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CELLULAR AND MOLECULAR
Laboratory of Molecular Pharmacology, Department of Pharmacy (E.G., E.P.); Division of Genetics, Cell & Developmental Biology, Department of Biology (P.K.); and Department of Radiation Oncology (D.K.), University of Patras, Patras, Greece.
Received August 6, 2002; accepted October 9, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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).
WR-2721 is the inactive form of the drug which is rapidly metabolized to the
active form WR-1065 by the membrane-bound alkaline phosphatase. WR-1065 is
further metabolized through intracellular oxidation to the symmetric disulfide
WR-33278, cysteamine, and mixed disulfides
(Capizzi, 1996). The active
metabolite WR-1065 and the symmetric disulfide WR-33278 resemble endogenous
polyamines, like spermine and spermidine, and may bind to and stabilize any
part of the DNA helix that is not protected by histones
(Grdina et al., 2000).
A major pharmacological benefit of amifostine ensues from the fact that it
protects normal tissues from the toxic effects of chemotherapeutic agents and
ionizing radiation, leaving the antitumor effects of these agents either
unchanged or enhanced (Santini,
2001). The selective protection of normal tissues by amifostine is
based on the higher amounts of alkaline phosphatase in normal compared with
tumor tissues. In addition, the activity of the enzyme is relatively lower in
tumor tissues, since the suitable pH for alkaline phosphatase is 6.6 to 8.2
and the environment of the tumors is more acidic
(Dorr, 1998). The fact that
amifostine enhances the antitumor effects of some chemotherapeutic agents and
ionizing radiation can be explained by the fact that proliferating tumor cells
maintain a high cellular polyamine content via ornithine
decarboxylase-controlled biosynthesis, thereby blocking WR-33278 cell import
by the induction of antizyme. However, the passive diffusion of WR-1065
accelerates polyamine degradation, resulting in lowered apoptotic threshold.
In normal cells, polyamine generation is not up-regulated and the disulfide is
actively imported into the cell, affording resistance to apoptosis induction
(List and Gerner, 2000;
Quinones et al., 2002).
Preclinical data indicate that amifostine can reduce the risk of secondary
cancers caused by radiation and certain forms of chemotherapy
(Dorr, 1998) and inhibits the
formation of spontaneous metastases
(Grdina et al., 2002).
Angiogenesis, the formation of new blood vessels from pre-existing ones, is
an active process that is dependent upon the balance of positive and negative
regulators. Vascular endothelial growth factor (VEFG) is one of the most
potent angiogenic growth factors and plays a significant role in both
development and homeostasis (Carmeliet and
Jain, 2000). Besides its role in activation of endothelial cells
during the initial steps of angiogenesis, VEGF is also very important for the
maintenance of the differentiated state of blood vessels
(Ferrara and Davis-Smyth,
1997). VEGF-induced proliferation, migration, differentiation of
endothelial cells, and angiogenesis are believed to be at least partly
mediated by nitric oxide (NO)
(Papapetropoulos et al., 1997;
Fukumura et al., 2001). NO
production is catalyzed by a family of enzymes, the NO synthases (NOS), which
exist in three isoforms, neuronal, inducible (iNOS), and endothelial
(Fukumura and Jain, 1998). VEGF
induces the expression of both endothelial NOS and iNOS in human umbilical
vein endothelial cells (Kroll and
Waltenberger, 1998).
The remodeling of the extracellular matrix (ECM) is pivotal for the
functioning of the endothelium during the different steps of angiogenesis. The
initial steps of angiogenesis involve degradation of the basement membrane and
the surrounding extracellular matrix, and proliferation and migration of
endothelial cells. During the final steps, endothelial cells differentiate and
secrete new basement membrane (Carmeliet
and Jain, 2000). Laminin is located mainly in the basement
membrane and plays a critical role in tube formation. Vessel formation also
involves the deposition of various collagens, including collagen types I, III,
IV, and V (Stromblad and Cheresh,
1996). The proteolytic enzymes that have mainly been implicated in
ECM remodeling and angiogenesis are the family of metalloproteinases (MMPs)
and the plasminogen/plasmin system
(Lijnen, 2002).
In the present study, we examined the action of amifostine on angiogenesis by using the in vivo model of chicken embryo chorioallantoic membrane (CAM). Our results indicate that amifostine inhibited angiogenesis through modulation of both the expression of important angiogenic genes and the composition of the ECM.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
Leg-horn fertilized eggs (Pindos, Ioannina, Greece) were incubated for 4 days
at 37°C, when a window was opened on the egg shell, exposing the CAM. The
window was covered with tape and the eggs were returned to the incubator.
Amifostine was diluted in a final volume of 50 µl of H2O and
applied at the 9th day of embryo development on an area of 1 cm2 of
the CAM, restricted by a plastic ring. To evaluate the effect of amifostine,
48 h after treatment and subsequent incubation at 37°C, CAMs were fixed in
situ, excised from the eggs, placed on slides, and left to air-dry. Pictures
were taken through a stereoscope equipped with a digital camera, and the total
length of the vessel network was measured, as previously described
(Papadimitriou et al., 2001).
The assay was carried out three times and each experiment contained 10 to 20
eggs per data point.
For the biochemical studies, amifostine was applied on the CAM as described
above, and after different time periods of incubation at 37°C, the CAMs
were excised, cut in pieces, washed three times in phosphate-buffered saline
(PBS), pH 7.4, and stored at -20°C until used
(Giannopoulou et al.,
2001).
Reverse Transcriptase-Polymerase Chain Reactions (RT-PCRs). Total
RNA isolation from CAMs was performed as previously described
(Giannopoulou et al., 2001).
Primers used for fibronectin, A1 chain of laminin (LM), collagen type IV
(C-IV), collagen type I (C-I), 
3 chain of integrin
3 (3), and proMMP-2 were designed
using the corresponding chicken sequences
(Giannopoulou et al., 2001).
The primers used for iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
were also designed using the corresponding chicken sequences
(Pipili-Synetos et al., 2000).
The RT-PCRs for all the above mRNAs were performed in a single step with 200
to 250 ng of total RNA, using the Access RT-PCR system (Promega, Madison, WI),
as previously described (Pipili-Synetos et
al., 2000; Giannopoulou et
al., 2001). The primers for VEGF were designed according to the
chicken sequence available (GenBank Accession Number AB011078). The sequences
of all the used primers and the anticipated product sizes are shown in
Table 1.
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The PCR primers for VEGF corresponded to sequences of exon 3 (sense) and
exon 7 (antisense) (Kim et al.,
2000) and amplified two splicing variants of avian VEGF,
VEGF190 (456 bp) and VEGF165 (381 bp)
(Sugishita et al., 2000). The
RT-PCRs for VEGF mRNA were performed in a single step using 200 to 250 ng of
total RNA and the One Step RT-PCR kit (QIAGEN, Hilden, Germany), as follows.
The reverse transcriptase reaction was performed by a mix of Omniscript and
Sensiscript Reverse Transcriptases for 30 min at 50°C, followed by an
incubation at 95°C for 15 min to activate the HotStarTaq DNA polymerase
and inactivate the reverse transcriptases. After the de-naturation step, 40
cycles of amplification (94°C for 1 min, 59°C for 1 min, and 72°C
for 1 min) were performed and ended with a final DNA synthesis step at
72°C for 10 min.
In all cases, PCRs were not in the saturating phase (data not shown). DNA contamination was excluded by performing PCRs in the absence of the reverse transcription step.
To further establish that the PCR products, although they had the expected size, represented the corresponding cDNAs for VEGF, the PCR products were purified from agarose gels and subjected to restriction enzyme analysis using PstI, which should cleave both products only at one site. The products of the enzyme cleavage were analyzed on 2% agarose gels and had the expected size (231 and 225 bp for VEGF190 and 225 and 156 for VEGF165).
The RT-PCR products of all the reactions were subjected to electrophoresis on 2% agarose gels containing 0.5 µg/ml ethidium bromide and photographed using a digital camera. The PCR product/GAPDH ratio of electrophoretic band values represents the expression of each gene at different time points after amifostine application.
Western Blot Analysis of ECM Proteins. CAM tissue was homogenized
using a glass-glass homogenizer in 50 mM Tris-HCl, pH 7.4, containing 3.4 M
NaCl, 1 mM phenylmethylsulfonyl fluoride, 4 mM EDTA, 2 mM
N-ethylmaleimide, and 1 µg/ml aprotinin, as previously described
(Giannopoulou et al., 2001).
The homogenates were centrifuged at 12,000g for 20 min at 4°C.
Pellets were once more homogenized and centrifuged as described, to ensure
complete removal of plasma proteins. The final pellet was resuspended in 50 mM
Tris, pH 7.4, containing 4 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2
mM N-ethylmaleimide, and the total protein concentration was
determined using the Bradford
(1976) method. Equal amounts of
total protein were loaded on 5% SDS-polyacrylamide gel electrophoresis
mini-gels, analyzed, and transferred to Immobilon-P membranes. Blocking was
performed by incubating the polyvinylidene difluoride membranes with 5% (w/v)
nonfat dry milk in Tris-buffered saline (TBS), pH 7.4, for 1 h at room
temperature and under continuous agitation. The membranes were then incubated
with anti-fibronectin (1:2500; Chemicon International, Temecula, CA), or
anti-laminin (1:2500; Chemicon International), or anti-collagen IV (1:500;
Chemicon International), or anti-collagen I (1:2,000; Chemicon International),
or anti-actin (1:500; Chemicon International), in 3% (w/v) nonfat dry milk in
TBS, 0.05% Tween 20 for 3 h at room temperature under continuous agitation,
and then with horseradish peroxidase-conjugated goat anti-rabbit IgG or rabbit
anti-goat IgG (when the anti-collagen IV antibody was used) at a dilution of
1:2500 in 3% (w/v) nonfat dry milk in TBS/0.05% Tween 20 or for 1.5 h at room
temperature under continuous agitation. Detection of immunoreactive bands was
performed by diaminobenzidine or, in the case of laminin, by enhanced
chemiluminescence (Amersham Biosciences UK, Ltd., Little Chalfont,
Buckinghamshire, UK), according to the manufacturer's instructions. The
antibodies used were monospecific and did not show any cross-reactivity (data
not shown). The pictures of the gels were digitized and the protein levels
that corresponded to each immunoreactive band were quantified using the
ImagePC image analysis software (Scion Corporation, Frederick, MD).
Detection of Apoptosis and Staining of CAM Paraffin Sections. CAM tissues were excised from the eggs 24 h after amifostine application, washed in PBS, fixed in saline-buffered formalin, dehydrated, and embedded in paraffin. Sections were cut at 4 µm thickness and placed on positively charged glass slides. After rehydration of the tissue sections, the slides were stained with standard hematoxylin-eosin staining.
Apoptosis was studied on CAM paraffin sections using a commercially available apoptosis kit (APOPTOS-I.S.; Ylem Srl, Roma, Italy), according to the manufacturer's instructions. Briefly, the slides were incubated with proteinase K for 15 min at room temperature, washed in double-distilled water twice for 5 min each, and covered with peroxidase blocking solution. After 20 min, the slides were washed in double-distilled water twice, and sections were preincubated for equilibration in TdT buffer (30 mM Tris-HCl, 140 mM sodium cacodylate, 1 mM cobalt chloride, pH 7.2) for 10 min at room temperature. For elongation and labeling of 3'-OH DNA termini, each section of the slide was incubated with 50 µl of reaction mixture, containing TdT enzyme and biotinylated dUTP in TdT buffer for 60 min at 37°C. The reaction was stopped by transferring the slides in PBS for 5 min at room temperature, and the slides were covered with blocking solution for 10 min at room temperature. For detection of poly(dUTP)-biotin complexes, the sections were incubated with diluted streptavidin-conjugated peroxidase for 30 min at room temperature. Then, the slides were washed twice for 5 min each in PBS, pH 7.4, and covered with freshly prepared diaminobenzidine solution. After 20 min, the slides were washed with distilled water, counterstained with hematoxylin, mounted in mounting fluid, viewed through a Zeiss microscope, and photographed using a digital camera.
Colorimetric Assay for the Determination of Active Plasmin.
Amifostine was applied on the CAM, in an area of 1 cm2, restricted
by a plastic ring, and after different time periods of incubation at 37°C,
the CAMs were excised, cut in pieces, washed three times in PBS, pH 7.4, and
homogenized in lysis buffer (0.1 M Tris-HCl, pH 7.4). Homogenates were
centrifuged at 20,000g for 20 min at 4°C. Total protein
concentration was determined in the supernatants, using the Bradford assay.
Total protein (100 µg) from each sample was diluted in 200 µl of lysis
buffer, placed in the wells of a 96-well microplate, and incubated at 37°C
for 1 h with the plasmin substrate Val-Leu-Lys-p-nitroanilide (0.6
mM/well) (Sigma, Athens, Greece). Substrate cleavage was determined by
monitoring the absorbance at 405 nm, using an enzyme-linked immunosorbent
assay microplate reader (Bio-Rad, Hercules, CA). The amounts of active plasmin
in each sample (milliunits/milligram of total protein) were determined using a
standard curve from assays with purified plasmin (Sigma)
(Baldi et al., 1996).
Statistical Analysis. The significance of variability between the results from each group and the corresponding control was determined by unpaired t test. Each experiment included triplicate measurements for each condition tested, unless otherwise indicated. All results are expressed as mean ± S.E.M. from at least three independent experiments.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Previous
studies in our laboratory have shown that there is active alkaline phosphatase
in the CAM, and the applied drug is metabolized in its active form within 15
min (Giannopoulou et al.,
2002a). To study the effect of amifostine on CAM angiogenesis,
different concentrations of the drug were applied on the CAM, as described
under Materials and Methods, and after 48 h, the total length of the
vessel network was measured. As shown in
Fig. 1A, amifostine decreased
angiogenesis in a dose-dependent and statistically significant manner. The
decrease was maximal at the dose of 0.25 µg/cm2 and was not due
to toxicity, as verified on CAM paraffin sections stained with
eosin-hematoxylin (Fig. 1B, a and
b) or treated with a kit for in situ detection of apoptosis
(Fig. 1B, c and d).
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Amifostine Decreased the mRNA Levels of VEGF190 and
VEGF165. VEGF is a multifunctional glycoprotein involved
in endothelial proliferation and migration, vasculogenesis, vascular
permeability and angiogenesis. Differential exon splicing of the VEGF gene
results in five main mRNA species which code for five secreted isoforms:
VEGF206, VEGF189, VEGF165,
VEGF145, and VEGF121
(Ferrara and Davis-Smyth,
1997). Avian VEGF isoforms exhibit more than 70% homology with the
corresponding human sequences and are also generated by alternative splicing
(Sugishita et al., 2000). As
mentioned under Materials and Methods, the PCR primers used in the
present study amplified two variants of avian VEGF, VEGF190 and
VEGF165 (Fig. 2). We
investigated whether amifostine affects the expression of these VEGF isoforms
by using a sensitive, semiquantitative RT-PCR, at several time points after
drug application. As shown in Fig.
3, amifostine decreased the mRNA levels of both VEGF190
and VEGF165 6 h after its application. The expression of both
isoforms remained decreased up to 48 h after amifostine was applied on the
CAM.
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Amifostine Decreased the mRNA Levels of iNOS. Our previous studies
have shown that iNOS is the only isoform of NOS detected in the chicken embryo
CAM (Pipili-Synetos et al.,
2000; Giannopoulou et al.,
2002b). To study the effect of amifostine on the iNOS mRNA, we
used a semiquantitative RT-PCR for the corresponding gene, as previously
described (Pipili-Synetos et al.,
2000). As shown in Fig.
4, the expression of the iNOS gene was decreased 24 h after
amifostine application on the CAM and remained decreased at later time
points.
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Amifostine Decreased the Deposition of ECM Proteins. In the present
work, we studied the effect of amifostine on ECM proteins that are implicated
in several steps of the angiogenic cascade
(Papadimitriou et al., 1993).
The ECM proteins were analyzed by Western blotting techniques. The protein
levels that corresponded to each immunoreactive band of fibronectin, B1 chain
of laminin, 1 chain of collagen I, and collagen IV were quantified
using image analysis software and were normalized to the corresponding levels
of actin. As shown in Fig. 5,
the protein levels of collagen I and laminin were decreased 6 h after
amifostine application and remained decreased up to 24 h. Fibronectin and
collagen IV were not affected by amifostine at any of the time points examined
in our study. To determine whether the above-described changes in the amounts
of laminin and collagen I were due to changes in the expression of the
corresponding genes, we tested the effect of amifostine on the levels of their
mRNAs by semiquantitative RT-PCRs, at several time points after drug
application. Amifostine did not affect the expression of any of the tested ECM
genes, including integrin 3, up to 48 h
after drug application (data not shown).
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Amifostine Decreased the mRNA Levels of proMMP-2. MMP-2 has been implicated in invasive processes, such as angiogenesis and tumor metastasis. In the present work, we studied the effect of amifostine on both MMP-2 expression and activity. By zymography, we found that amifostine did not affect either the protein amounts or the activity of MMP-2 in the chicken embryo CAM (data not shown). However, the expression of the proMMP-2 gene was decreased 6 h after amifostine application and remained decreased at later time points (Fig. 6).
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Amifostine Increased the Activity of Plasmin. Plasmin is a protease
that hydrolyzes many extracellular proteins
(Pepper, 2001). In the present
work, we examined the effect of amifostine on plasmin activity at several time
points after drug application on the chicken embryo CAM, using a colorimetric
assay as described under Materials and Methods. We found that
amifostine increased the activity of plasmin within the first 6 h after its
application (Fig. 7).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Grdina et al., 2000).
Amifostine can affect several cellular processes, such as the activation of
certain transcription factors, the expression levels of genes, and the
activities of certain proteins (Murley at al.,
1997,
2001;
Romano et al., 1999;
Shen et al., 2001). In the
present study, we showed that amifostine inhibited angiogenesis in the chicken
embryo CAM by modulating both the expression of important angiogenic genes and
the composition of the ECM.
The effect of amifostine on angiogenesis has not been widely studied. To
our knowledge, there is only one short study suggesting increased angiogenesis
and endothelial cell proliferation after amifostine application on the yolk
sac membrane of the chicken embryo
(Plasswilm et al., 1999).
However, only one very high dose of amifostine (26 µg/cm2) was
applied in that study. In the present work, we showed a dose-dependent
inhibition of angiogenesis in the chicken embryo CAM, which is supported by
the effects of amifostine on several molecules implicated in angiogenesis. In
agreement with an antiangiogenic effect of amifostine are studies that show
that it down-regulates cell cycle progression
(North et al., 2000),
represses c-myc gene expression
(Liu et al., 1997), inhibits
topoisomerase II activity (Snyder and
Grdina, 2000), increases the plasma levels of angiostatin
(Grdina et al., 2002), and
decreases MMP-2 activity (Grdina et al.,
2002).
VEGF is considered the key angiogenic factor, expressed under both
physiological and pathological conditions of angiogenesis, and many promoters
or inhibitors of angiogenesis modulate VEGF and/or its receptors
(Ferrara and Davis-Smyth,
1997). This is the first study that shows that CAM cells express
at least two VEGF isoforms, VEGF165 and VEGF190. The
former is found both secreted and bound to the ECM, whereas the latter is
mainly sequestered on cell surfaces and the ECM. Although VEGF165
is considered the most potent mitogen for endothelial cells,
VEGF190 can also be mitogenic
(Ferrara and Davis-Smyth,
1997). What is the exact role and distribution of the two VEGF
isoforms in the chicken embryo CAM or the identity of the cells that produce
them need further investigation. Amifostine decreased the mRNA expression of
both VEGF isoforms, and this may at least partly contribute to its
antiangiogenic effect on the CAM.
VEGF acts through up-regulation of NO
(Papapetropoulos et al., 1997;
Kroll and Waltenberger, 1998;
Fukumura et al., 2001). iNOS
is the only NOS isoform detected in the chicken embryo CAM
(Pipili-Synetos et al., 2000)
and is produced by blood cells
(Giannopoulou et al., 2002b).
Amifostine decreased the mRNA levels of CAM iNOS 24 and 48 h after its
application. Whether this effect of amifostine is direct or secondary to VEGF
inhibition, which is evident at earlier time points than that of iNOS, is not
known. Moreover, the reduction of iNOS mRNA by amifostine might be an indirect
effect due to an increase in the expression of superoxide dismutase
(Murley et al., 2001).
Changing the redox status of CAM cells may lead to inhibition of iNOS
expression (C. Polytarcho and E. Papadimitriou, unpublished data;
Saito et al., 2001).
MMPs degrade the ECM and facilitate angiogenesis. In the present study we
showed that amifostine decreased proMMP-2 mRNA levels, although it did not
affect the total amounts or the activity of MMP-2 in CAM protein extracts. A
decrease of MMP-2 protein amounts and activity has been observed in tumor
cells (Grdina et al., 2002),
as well as in human endothelial cells (E. Giannopoulou and E. Papadimitriou,
unpublished observations). The discrepancy between the effect of amifostine on
the mRNA and the protein levels of MMP-2 could be due to a concomitant effect
of amifostine on the amounts of membrane type 1 MMP or the inhibitor of MMP-2,
tissue inhibitor of metalloproteinase 2. Alternatively, platelets also release
MMP-2, which may contribute a large amount of the total MMP-2 of the tissue,
masking the effect of amifostine on the endothelial MMP-2. In this case, there
may be a locally decreased amount or activity of MMP-2 on the surface of
migrating endothelial cells, in agreement with decreased angiogenesis
(Stromblad and Cheresh,
1996).
ECM protein remodeling is very important for the control of angiogenesis
and maturation of vessels (Papadimitriou
et al., 1993; Pepper,
2001). In the present study it was shown that amifostine decreased
the deposited amounts of laminin and collagen type I and, to a lesser extent,
collagen type IV, without affecting the expression of the corresponding genes.
These data suggest that it affects ECM deposition through an effect on
mechanisms that control the formation of the matrix.
In the chicken embryo CAM, laminin seems to play a significant role in both
the early stages of angiogenesis, when proliferation and migration of
endothelial cells takes place, and during differentiation and maturation of
the new vessels (Ausprunk et al.,
1991; Papadimitriou et al.,
1993). Since laminin is detected only in the basement membrane of
CAM vessels (Ausprunk et al.,
1991), a decrease in the amounts of deposited laminin corresponds
well with a decreased number of blood vessels. However, it cannot be
established from the present study whether this decrease in the deposition of
laminin is the cause or the effect of the reduced number of vessels after
amifostine application. The decrease in laminin deposition could be due to
activation of proteolytic enzymes, such as plasmin
(Pepper, 2001). Amifostine
increased the activity of plasmin in the CAM and this may, at least partly, be
the mechanism through which it affects the deposited amounts of laminin.
Collagens play an important role in angiogenesis. Inhibition of
biosynthesis, or deposition or cross-linking of collagen chains inhibits
angiogenesis (Ingber and Folkman,
1988). Amifostine decreased the deposition of collagen, without
affecting its biosynthesis. These data suggest that amifostine may affect
collagen cross-linking. Lysyl oxidase is the enzyme that initiates the
covalent cross-linking of extracellular collagen molecules, converting these
to insoluble fibers (Kagan,
2000). Lysyl oxidase is a copper-dependent amine oxidase and is
inhibited by a variety of compounds bearing primary amine functions, such as
-aminoproprionitrile, -bromoethylamine, ethylenediamine, and
others (Kagan, 2000).
Amifostine and its metabolites resemble polyamines and, having amine
functions, they may inhibit lysyl oxidase and, thus, collagen cross-linking.
In the same line, since amifostine is metabolized by copper-dependent amine
oxidases to acrolein and cysteamine (Meier
and Issels, 1995), it may act as an antagonistic substrate for
lysyl oxidase. The product of this reaction, cysteamine, may also act as an
inhibitor of lysyl oxidase.
In summary, amifostine affects ECM remodeling and the expression of several
angiogenic molecules in a way that inhibits angiogenesis. Taking into account
that embryonic angiogenesis may differ in several aspects from human tumor
angiogenesis in the adult, further studies are in progress using other
angiogenesis models. An antimetastatic
(Grdina et al., 2002) and an
antiangiogenic (this study) action of amifostine, in addition to its
radioprotective effects, further support the use of amifostine in combination
with radiotherapy for increased therapeutic efficacy.
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Footnotes |
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ABBREVIATIONS: WR-2721, amifostine; VEGF, vascular endothelial growth factor; NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; ECM, extracellular matrix; MMP, matrix metalloproteinase; CAM, chorioallantoic membrane; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair; TBS, Tris-buffered saline; TdT, terminal deoxynucleotidyl transferase; WR-1065, S-2-(3-aminopropylamino)ethanethiol; WR-33278, symmetric disulfide of WR-1065.
Address correspondence to: Dr. E. Papadimitriou, Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, GR 26504 Greece. E-mail: epapad{at}upatras.gr
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