytes (>99%) and Percoll-purified
monocytes were isolated under endotoxin-free conditions from buffy
coats of healthy donors (17). After purification,
leukocytes were suspended at 107/ml in RPMI 1640
medium (Biowhittaker, Walkersville, MD) supplemented with 10%
low-endotoxin FBS (<0.009 ng/ml; Biochrom, Berlin, Germany), and
incubated for 3 h in polypropylene tubes at 37°C, 5%
CO2 atmosphere, before stimulation. In selected
experiments, preincubation was conducted in the presence or absence of
either 100 U/ml IFN-
(Hoffman-La Roche, Basel, Switzerland) or 1000
U/ml IFN-
(Roferon-A; Hoffmann-La Roche; Ref. 18).
Cells were then washed once with PBS, suspended in serum-free culture
medium (SFCM), and routinely stimulated for 15 min with 100 nM fMLP
(Sigma-Aldrich, St. Louis, MO) after a previous 5-min preincubation
with 5 µg/ml cytochalasin B (CB) (19). In selected
experiments, neutrophils were stimulated either for 15 min with IL-8
(provided by Dr. M. Ceska, Sandoz, Vienna, Austria), growth-related
gene product- (GRO), or for 1 h with TNF-, GM-CSF
(Peprotech, Rocky Hill, NJ), and LPS (from Escherichia coli,
serotype 026:B6, Sigma-Aldrich). After stimulation, SFCM was harvested
and stored at -20°C. The human pancreatic carcinoma cell line ASPC1
(from American Type Culture Collection, Manassas, VA) was maintained in
RPMI 1640 supplemented with 10% FBS and used to generate SFCM, as
described (20). All reagents used were of the highest
available grade and were dissolved in pyrogen-free water for
clinical use.
Angiostatin generation
A total of 100 µg/ml of human Glu-plasminogen (HPg; purchased
from Enzyme Research Laboratories, South Bend, IN) were added to the
SCFM harvested from the leukocyte cultures and then incubated at 37°C
under continuous shaking for 14 h. Small aliquots of the latter
mixtures were then withdrawn and assessed for angiostatin
generation by Western blot analysis. In some experiments, HPg cleavage
was also conducted in the presence of the following proteinase
inhibitors (added 1 h before HPg): 5 mg/ml 1-anti-trypsin,
1 mM N-methoxysuccinil-Ala-Ala-Pro-Val chlormethyl ketone
(IE), 0.3 µM aprotinin, 25 mM EDTA (Merck, Frankfurt,
Germany), 100 µg/ml plasminogen activator inhibitor-1 (PAI)
(Calbiochem, San Diego, CA). Purified NE, cathepsin G (CG) (ICN
Biochemicals, Aurora, OH), and MMP-9 (Roche Molecular
Biochemicals, Mannheim, Germany) were also incubated with HPg in the
absence or the presence of the specific inhibitors as
controls. Samples (100 ng/lane) were electrophoresed under nonreducing
conditions on 10% polyacrylamide gels in Tris-glycine running buffer
and subsequently transferred to nitrocellulose membranes by
electroblotting. Membranes were first blocked for 1 h at room T in
TBS/T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20)
containing 5% BSA, and then incubated overnight at 4°C in the
presence of a 2–2.5 µg/ml of mAbs directed against kringles
1–3 (K1–3) of HPg (from American Diagnostica, Greenwich, CT, or clone
10-V-1 from Calbiochem). Ab binding was detected by using
HRP-conjugated anti-mouse IgG (1/3000 dilution in TBS/T containing
3% BSA) and revealed using the chemiluminescence system (ECL; Amersham
Pharmacia Biotech, Piscataway, NJ).
Lysine-Sepharose purification of angiostatin
To obtain sufficient amounts of PMN-derived angiostatin to be used for the various biological assays (see Results), 1.5 mg HPg was incubated for 14 h at 37°C with 10 ml of SFCM harvested from CB plus fMLP-stimulated PMN. The reaction products were then loaded to a column containing 5 ml of lysine Sepharose 4B (Amersham Pharmacia Biotech), previously equilibrated with binding buffer (50 mM phosphate buffer, pH 7.5). The column was washed with 10 vol of binding buffer, 5 vol of 0.5 M NaCl dissolved in binding buffer to remove nonspecifically bound proteins, and finally eluted with 0.2 M 6-aminocaproic acid to recover angiostatin, according to the protocol described by O’Reilly et al. (21). Fractions (0.5 ml) were collected, pooled, and 6-aminocaproic acid removed by gel filtration on a PD-10 desalting column (Amersham Pharmacia Biotech) previously equilibrated with either serum-free RPMI or distilled water. The resulting products were quantified by measuring their absorbance at 280 nm, filter-sterilized, divided into working aliquots, and stored at -80°C. Angiostatin preparations were subjected to Western blot analysis, and purity evaluated to be >95% by silver staining of polyacrylamide gels (7).
Measurement of NE activity
The amount of bioactive NE released from stimulated PMN was estimated by measuring the rate of hydrolysis of the NE specific chromogenic substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) (22). Briefly, 100 µl of either purified NE or PMN-derived SFCM were dispensed into 96-well plates and incubated for 5 min at 37°C. A total of 100 µl of substrate solution (1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide diluted in 100 mM Tris-HCl buffer, pH 8) were then added, and substrate hydrolysis was monitored for up to 1 h in an automated microplate spectrophotometric reader (Spectra Count, BS1000; Packard Instrument, Meriden, CT) for the release of p-nitroanilide. Each sample was run in duplicate. The amount of bioactive elastase detected in SFCM was calculated according to a calibrated standard curve.
Endothelial cell proliferation assay
HUVEC were isolated from the umbilical vein as described (23) and grown in M199 medium (Sigma-Aldrich) supplemented with 20% FBS, antibiotics, 5 U/ml sodium heparin (Bristol-Myers Squibb, Latina, Italy), and 30 µg/ml endothelial growth supplement (Sigma-Aldrich). For the cell proliferation assay, low passage (p2-p4) HUVEC were harvested by trypsinization and seeded at 6,250 cells/cm2 in 96-well plates in complete medium in the absence of endothelial growth supplement. After 24 h, cells were added with fresh medium containing FGF2 and VEGF (both at 30 ng/ml) in 5% FBS in the absence or in the presence of affinity-purified neutrophil-generated angiostatin (PMN-Angio K1–3) or commercial angiostatin K1–3 (Calbiochem). After a further 72 h of incubation, cells were trypsinized and counted.
In vivo angiogenesis
The Matrigel model of angiogenesis in vivo (24) as
modified by Albini et al. (25) was used. A potent
angiogenic mixture of VEGF (100 ng/ml) and TNF- (2 ng/ml) was added
to liquid Matrigel containing 26 U/ml of heparin (VEGF plus TNF plus
heparin) at 4°C with or without 0.5 µg/ml commercial
angiostatin or PMN-Angio K1–3, to a final volume of 600 µl. The
Matrigel suspension was slowly injected s.c. into the flanks of C57
mice using a cold syringe, where the gel rapidly polymerizes to form
solid implants. After 4 days, gels were collected, weighed, and
subjected to analysis of the hemoglobin content as an estimate of
vascularization as previously described (25). Portions of
some samples were paraffin embedded and stained with H&E for
histological analysis or with anti-Factor VIII mAb for
histochemical identification of vessels.
Chick embryo CAM assay
Fertilized white leghorn chick eggs were incubated under conditions of constant humidity at 37°C. On the third day of incubation, a square window was opened in the egg shell after removal of 2–3 ml of albumen so as to detach the developing CAM from the shell. The window was sealed with a glass of the same size, and the eggs were returned to the incubator. At day 8, 1-mm3 sterilized gelatin sponges (Gelfoam; Upjohn Company, Kalamazoo, MI) adsorbed with rFGF2 (500 ng/embryo) dissolved in 2 µl of PBS were implanted on the top of growing CAMs (26). Immediately after implantation, sponges were treated with 1 µg of PMN-Angio K1–3 preparations. Angiostatin treatment was repeated daily until day 10. Sponges containing vehicle or FGF2 alone were used as negative and positive controls, respectively. CAMs were examined under a stereomicroscope and blood vessels around the sponges were counted at day 11.
Statistical analysis
Data are expressed as means ± SD. Statistical evaluation was performed by the Student’s t test, and considered significant if p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, we analyzed human PMN for their in vitro
capacity to generate enzymatic activities able to excise angiostatin
from HPg. For this purpose, SFCM harvested from PMN stimulated under
conditions known to induce optimal degranulation (CB plus 100 nM fMLP
for 15 min) was incubated with 100 µg/ml HPg at 37°C for 14 h.
Reaction products were then analyzed under nonreducing conditions by
Western blot, using mAbs recognizing the kringle 1–3 (K1–3) domains
of HPg (Fig. 1
A). Only a
prominent band at 88 kDa corresponding to HPg (Fig. 1A,
lane 1) was detected in the digestions performed with SCFM
derived from either resting (Fig. 1A, lane 2) or
CB-treated neutrophils (data not shown). In contrast, SFCM recovered
from CB plus fMLP-stimulated neutrophils completely degraded HPg,
generating three major protein bands with molecular masses at
32 kDa (Fig. 1A, lane 3), which corresponded
to the molecular masses of angiostatin K1–3 (Fig. 1A,
lane 7), but not of angiostatin K1–4 (Fig. 1A,
lane 6). Under these experimental conditions, end-generation
of angiostatin K1–3 peptides consisted of multiple steps. At earlier
times of incubation, several transient bands of low intensity in the
plasmin H chain and angiostatin K1–4 molecular mass range (66 and 45
kDa, respectively) were observed (data not shown). Importantly,
formation of angiostatin K1–3 fragments was also induced by SFCM
obtained from PMN stimulated with fMLP in the absence of CB (Fig. 1B, lane 3), IL-8 (Fig. 1C,
lanes 2 and 3), GRO (Fig. 1C,
lanes 5 and 6), and GM-CSF (Fig. 1D);
though the extent of HPg degradation and angiostatin K1–3 formation
depended on the concentration of the stimulus used. In contrast, other
inflammatory compounds such as IFN- (Fig. 1B, lane
4), IFN- (Fig. 1B, lane 6), LPS, or
TNF- (Fig. 1D) did not induce HPg degradation. However,
under the same experimental conditions, either LPS or TNF- triggered
IL-8 release (data not shown) as expected (15), whereas
both IFN- and IFN enhanced agonist-stimulated NE secretion (see
below). Consistent with the evidence that PMN generate
angiostatin K1–3, incubation of HPg with SFCM harvested from
autologous monocytes proved ineffective in degrading HPg (Fig. 1A, lanes 4 and 5).
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In addition to MMP-9 and urokinase that are known to generate
angiostatin (5, 7), PMN also contain other protineases,
including CG and NE, able to degrade HPg into proteolytic fragments
(27). To identify the type of enzymes present in the
PMN-derived supernatants that are responsible for the generation of
angiostatin peptides, HPg and either SFCM collected from stimulated
neutrophils or as controls, purified enzymes were mixed with selective
proteinase inhibitors before a 14-h incubation and Western blot
analysis. Fig. 2 shows that, among many
others, only 1-anti-trypsin (Fig. 2A, lane
2), a serine proteinase inhibitor blocking the enzymatic
activities of both elastase (Fig. 2A, lane 9) and
CG (Fig. 2B, lane 3), as well as IE (Fig. 2A, lane 3), a specific elastase inhibitor (Fig. 2A, lane 10) which does not affect CG activity
(Fig. 2B, lane 2), completely inhibited the
conversion of HPg to angiostatin exerted by SFCM derived from
stimulated PMN. Similar to neutrophil-derived SCFM (Fig. 2A,
lane 1), purified NE generated angiostatin K1–3 fragments
(Fig. 2A, lane 8), whereas purified CG incubated
with HPg only yielded angiostatin K1–4 fragments (Fig. 2B,
lane 1). Based on these findings and on the specific effect
of IE, we conclude that NE is the enzyme responsible for the generation
of angiostatin K1–3 by neutrophil-derived SFCM. This conclusion was
consistent with the effects of the other inhibitors tested. For
example, while EDTA, a metalloprotease inhibitor, was unable to inhibit
the angiostatin-generating capacity of neutrophil-derived SFCM (Fig. 2A, lane 4), it effectively blocked the cleavage
of HPg determined by recombinant exogenous MMP-9 (Fig. 2C,
lane 2). Similarly, PAI and aprotinin did not affect the
generation of angiostatin by PMN-derived SFCM (Fig. 2A,
lanes 5 and 6), but effectively blocked the
capacity of SFCM from ASPCI cells to generate angiostatin (Fig. 2D, lanes 2 and 3) that is mediated by
urokinase (20). Finally, measurement of NE activity (Table I) revealed that the amount of bioactive
elastase released by PMN under the various stimulatory conditions was
in line with previous findings (18, 28, 29, 30, 31, 32) and correlated
with the capacity of SCFM to convert HPg into angiostatin K1–3 (Fig. 1
). In general, we observed that concentrations of NE >3 µg/ml were
required to obtain a complete degradation of 100 µg/ml HPg in a 14-h
incubation time. Moreover, both IFN- and IFN- significantly
primed PMN for an enhanced NE secretion in response to 100 nM fMLP, 100
ng/ml IL-8, and GRO (Table I), and as a consequence, rendered the
related SCFM much more efficient in degrading HPg and generating
angiostatin K1–3 (Fig. 1B, lanes 5 and
7 for fMLP, and data not shown for IL-8 and GRO).
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We subsequently determined whether angiostatin K1–3 generated by
PMN-derived SFCM exerted anti-angiogenic activities. Large
preparations of SFCM harvested from PMN stimulated with CB plus fMLP
and incubated with HPg were affinity-purified (21) (Fig. 3, insert, lane 3).
Initially, we tested the effect of PMN-Angio K1–3 on the proliferation
of HUVEC cells induced by FGF2 plus VEGF. As shown in Fig. 3, PMN-Angio
K1–3 suppressed the FGF2 plus VEGF-induced HUVEC proliferation in a
dose-dependent manner, being at 20 µg/ml even more effective than
equivalent amounts of commercial angiostatin K1–3 in the same assay
(data not shown). Subsequently, we examined whether PMN-Angio K1–3
possessed the ability to inhibit VEGF-induced angiogenesis in vivo,
using the Matrigel implant model (25). VEGF and TNF-
(VTH) produced a potent angiogenic response in vivo that was strongly
inhibited by the addition of 0.5 µg/ml PMN-Angio K1–3 (Fig. 4). Histological examination of
VTH-containing implants demonstrated the formation of dilated,
hemorrhagic vessels and a massive cellular infiltrate through the
Matrigel (Fig. 4A). Gels containing commercial angiostatin
(Fig. 4C), as well as implants containing neutrophil-derived
angiostatin (Fig. 4D) showed reduced infiltrating cells with
the formation of a few small blood vessels. Furthermore, measurement of
the hemoglobin content as an indicator of the angiogenic response
showed that the mean hemoglobin level was significantly lower in VTH
plus angiostatin-containing pellets (p < 0.03,
Student’s t test) than in gels with VTH alone (Fig. 4E). Finally, PMN-Angio K1–3 was also found to inhibit
angiogenesis induced by rFGF2 in the chick embryo CAM assay (Fig. 5). Indeed, daily administration of
PMN-Angio K1–3 prevented neovascularization triggered by FGF2 without
affecting the physiological blood vessel formation observed in
FGF2-untreated CAMs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(15), IL-8, GRO
(15), and FGF2 (34); whereas the
neutrophil-derived factors recognized to be anti-angiogenic
molecules are IL-12 (15), inducible protein 10, monokine
induced by IFN- (18), and cationic proteins, such as
bactericidal/permeability-increasing protein (16).
Similarly, PMN represent a particularly rich source of
proteinases (16), which are known to exert contrasting
functions in the context of the angiogenic process. On one hand, these
enzymes degrade extracellular matrix molecules, modulate mechanical
structures, and liberate extracellular matrix molecules-bound growth
factors, which are all activities fundamental for the initiation of the
angiogenic process. In contrast, proteinases are involved
in the generation of several endogenous angiogenic inhibitors from
large precursor proteins (2). However, despite this body
of information, it is not clear yet what the actual role of PMN in the
regulation of angiogenesis might be (35, 36), other than
their ability to induce angiogenesis in vitro (37, 38),
and to indirectly mediate the proangiogenic effects of Fas, laminin,
and Tat in vivo (39, 40, 41).
In this study, we provide evidence that supernatants from human
neutrophils activated with proinflammatory agonists including fMLP, a
bacterial derived-peptide, chemokines such as IL-8 and GRO, and
growth factors such as GM-CSF, contain proteolytic activities that
mediate the conversion of purified plasminogen into angiostatin-like
fragments corresponding to the known angiostatin K1–3. The latter has
been originally identified as a product of the in vitro digestion of
HPg with porcine pancreatic elastase (11), and has been
subsequently demonstrated to be the portion of angiostatin K1–4
retaining the strongest inhibitory activity on endothelial cell growth,
as well as the strongest tumor-suppressing activity (11, 42). To our knowledge, these findings are the first
identification of a nonneoplastic human cell type able to generate
angiostatin K1–3. Through the use of specific protease inhibitors and
several other criteria, we identified NE as the major enzyme
responsible for the angiostatin-generating capacity of
neutrophil-derived supernatants. Furthermore, cytokines such as IFN-
and IFN- that enhanced the NE-secretory capacity of stimulated
neutrophils also potentiated the ability of PMN to generate angiostatin
K1–3. Moreover, supporting the specific role of NE, freshly isolated
monocytes, stimulated as neutrophils, did not show any
angiostatin-forming capacity. Although we cannot exclude that other
more selective agonists might induce monocytes to generate plasminogen
degrading activities, it is worth remarking that monocytes only express
small amounts of NE (43) that are completely lost during
differentiation into macrophages and replaced with the macrophage
metalloelastase (MMP-12) (43, 44).
The angiostatin K1–3 generated by neutrophils was then enriched by
affinity purification and also shown to be biologically active on the
basis of its ability to inhibit endothelial cell
proliferation in vitro, and to inhibit angiogenesis induced by VEGF as
well as by FGF2 in the chick embryo assay in vivo. The ability of human
neutrophils to produce biologically active angiostatin-like fragments
extends the previous findings on the production of angiostatin by other
phagocytes, namely murine macrophages in a model of Lewis Lung
Carcinoma (12), after thioglycollate-elicited peritoneal
inflammation (13), and after
TGF-
1-activation (13).
Interestingly, angiostatin generation by macrophages was shown to be
dependent on either MMP-12 activity (12), or as a
consequence of the proteolytic regulation of membrane bound plasmin
(13), and different from neutrophils, they mainly
generated angiostatin K1–4.
Even though the physiological role of PMN-generated angiostatin remains
elusive, it potentially has broad implications. Most of the
physiological events and pathological inflammatory disorders where
angiogenesis plays a key role, such as wound healing, rheumatoid
arthritis, cardiovascular diseases, psoriasis, and diabetic retinopathy
(1) are processes in which neutrophils represent a
prominent cellular component (45). The data shown in this
study together with previous observations (15) suggest
that the contribution of neutrophils to angiogenesis regulation most
likely depends upon a fine balance of proangiogenic and
anti-angiogenic factors. It is plausible that PMN, once recruited
into an inflammatory site, may secrete either pro- or
anti-angiogenic molecules depending on the environmental stimuli.
For instance, previous in vitro findings demonstrated that if
neutrophils sense LPS or TNF-, they are induced to produce mainly
proangiogenic factors, such as VEGF or IL-8 (15). In
contrast, whether PMN encounter IFN- or IFN- simultaneously with
LPS or TNF-, then they are triggered to additionally and
specifically release anti-angiogenic molecules, including IL-12,
inducible protein 10, monokine induced by IFN-, and IFN-inducible T
cell -chemoattractant (15, 18). Our present data
are in complete agreement with these observations, given that LPS or
TNF- did not provoke any release of HPg-degrading activity, whereas
both IFN- or IFN- dramatically enhanced the release
of NE by neutrophils stimulated with fMLP, IL-8, and GRO; therefore,
strongly enhancing the angiostatin formation. Taken together, these
data not only suggest that generation of angiostatin by neutrophils
might play a negative feedback role in the neovascularization processes
induced by proangiogenic peptides such as IL-8 and GRO
(46), but also indicate that PMN may contribute to the
anti-angiogenic activities of both IFN- and IFN- in vivo
(47, 48, 49).
In addition to inflammatory processes, neutrophils may also contribute
to the generation of angiostatin in cancer. Although PMN are usually
but a marginal component of both human and animal tumors, recent
studies using cytokine gene transfer strategies have suggested that
they are active in immunosurveillance against several tumor types
(50). It has been shown that many cytokines and chemokines
released by engineered tumor cells, for instance IL-1, IL-2, IL-12,
IFN-, IFN-, IFN-, G-CSF, IL-8, and GRO (50)
quickly recruit a massive local reaction that leads to the rejection of
engineered tumor cells and the establishment of a significant immunity
against the wild-type parental tumor. PMN play a key role in all of
these cytokine-induced tumor rejections, often in cooperation with
CD8+ T lymphocytes. Although recruited PMN may
produce several cytotoxic mediators, including reactive oxygen species,
proteases, membrane-perforating agents, and soluble mediators of cell
killing such as TNF-, the formation of angiostatin K1–3 mediated by
the release of NE might additionally contribute to the anti-tumor
activities observed.
In conclusion, our results attribute a novel and unsuspected role of angiogenesis inhibition to neutrophils. Their ability to release large amounts of elastase and other proteinases, classically associated with defense purposes (45) or tissue injury (51, 52), might actually have an additional biological function. Based on the findings presented here, proteinase release by PMN might in fact contribute to generation of endogenous inhibitors of angiogenesis in vivo from large precursors having distinct functions, as has already been observed for degradation of collagen XVIII and serpin anti-thrombin in vitro (53, 54). Whether NE or other enzymatic activities released by activated neutrophils participate to the formation of anti-angiogenic factors (such as fragments of fibronectin, Kringle 5 of plasminogen, restin) has not yet been established and is currently under investigation in our laboratories.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marco A. Cassatella, Section of General Pathology, Department Pathology, University of Verona, Strada Le Grazie 4, 37134 Verona, Italy. E-mail address: marco.cassatella{at}univr.it
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; NE, neutrophil elastase; VEGF, vascular endothelial growth factor; FGF2, basic fibroblast growth factor; CAM, chorioallantoic membrane; SFCM, serum-free culture medium; CB, cytochalasin B; GRO, growth-related gene product-; HPg, human Glu-plasminogen; PAI, plasminogen activator inhibitor-1; CG, cathepsin G; MMP, matrix metalloproteinase; IE, N-methoxysuccinil-Ala-Ala-Pro-Val chlormethyl ketone.
Received for publication January 8, 2002. Accepted for publication April 1, 2002.
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P. Scapini, M. Morini, C. Tecchio, S. Minghelli, E. Di Carlo, E. Tanghetti, A. Albini, C. Lowell, G. Berton, D. M. Noonan, et al. CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A J. Immunol., April 15, 2004; 172(8): 5034 - 5040. [Abstract] [Full Text] [PDF]
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 G. B. Perchick and H. N. Jabbour Cyclooxygenase-2 Overexpression Inhibits Cathepsin D-Mediated Cleavage of Plasminogen to the Potent Antiangiogenic Factor Angiostatin Endocrinology, December 1, 2003; 144(12): 5322 - 5328. [Abstract] [Full Text] [PDF]
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