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Regular Articles |
From the Laboratory of Molecular Pathology and
Ultrastructure,*
Regina Elena Cancer Institute, Rome; and
the Laboratory of Biology and Treatment of
Metastasis,
Mario Negri Institute for
Pharmacological Research, Bergamo, 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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The aim of our study was to investigate the role of ET-1 in the angiogenic process by evaluating the effect of ET-1 at the different stages of neovascularization including proliferation, migration, invasion, protease production, and morphogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, in the present study, we evaluated the receptor subtype mediating the angiogenic effects of ET-1 on endothelial cells and investigated whether specific antagonists inhibit these ET-1-induced responses. There is increasing evidence that the nature of the response elicited by a specific angiogenic factor is contextual, ie, depends on the presence or absence of other regulatory molecules in the pericellular environment of the responding cell.20 Because ET-1, predominantly through ETAR, stimulates the synthesis of VEGF in vascular smooth muscle cells and the VEGF-mediated migration and proliferation of endothelial cells,21 we investigated whether ET-1 would be able to induce an angiogenic phenotype in endothelial cells in association with VEGF. Finally, using the model of in vivo angiogenesis in the pellet of reconstituted basement membrane (Matrigel), we evaluated the ability of ET-1 to stimulate the formation of new vessels. The results clearly demonstrate that ET-1 is able to induce angiogenic responses in cultured endothelial cells through the ETB receptor and stimulates neovascularization in vivo in concert with VEGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human endothelial cells were isolated from human umbilical vein as previously described22 and maintained in M199 supplemented with 10% fetal calf serum (FCS), 10% newborn calf serum, 0.1 mg/ml endothelial growth supplement (crude extract from bovine brain), 0.1 mg/ml heparin, and 20 mmol/L HEPES. All culture reagents were from Gibco (Paisley, Scotland).
Thymidine Incorporation Assay
Cells were seeded in 96-well plates at 80% confluence (1 x 104cells/well) and incubated in 0.5% FCS medium for 24 hours. Mitogenic stimuli (ET-1, Peninsula Laboratories, Belmont, CA; VEGF165, R&D Systems, Minneapolis, MN) were then added, and after 18 hours 1 µCi of [methyl]-[3H] thymidine (6.7 Ci/mmol; DuPont, New England Nuclear Research Products, Wilmington, DE) was added to each well. ET receptor antagonists (Peninsula Laboratories) were incubated 15 minutes before the addition of ET-1. The antagonists had no effect on basal cellular proliferation. The effects of ET-1 were compared with the basal control condition of 0.5% FCS medium. Six hours later the culture media were removed and the cells were washed three times with phosphate-buffered saline, treated with 10% trichloroacetic acid for 15 minutes, washed twice with 100% ethanol, and solubilized in 0.4 N sodium hydroxide. The cell-associated radioactivity was then determined by liquid scintillation counting. Responses to all agents were assayed in sextuplicate and results were expressed as means of three separate experiments.
Chemoinvasion and Chemotaxis Assay
Chemotaxis was conducted in a 48-well modified Boyden chamber (NeuroProbe, Pleasanton, CA) as previously described.23 Eight micrometer pore size polyvinylpyrrolidone-free polycarbonate Nucleopore filters (Costar, New York, NY) were coated with gelatin by immersing them overnight in a solution of 100 µg/ml gelatin in 0.1% acetic acid and then dried. The filter separated the attractants from the upper part of the chamber in which HUVECs were added. For chemoinvasion the filter was coated with an even layer of Matrigel (0.5 mg/ml; Becton Dickinson, Milan, Italy), as previously described.24 HUVECs were harvested in trypsin/ethylenediaminetetraacetic acid solution, collected by centrifugation, and resuspended in Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine serum albumin. The lower compartment of the chamber was filled with chemoattractants or inhibitors (27 µl/well). Cells (5 x 105 cells/ml) were placed in the upper compartment (55 µl/well). ET receptor antagonists (Peninsula Laboratories) were previously added to the cells and preincubated for 15 minutes at 37°C and had no effect on basal cellular migration. After 4 hours (chemotaxis) or 6 hours (chemoinvasion) of incubation at 37°C, filters were stained with Diff-Quick (Merz-Dade, Dudingen, Switzerland) and the migrated cells in 10 high-power fields were counted. Each experimental point was analyzed in triplicate.
Gelatin Zymography
For analysis by zymography, HUVECs at 80% confluence were cultured for 24 hours with M199 with 0.5% FCS and then stimuli were added for an additional 24 hours. The supernatants were collected, concentrated, and electrophoresed (40 µg of protein) for analysis in 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels co-polymerized with 1 mg/ml gelatin. The gels were then washed for 30 minutes at 22°C in 2.5% Triton X-100 to remove sodium dodecyl sulfate and then incubated in 50 mmol/L Tris, pH 7.6, 1 µmol/L ZnCl2, 5 mmol/L CaCl2 for 18 hours at 37°C. After incubation the gels were stained with 0.1% Coomassie blue. Enzyme-digested regions were identified as white bands on a blue background and quantified by computerized image analysis of the band. Molecular sizes were determined from the mobility using gelatinase zymography standards (Bio-Rad Laboratories, Richmond, CA).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was isolated by the guanidium thiocyanate-phenol-chloroform extraction method. The RT-PCR was performed using a GeneAmp RNA PCR kit (Perkin-Elmer Corp., Norway, CT). Briefly, 1 µg of mRNA was used for cDNA synthesis using 50 µmol/L oligo d(T)16 as reverse transcriptase primer at 42°C for 15 minutes. The cDNA were amplified in a final reaction volume of 100 µl containing 0.15 µmol/L oligonucleotide primers. Sequence of the primer set used for metalloproteinase (MMP)-2 was 5'-TTTGGACTGCCCCAGACAGG-3' and 5'-GCTGCGGCCAGTATCAGTGC-3' (518-bp fragment).25 All 5' primers covered splice junctions, thus evaluating the amplification of genomic DNA. In all experiments, two control reactions, one that did not contain any RNA and the other containing mRNA but no reverse transcriptase, were included. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Gibco) was used as an internal control. The semiquantitative analysis was performed essentially as described by Rieckmann et al.26 Ten µl of the amplified products were analyzed on a 1.5% agarose gel containing ethidium bromide (Bio-Rad) and photographed. Densitometric scanning was performed with a Mustek MFS-6000CX apparatus, and data were analyzed with Phoretix 1D software and normalized to those of GAPDH. The mRNA values are expressed as relative units calculated according to the following formula: density of the MMP-2 amplification product/density of the GAPDH amplification product x 100. To compare the results of different experiments, optimal cycle conditions for linear amplification were determined by a semiquantitative assay of the amplified products at different cycles. Thirty-six cycle products (94°C for 1 minute, 54°C for 1 minute, and 72°C for 1 minute), which were within the linear logarithmic phase of the amplification curve, were chosen for comparative analysis.
In Vitro Angiogenesis
Morphogenesis assay on Matrigel was performed as previously described.27 HUVECs cultured for 24 hours in M199 with 0.5% FCS were then plated at 3.5 x 105 cells/well in 24-well plates precoated with 250 µl of Matrigel (10.7 mg/ml; Becton Dickinson) in M199 with 0.5% FCS in the absence or in the presence of ET-1 (10 nmol/L) or VEGF (1 ng/ml). After 24 hours of incubation in a 5% CO2-humidified atmosphere at 37°C, the cell three-dimensional organization was examined under an inverted photomicroscope. Each treatment was performed in triplicate wells.
In Vivo Angiogenic Assay
The method described by Passaniti et al28 has been used with minor modification.29 Briefly, 0.5 ml Matrigel (10.7 mg/ml) alone or containing angiogenic stimuli was injected subcutaneously into C57BL/6N male mice (Charles River, Calco, Como, Italy). After 7 days animals were sacrificed and the gels were recovered. The angiogenic response was evaluated by macroscopic analysis and by measurement of the hemoglobin content. Hemoglobin was mechanically extracted by mincing the pellet. The hemoglobin content was measured using Drabkin reagent kit 525 (Sigma Chemical Co., St Louis, MO) and the final concentration of hemoglobin was calculated from a standard calibration curve.
Statistical Analysis
All statistical analysis were performed by the Inplot software system (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR analysis, using HUVEC total cellular RNA and specific ET-1 and its receptor primers, demonstrated the presence of endogenous transcripts of the appropriate molecular size, thereby confirming that HUVECs express mRNA for ET-1 and for the ETB receptor as the major population, whereas the expression of ETA mRNA was weaker (data not shown).
ET-1 induced a concentration-dependent proliferation of HUVEC with a
maximal effect of ET-1 at 10 nmol/L (50% increase over 0.5% FCS
medium, used as control) (Figure 1)
. The
maximal proliferative effect induced by 10 nmol/L of ET-1 was
equipotent with that elicited by 1 ng/ml VEGF. Additive proliferative
effects were observed when adding ET-1 (10 nmol/L) to VEGF (1 ng/ml) as
demonstrated by a significant (P < 0.001)
increase in DNA synthesis. To elucidate the subtype of the ET receptor
involved in HUVEC proliferation, we co-incubated endothelial cells with
ET-1 and the ETAR antagonist, BQ 123, and the
ETBR antagonist, BQ 788. The stimulatory action
of 10 nmol/L ET-1 was completely blocked in the presence of 1 µmol/L
BQ 788. In the same experiment, the addition of the
ETAR antagonist (BQ 123 1 µmol/L) caused only a
slight decrease in [3H]thymidine incorporation
by HUVECs. Endothelial cell proliferation induced by bFGF was not
inhibited by either the addition of 1 µmol/L BQ 788 or BQ 123,
indicating that the inhibitory effect induced by the
ETBR and ETAR antagonist
was specific and was not because of cytotoxicity (data not shown).
Taken together, these data confirm that mitogenic signaling by ET-1 is
mediated mainly by the ETBR subtype.
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To test whether ET-1 could affect endothelial cell motility,
HUVECs were incubated in a Boyden chamber with ET-1 for 4 hours. ET-1
induced a dose-dependent increase in HUVEC migration, as shown in Table 1
. A significant
(P 
0.02) 50% increase in the number of
migrated cells was detected with ET-1 (10 nmol/L), similar to that
observed in the presence of VEGF (1 ng/ml). When endothelial cells were
treated with ET-1 in the presence of VEGF, additive chemotactic effects
were observed, as shown in Figure 2
. To
elucidate the type of ET receptor involved in the stimulation of HUVEC
migration, experiments were performed with an antagonist of the
ETA receptor and agonist and antagonist of
ETB receptors. Ten nmol/L of selective
ETB agonists, ET-3 and S6c, were able to
significantly (P 0.02) enhance endothelial
cell migration. In the same experiment, a selective
ETBR antagonist, BQ 788, strongly inhibited the
stimulatory action of ET-1, whereas the addition of BQ 123 only induced
a partial decrease. Taken together, these data indicate that ET-1
induces endothelial cell migration and that chemotactic signaling is
mediated mainly by the ETB receptor subtype.
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To form new blood vessels, endothelial cells have to migrate and
cross basement membranes. The invasive capacity of HUVECs in response
to ET-1 was investigated by measuring the invasion of a Matrigel layer
in a Boyden chamber assay. Addition of ET-1 induced a dose-dependent
increase in HUVEC invasion through Matrigel, as shown in Table 1
.
Maximal stimulation, corresponding to a 50% increase in the number of
migrated cells, was obtained at 100 nmol/L ET-1. Furthermore, ET-1 (100
nmol/L) potentiated the VEGF-induced invasive effects (1 ng/ml).
Addition of BQ 788 significantly (P 0.001)
affected endothelial cell invasion, whereas BQ 123 did not induce a
significant inhibition (Figure 3)
indicating that this response to ET-1 is mediated by the
ETB receptor. Phenanthroline, used as a reference
inhibitor of invasiveness, completely blocked the invasive capacity of
HUVECs (data not shown). These data demonstrate, for the first time,
that ET-1 is able to induce endothelial cell invasion through
ETBR and to cooperate with VEGF inducing the
invasion through the basement membrane.
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To invade through the basement membrane, endothelial cells must
degrade various constituents of the interstitial stroma and basement
membrane. MMP-2 is a major extracellular matrix proteolytic enzyme and
it is secreted when endothelial sprouting takes place, thus enhancing
endothelial cell migration across the matrix. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gelatin zymography of
conditioned media of HUVECs not exposed to angiogenic stimuli,
demonstrated a characteristic gelatinase activity corresponding to the
72-kd form of proMMP-2. When the cells were grown in the medium
supplemented with different concentrations of ET-1 for 24 hours, the
secretion of MMP-2 was increased in a dose-dependent manner (data not
shown). A low level of the MMP-9 was also detected. Soft laser scanning
of the band, in fact, showed a 2.5-fold increase in the conditioned
media of HUVECs treated with ET-1 (100 nmol/L), equipotent to that
induced by VEGF (1 ng/ml) when compared with nonstimulated cultures
(Figure 4)
.30
An ET-1
concentration of 100 nmol/L is saturating in terms of MMP-2 production
in HUVECs, as observed in a dose-response experiment (not shown). The
addition of VEGF to ET-1 was not able to induce a further increase in
the MMP-2 zymogram induced by ET-1 alone (data not shown). To determine
whether the observed effects of ET-1 and VEGF on the MMP-2 production
were transcriptionally regulated, we established a sensitive RT-PCR
analysis to detect mRNA transcript for the MMP-2 gene. The RT-PCR
amplified cDNA fragments for MMP-2 were detectable in all samples as a
single band at the expected size (Figure 4B)
. Primers for the
amplification of the GAPDH gene were used as control. Densitometric
analysis of these bands and comparison with the intensity of the bands
of the GAPDH expression, indicated an up-regulation of MMP-2 mRNA after
4 hours of stimulation with ET-1 (100 nmol/L, Figure 4C
). These results
correlated closely with the protein expression observed by zymography.
Hence, RT-PCR analysis showed that MMP-2 mRNA levels correlated well
with the secreted protein levels, suggesting that MMP-2 up-regulation
induced by ET-1 and VEGF was at the transcriptional level.
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To examine whether ET-1 induces morphogenetic changes resembling
capillary like-structure tube formation, HUVECs were plated on
Matrigel. HUVECs in the presence of 0.5% FCS exhibited small round
shapes, isolated cells, and did not spread (Figure 5A)
. Treatment with different doses of
ET-1 for 24 hours resulted in morphological changes (data not shown).
At the optimal dose of 10 nmol/L of ET-1, the cells became elongated,
forming thin cords of interconnecting cells (Figure 5B)
. Similar
effects were also observed with 1 ng/ml VEGF (Figure 5C)
.31
Co-addition of ET-1 and VEGF resulted in a marked
increase in morphogenesis in vitro, compared to that
observed with ET-1 and VEGF alone, inducing HUVEC elongation and
branching to form a network of capillary-like structures (Figure 5D)
.
These data demonstrate that ET-1, like VEGF, is able to mediate
morphogenetic effects and that it cooperates with VEGF to enhance HUVEC
differentiation into vascular structures, which would be necessary
in vivo for the sprouting of endothelial cells and tube
formation.
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The effect of ET-1 on angiogenesis in vivo was tested
using an experimental model in which angiogenesis was induced by
factors embedded in a pellet of Matrigel.29
The gels were
then injected subcutaneously in mice and after 7 days, the degree of
vascularization was evaluated. Quantitation of angiogenesis by
hemoglobin content showed that the addition of ET-1 (100 nmol/L) or
VEGF (up to 300 ng/ml) to the Matrigel did not induce an increased
angiogenic response compared to the negative control (Matrigel plugs
containing vehicle). At variance, ET-1 added with VEGF to the pellets
caused a significant increase of the angiogenic response compared to
ET-1 or VEGF alone. The angiogenic response of Matrigel
neovascularization was comparable to that elicited by the prototypic
angiogenic factor bFGF (300 ng/ml) used as positive control (Table 2)
. These results were confirmed by
histological analysis of Matrigel pellets, demonstrating that the
addition of both ET-1 and VEGF to the Matrigel resulted in the
induction of cellularity and in the formation of cords, tubules, and
several blood-filled channels containing red blood cells (Figure 6B)
, whereas Matrigel pellets without
angiogenic stimuli presented only few infiltrating single elongated
cells (Figure 6A)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ET-1 is a potent mitogen for vascular smooth muscle cells as well as for endothelial cells and is produced by neoplastic cells.8,12,13 Several investigators have provided functional evidence of the expression of the ETB receptor in HUVECs, by amplifying the ETBR numbers through the use of phosphoramidon, demonstrating the presence of 100% ETBR in HUVEC.33 Thus, HUVEC can actively produce and secrete ET-1 and simultaneously express ETB receptor (Kd = 17 pmol/L) as the major population, indicating an autocrine role for synthesized ET-1. Moreover ET-1, through a positive autocrine feed-back action, increases its own synthesis in HUVECs acting via ETBR.34,35
Previous observations demonstrated that ET-1 and ET-3, acting through ETB receptors, have a dose-dependent stimulatory proliferative and migratory effect on endothelial cells isolated from bovine adrenal capillaries and HUVECs.8-11 ET-1 has also been shown to accelerate endothelial wound healing via the ETB receptor.9 These findings suggested that ET-1 may exert an angiogenic activity,36 as also seen in the case of well-vascularized brain tumors such as gliomas or astrocytomas in which the expression of ET-1 correlates with tumor vascularity and malignancy.17
We have demonstrated that ET-1 induces a proangiogenic phenotype in human endothelial cells. This phenotype includes both early (ie, increase in cell proliferation and migration and MMP-2 production) and late angiogenic events (differentiation into vascular cords). During the formation of new blood vessels, endothelial cells are stimulated to migrate, proliferate, and invade surrounding tissue to form capillaries. ET-1 and VEGF, the two growth factors that we studied, both induced these angiogenic effects, and by acting in concert have a potent additive effect on different stages of the angiogenesis process. The ability of ET-1 to induce a significant increase of MMP-2, released by endothelial cells, may represent an important step in ET-1-mediated vascularization, because MMP-2, a major extracellular matrix proteolytic enzyme, is secreted when endothelial sprouting takes place, thus enhancing endothelial cell migration across the extracellular matrix.37 Endothelial cells exposed to ET-1 migrate throughout the Matrigel surface and align to form vascular cord-like structures, indicating that ET-1 is able to stimulate morphogenesis in cultured endothelium. The most striking effect was seen in combination with VEGF: when added simultaneously, VEGF and ET-1 induced a marked increase in the tube formation in cultured endothelial cells.
Overall, our observations demonstrate that ET-1 stimulates vasoproliferative processes and at maximally effective concentrations its effects were additive to that of VEGF. The mechanisms underlying the cooperation between ET-1 and VEGF are not well defined. In our study, the predominately additive actions of ET-1 and VEGF on angiogenic events at saturating agonist concentrations reflect the independence between their individual signaling mechanisms.
Recent studies, exploring a potential interaction between VEGF and ET-1 in vascular cells, evidenced that VEGF enhanced ET-1 mRNA expression and ET-1 secretion in endothelial cells.38 Similarly, in vascular smooth muscle cells, ET-1, predominantly through ETAR, enhances VEGF mRNA expression and VEGF secretion and stimulated the VEGF-induced endothelial cell proliferation and invasion.21,39 This indicated that VEGF and ET-1 have reciprocal stimulatory interactions, resulting in concomitant proliferation of endothelial and vascular smooth muscle cells. In pathological conditions such as cancer, VEGF and ET-1 may be up-regulated by various stimuli including hypoxia, growth factors, and inflammatory cytokines.39 Because hypoxia constitutes a potent stimulus for VEGF and ET-1 production, it is reasonable to hypothesize that in tumor tissues, acute or chronic hypoxia may stimulate VEGF production through both a direct and indirect effect, the latter involving ET-1 secretion as well.40 Furthermore, because ET-1 functions as an antiapoptotic factor for endothelial cells and vascular smooth muscle cells,41,42 this peptide may also contribute to endothelial cell integrity acting as a survival factor for newly formed blood vessels.
Previous observations had shown that ET-1 significantly increased angiogenesis evaluated by blood vessel growth in the rat aortic ring assay, and that erythropoietin-induced neovascularization is partially because of the enhanced autocrine release of ET-1 induced by erythropoietin, because the stimulation of angiogenesis by erythropoietin had been blunted by the ET-1 antibody.43,44 On the contrary, other data show that ET-1 is unable to stimulate blood vessel growth in the chick embryo chorioallantoic membrane45 and in a rat sponge model.46 Having shown that ET-1 cooperates with VEGF in the induction of an angiogenic phenotype in HUVECs in several in vitro assays, it was important to investigate the in vivo relevance of these properties. In this study, ET-1 in association with VEGF has a clear angiogenic activity in the Matrigel in vivo assay, comparable to that promoted by bFGF, demonstrating that ET-1 enhances formation of new vessels in vivo.
These results demonstrated that ET-1 like VEGF, induces angiogenic responses, including endothelial cell protease production, migration, and invasion, via a direct effect on the endothelial cells, and by acting in concert, these two factors have an additive effect on the induction of angiogenesis in vitro and in vivo, suggesting that the interactions between VEGF and ET-1 play an important role in the control of angiogenesis.
In tumors, such as in ovarian carcinoma, ET-1 and VEGF apparently play a complementary and coordinated role during neovascularization and malignant ascite formation.47 In these cells, we demonstrated that ET-1 stimulated VEGF production through ETAR in a manner equipotent to hypoxia, a recognized potent stimulus of VEGF production. Moreover, we demonstrated that elevated levels of ET-1 released by ovarian carcinoma cells in ascitic fluids and in culture media were primarily responsible for endothelial cell migration. The significant inhibition of migration obtained co-incubating endothelial cells with ETBR antagonists and with antibody to VEGF strongly indicated that ET-1, together with VEGF, could modulate angiogenesis.
Altogether, these results provide evidence of the role of ET-1 in neovascularization via a mechanism requiring the activation of the ETB receptor on endothelial cells. Thereafter, the tumor promoting effect of ET-1 could be mediated through direct angiogenic effects on endothelial cells and in part through the VEGF stimulation in the existing tumor.
Because angiogenesis is not only controlled by the presence of VEGF, but may be mediated by several angiogenic factors, we identified ET-1 and its receptors as angiogenic regulators that could represent novel targets for anti-angiogenic therapies. If an angiogenic response is sustained by both VEGF and ET-1, neutralization of one of these two factors may greatly decrease that response by suppressing the contextual effect. New therapeutic strategies using specific antagonists for ET receptor,48 which have no genotoxic effects, provide an additional approach for the treatment of tumors characterized by active angiogenesis, and of other angiogenesis-dependent diseases.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Associazione Italiana Ricerca sul Cancro and Fondazione Italiana per la Ricerca sul Cancro. D. S. and L. R. are recipients of fellowships from Fondazione Italiana Ricerca sul Cancro.
Accepted for publication July 24, 2000.
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