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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2627-2636
Human Erythropoietin Induces a Pro-Angiogenic Phenotype in Cultured
Endothelial Cells and Stimulates Neovascularization In Vivo
By
Domenico Ribatti,
Marco Presta,
Angelo Vacca,
Roberto Ria,
Roberta Giuliani,
Patrizia Dell'Era,
Beatrice Nico,
Luisa Roncali, and
Franco Dammacco
From the Institute of Human Anatomy, Histology and Embryology, and
the Department of Biomedical Sciences and Human Oncology, University of
Bari School of Medicine, Bari, Italy; and the Unit of General Pathology
and Immunology, Department of Biomedical Sciences and Biotechnology,
University of Brescia School of Medicine, Brescia, Italy.
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ABSTRACT |
Hematopoietic and endothelial cell lineages share common
progenitors. Accordingly, cytokines formerly thought to be specific for
the hematopoietic system have been shown to affect several functions in
endothelial cells, including angiogenesis. In this study, we
investigated the angiogenic potential of erythropoietin (Epo), the main
hormone regulating proliferation, differentiation, and survival of
erythroid cells. Epo receptors (EpoRs) have been identified in the
human EA.hy926 endothelial cell line by Western blot analysis. Also,
recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2)
phosphorylation, cell proliferation, and matrix metalloproteinase-2
(MMP-2) production in EA.hy926 cells and significantly enhances their
differentiation into vascular structures when seeded on Matrigel. In
vivo, rHuEpo induces a potent angiogenic response in the chick embryo
chorioallantoic membrane (CAM). Accordingly, endothelial cells of the
CAM vasculature express EpoRs, as shown by immunostaining with an
anti-EpoR antibody. The angiogenic response of CAM blood vessels to
rHuEpo was comparable to that elicited by the prototypic angiogenic
cytokine basic fibroblast growth factor (FGF2), it occurred in the
absence of a significant mononuclear cell infiltrate, and it was not
mimicked by endothelin-1 (ET-1) treatment. Taken together,
these data demonstrate the ability of Epo to interact directly with
endothelial cells and to elicit an angiogenic response in vitro and in
vivo and thus act as a bona fide direct angiogenic factor.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RECENT STUDIES HAVE indicated that
several cytokines and interleukins (ILs) formerly thought to be
specific for the hematopoietic system, including granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), IL-3, IL-4, IL-6, and IL-8, are
also capable of affecting certain functions of endothelial
cells.1-9 This responsiveness of both vascular and
hematopoietic systems to some cytokines and ILs may reflect the common
ontogenesis of endothelial and hematopoietic cells. Indeed,
differentiation of vascular endothelium during embryonic development is
closely linked to the appearance of primitive hematopoietic cells,
suggesting that both cell lineages share a common progenitor, the
hemangioblast.10,11 This is supported by the finding that
cell surface antigens present on endothelium are also expressed by
hematopoietic cells, including quail QH1 and MB1
antigens12,13 and human and murine CD31 and CD34
antigens.14,15 Accordingly, deletion of the
endothelial-specific vascular growth factor receptor 2 (VEGFR2) by gene
targeting has shown that both endothelial and hematopoietic cells are
absent in homozygous null mice.16 VEGFR2
expression also defines a population of early hematopoietic
precursors17 and VEGFR2+ cells isolated from
the chick embryo mesoderm at the gastrulation stage give rise to either
hematopoietic or endothelial cell colonies.18 Moreover,
endothelial cell precursors have been isolated from the peripheral
blood on the basis of cell surface antigen expression.19
Erythropoietin (Epo) was formerly believed to exert its hematopoietic
effects by stimulating the proliferation of early erythroid precursors
and the differentiation of later precursors of the erythroid
lineage.20 However, the appearance of side effects arising
from the vascular system, including hypertension and
thrombosis21 during the therapeutic use of recombinant
human Epo (rHuEpo), has prompted investigation of a possible
interaction of Epo with endothelial cells. The Epo receptor (EpoR), in
fact, has been demonstrated in endothelial cells in vitro and in
vivo.22 rHuEpo also stimulates cell migration,
proliferation, endothelin-1 (ET-1) release, and an increase in
cytosolic free calcium concentration in endothelial cell
cultures.23-26
Angiogenesis, the formation of new capillaries from pre-existing
vessels, is a process involved in vascularization of organs of
ectodermal or mesenchymal origin during embryonic
development.27 In the adult, the proliferation rate of
endothelial cells is very low compared with other cell types in the
body. Physiological exceptions in which angiogenesis occurs under tight
regulation are found in the female reproductive system and during wound
healing. Uncontrolled endothelial cell proliferation has pathological
implications and plays a pivotal role in tumor progression as well as
in inflammatory and viral diseases.28 During
neovascularization, endothelial cells change their genetic program and
express an angiogenic phenotype that includes the production of
proteases, cell migration, and proliferation followed by
redifferentiation, thus resulting in the formation of new blood
vessels.27
Several angiogenic growth factors have been characterized so far,
including members of the VEGF and fibroblast growth factor (FGF)
families.29,30 Interestingly, hematopoietic stimulators, including G-CSF and GM-CSF,1,2 have also been shown to
induce an angiogenic response in endothelial cells, suggesting that
endothelial cell growth and survival may contribute to the maintenance
of bone marrow microenvironment and hematopoiesis. Similar to
well-known angiogenic growth factors, Epo affects some properties of
endothelial cells in culture, eg, cell proliferation and motility (see
above), that are related to neovascularization in vivo, thus suggesting that it acts as an angiogenic factor. Nevertheless, to our knowledge there is no direct experimental evidence of the ability of Epo to
induce neovascularization in vivo. This appears to be of particular importance when considering that IL-4 exerts an anti-angiogenic activity in vivo31 despite its ability to induce cell
proliferation and protease production in cultured endothelial
cells.5,32 We have therefore investigated the ability of
rHuEpo to induce a pro-angiogenic phenotype in cultured endothelial
cells, namely an increase in cell proliferation, protease production
and morphogenesis, and the stimulation of new blood vessel formation in
the chick embryo chorioallantoic membrane (CAM). The results
unequivocally demonstrate that rHuEpo is a potent angiogenic factor
both in vitro and in vivo.
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MATERIALS AND METHODS |
Cells.
Immortalized EA.hy926 endothelial cells, derived from the fusion of
human umbilical vein endothelial cells (HUVECs) with A549 lung
carcinoma cells,33 were maintained in Dulbecco's modified Minimal Essential Medium (DMEM) supplemented with 10% fetal calf serum
(FCS) and 1% glutamine. HUVECs were grown in M199 medium supplemented
with 10% FCS.
Immunoprecipitation with anti-Janus Kinase-2 (JAK-2) and Western
blot analysis of EpoR and JAK-2 phosphorylation.
Confluent cell cultures were lysed in phosphate-buffered saline (PBS)
containing 2% Triton-X100. Cellular extracts (25 µg) were
resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subjected to 8% SDS-PAGE. Proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (NEN, Boston, MA) and probed
with anti-EpoR rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz,
CA). The membrane was then incubated sequentially with horseradish
peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) and
with Renaissance chemiluminescence reagents (Du Pont de Nemours,
Boston, MA) according to the manufacturer's instructions and exposed
to Reflection film (Du Pont de Nemours). An EpoR synthetic peptide
(Santa Cruz Biotechnology) was used as positive control.
For evaluation of JAK-2 phosphorylation, EA.hy926 cells were cultured
in serum-free medium for 16 to 18 hours and exposed to 0.5 to 30 U/mL
rHuEpo (Eprex; Janssen-Cilag, Cologno Monzese, Milan, Italy) for
different periods of time. They were then lysed in ice-cold lysis
buffer (20 mmol/L HEPES, pH 7.2, 150 mmol/L NaCl, 1% Triton-X100, 10%
glycerol, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 1 µg/mL
leupeptin, 100 U/mL aprotinin, 1 mmol/L phenylmethyl sulfonyl fluoride
[PMSF], 1 mmol/L NaVO4, 2 mmol/L NaPPi) for 20 minutes on ice. Lysates were centrifuged at 12,000 rpm for 15 minutes and incubated with rabbit preimmune serum (Santa Cruz Biotechnology) and 50 µL of 50% protein A slurry (Sigma Chemical Co,
St Louis, MO). Supernatants were collected and incubated overnight at
4°C with anti-JAK-2 rabbit antiserum (Santa Cruz Biotechnology) and 50 µL of 50% protein A slurry. Immunoprecipitates were washed with 20 mmol/L HEPES, pH 7.2, 150 mmol/L NaCl, 10% glycerol, 1% Triton-X100; resuspended in SDS-PAGE sample buffer; subjected to 8%
SDS-PAGE; and probed with anti-phosphotyrosine monoclonal antibody
(Transduction Laboratories, Lexington, UK) as described above.
The membrane was then stripped in 6.25 mmol/L Tris/Cl (pH 6.8) SDS, 100 mmol/L  mercaptoethanol for 30 minutes at 50°C and then washed
in TBS-Tween 20 three times and incubated in blocking buffer for 1 hour. The membrane was incubated with the anti-EpoR rabbit antiserum
for 1 hour at room temperature, washed again, and then incubated with
antirabbit antibody horseradish peroxidase-conjugate for 1 hour. The
filter was washed in TBS 0.1% Tween 20 and incubated with enhanced
chemiluminescence (NEN). The signal was shown by exposure to Kodak
biomax film (Eastman Kodak, Rochester, NY). After one more
stripping, the same procedure was applied to the same membrane for the
anti-JAK-2 antiserum.
Cell proliferation assay.
EA.hy926 cells were plated at 2 × 103 cells per well
in 96-well plates precoated with 1% gelatin. After 24 hours, medium
was removed and replaced every other day with fresh DMEM containing 0.25% FCS and supplemented 1:1 (vol:vol) with RPMI-1640 medium alone
or containing increasing concentrations (from 0.5 to 40 U/mL) of
rHuEpo. Experiments were performed in quadruplicate. The cell number
was measured at day 6 of growth34 by the colorimetric method of Kueng et al.35 Briefly, cells were fixed for 20 minutes at room temperature with 2.5% glutaraldehyde, stained with
0.1% crystal violet in 20% methanol, and solubilized with 10% acetic acid. Wells were read at 595 nm with a microplate reader (Model 3550;
Bio-Rad Laboratories, Richmond, CA) and the cell number was calculated
from an appropriate calibration curve. Values are expressed as mean ± 1 standard deviation (SD).
Matrix metalloproteinase (MMP) SDS-PAGE zymography.
EA.hy926 cells at 80% confluence were cultured for 24 hours in
serum-free medium in the absence or in the presence of 0.5, 1.0, or 2.0 U/mL of rHuEpo. After incubation, the conditioned medium was collected,
sequentially centrifuged at 1,200 and 12,000 rpm for 10 minutes,
filtered through 0.54-µm pore-size filters (Costar, Cambridge, MA),
and stored at  80°C until use. Gelatin-zymography was
performed to visualize the MMP activity present in the
samples.36 Five micrograms of protein were applied to 7.5%
SDS-PAGE gels copolymerized with type A gelatin from porcine skin
(Sigma Chemical Co) at a final concentration of 0.6 mg/mL. After
electrophoresis, gels were washed in 2.5% Triton-X100 for 1 hour to
remove SDS, incubated for 18 hours at 37°C in collagenase buffer,
and stained in 0.1% Coomassie brilliant blue. Gelatinolytic activity
was visualized as a transparent band against a blue background and
quantified by computerized image analysis of the band.
Matrigel morphogenetic assay.
This was performed as described previously.36 Briefly,
EA.hy926 cells were plated at 2 × 105
cells per well in 24-well plates precoated with 300 µL of
Matrigel (8.1 mg/mL; Becton Dickinson Italia, Milan, Italy) in DMEM
added with 0.1% BSA in the absence or in the presence of 0.5, 1.0, or 2.0 U of rHuEpo. After 6 hours of incubation in 5% CO2
humidified atmosphere at 37°C, the cell three-dimensional
organization was examined under an inverted phase contrast
photomicroscope. Each treatment was performed in triplicate wells.
Chick embryo chorioallantoic membrane (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 to 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, 1mm3 sterilized gelatin sponges
(Gelfoam; Upjohn Co, Kalamazoo, MI) adsorbed with rHuEpo or ET-1
(Peninsula Laboratories, Belmont, CA) dissolved in 2 µL of PBS were
implanted on the top of growing CAMs under sterile conditions within a
laminar flow hood.37 rHuEpo was delivered at 1.0 to 10.0 U
per implant and ET-1 at 10 6 to
108 mol/L per implant, whereas sponges containing
vehicle alone or 1 µg of FGF2 were used as negative and positive
controls, respectively. CAMs were examined daily until day 12 and
photographed in ovo under a Zeiss stereomicroscope SR equipped with the
MC 63 Camera System (Zeiss, Oberkochen, Germany). At day 12, CAMs were
processed for light microscopy. Briefly, embryos and their membranes
were fixed in ovo in Bouin's fluid, and then sponges and the
underlying and immediately adjacent CAM portions were removed and
processed for embedding in paraffin. Eight-micrometer serial sections,
cut according to a plane parallel to the surface of the CAM, were stained with 0.5% aqueous solution of toluidine blue (Merck
Biochemica, Darmstadt, Germany) and observed under a Leitz-Dialux 20 light microscope (Leitz, Wetzlar, Germany). At day 12, some CAMs were also processed for electron microscopy. Briefly, the embryos and their
membranes were fixed in ovo in 3% phosphate-buffered glutaraldehyde, dehydrated in serial alcohols, postfixed in 1% phosphate-buffered OsO4, and embedded in Epon 812. Ultrathin sections were cut
on an LKB V ultramicrotome (LKB, Bromma, Sweden) according
to a plane perpendicular to the surface of the CAM. The sections
were stained with uranyl acetate followed by lead citrate and examined
under a 9A Zeiss electron microscopy.
The angiogenic response and the infiltration of mononuclear cells were
assessed by a planimetric method of point counting.38,39 Briefly, every third section within 30 serial slides from an individual specimen was analyzed by a 144-point mesh inserted in the eyepiece of
the Leitz-Dialux 20 photomicroscope. Six randomly chosen 250× fields of each section were used to count the total number of the
intersection points that were occupied by vessels transversally cut
(diameter ranging from 3 to 10 µm). Mean values ± SD were determined for each analysis. The vascular density was indicated by the
final mean number of the occupied intersection points. The statistical
significance of differences between the mean values of the intersection
points in the experimental and control CAMs was determined by the
Student's t-test for unpaired data.
Immunohistochemistry.
The antibodies used in this study were (1) anti-factor VIII polyclonal
rabbit antibody (Dako) and (2) polyclonal rabbit anti-EpoR antibody
(Santa Cruz Biotechnology). Eight-micrometer acetone-fixed cryostat CAM
sections, treated with 7.5% H2O2 to destroy
endogenous peroxidase, were stained with a three-step
avidin-biotin-immunoperoxidase, as described
elsewhere.40 Briefly, incubation with primary
antibodies and then biotin-labeled swine antirabbit Ig (Dako) and
avidin-horseradish-peroxidase conjugate (Vector Inc, Burlingame, CA)
was followed by red-staining with a 3-amino-9-ethylcarbazole (Sigma
Chemical Co) solution and counterstaining with Gill's hematoxylin no.
2 (Polysciences Inc, Warrington, PA) and was mounted in buffered
glycerin. A preimmune rabbit serum (Dako) replacing the antibodies
served as negative control.
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RESULTS |
EA.hy926 endothelial cells express the biologically active EpoR.
The immortalized EA.hy926 endothelial cell line retains several
endothelial characteristics.33 As a preliminary experiment, the ability of EA.hy926 cells to express EpoR was investigated. To this
purpose, an EA.hy926 cell extract was probed with anti-EpoR antibodies.
As shown in Fig 1A, these antibodies
recognize a molecular weight (Mr) 78,000 immunoreactive protein in the
extract of EA.hy926 cells as well as HUVEC extracts used as an
additional positive control.22 To assess whether the EpoR
expressed by EA.hy926 cells is functionally active, the ability of Epo
to stimulate JAK-2 phosphorylation41 was investigated.
EA.hy926 cell cultures were treated with 0.5 to 30 U/mL rHuEpo for
periods ranging from 30 seconds to 1 hour. Cell extracts were
immunoprecipitated with anti-JAK-2 antibodies and sequentially probed
with anti-phosphotyrosine, anti-EpoR, and anti-JAK-2 antibodies in a
Western blot (Fig 1B through D). rHuEpo (30 U/mL) causes a transient
increase in JAK-2 phosphorylation, with a maximal effect being observed
5 minutes after stimulation. Similar results were obtained when cells
were stimulated with 5, 10, and 15 U/mL rHuEpo (data not shown).
Moreover, EpoR coprecipitates with phosphorylated JAK-2, thus
demonstrating the formation of a EpoR/phospho-JAK-2 complex in
rHuEpo-treated cells (Fig 1C).
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| Fig 1.
EpoR expression and rHuEpo-dependent JAK-2 activation in
EA.hy926 cells. (A) Twenty-five-microgram aliquots of the extracts of
confluent EA.hy926 cells and HUVECs were run on 8% SDS-PAGE gel and
probed with anti-EpoR rabbit antiserum. EpoR synthetic peptide was used
as a positive control. (B through D) EA.hy926 cells were incubated with
30 U/mL rHuEpo in serum-free conditions for the indicated periods of
time. Cell extracts were then immunoprecipitated with anti-JAK-2
antibody. Immunoprecipitates were subjected to 8% SDS-PAGE and probed
with anti-phosphotyrosine antibody in a Western blot (B). After
stripping of the membrane, immunoprecipitates were probed with
anti-EpoR antibody (C). Note that EpoR coprecipitates with
phosphorylated JAK-2. Uniform loading of the gel was shown by
probing the membrane with anti-JAK-2 antibody (D).
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rHuEpo induces a pro-angiogenic phenotype in EA.hy926 endothelial
cells.
Several angiogenic growth factors induce a pro-angiogenic phenotype in
endothelial cell cultures. This phenotype includes, among other
responses, endothelial cell proliferation and protease production.42 To evaluate the mitogenic capacity of Epo,
EA.hy926 cells were seeded at 2.5 × 103 per well in
96-well plates and treated every other day with fresh medium containing
0.25% FCS in the absence or in the presence of 0.5 to 40 U/mL rHuEpo.
On day 6, cells were counted. In agreement with previous
studies23 and our own observations on HUVECs and bovine
adrenal capillary endothelial cells, rHuEpo exerted a significant increase in EA.hy926 cell proliferation
(Fig 2). Maximal stimulation, corresponding
to an eightfold increase in cell number, was observed at 5 U/mL rHuEpo.
No further increase in cell number was observed at higher rHuEpo
concentrations.
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| Fig 2.
Effects of rHuEpo on EA.hy926 cell proliferation. Cells
were seeded at 2 × 103 cells per well. After 24 hours,
medium was removed and replaced every other day with fresh medium
containing 0.25% FCS and supplemented 1:1 (vol:vol) with RPMI-1640
medium containing increasing concentrations of rHuEpo. The cell number
was counted at day 6 of growth. The experiment was performed in
quadruplicate and values are shown as the mean ± SD.
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EA.hy926 cell cultures maintained in serum-free medium for 24 hours
secreted significant amounts of the cleaved, activated form of MMP-2,
as shown by the presence of a gelatinolytic band with an apparent Mr
equal to 62,000 when their conditioned medium is analyzed by gelatin
SDS-PAGE zymography (Fig 3A). When the cells were grown for 24 hours in the same medium supplemented with
rHuEpo, MMP-2 activity increased. Soft laser scanning of the band, in
fact, showed a threefold increase in the conditioned medium of EA.hy926
cells treated with 2 U/mL rHuEpo when compared with nonstimulated
cultures (Fig 3B).
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| Fig 3.
Effect of rHuEpo on MMP-2 production in EA.hy926 cells.
Subconfluent EA.hy926 cells were cultured for 24 hours in serum free
medium in the absence (SFM) or in the presence of the indicated
concentrations of rHuEpo. After incubation, the conditioned medium was
analyzed by gelatin-zymography as described in Materials and Methods.
(A) One representative experiment showing the presence of a Mr 62,000 gelatinolytic band corresponding to activated MMP-2 in the conditioned
medium of control (a), 1 U/mL rHuEpo (b), and 2 U/mL rHuEpo (c) treated
cells. M, molecular weight markers. (B) Quantitation of MMP-2 activity
by computerized image analysis of the gelatinolytic bands. Data are the
mean ± SD of eight independent experiments (statistical analysis by
Student's t-test).
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Cultures of endothelial cells on Matrigel resulted in the formation of
vascular tubes and cord-like structures connecting cellular nodes, a
phenomenon known as angiogenesis in vitro.43-47 This assay
has been extensively used to study positive and negative regulators of
angiogenesis.48 As shown in Fig
4A, EA.hy926 cells remained spherical and isolated when seeded on
Matrigel in the presence of serum-free medium, with small cellular
nests and short tubes being rarely observed. By contrast, rHuEpo caused
a dose-dependent morphogenetic effect (Fig 4B through D). At 2 U/mL
rHuEpo, EA.hy926 cells migrated throughout the Matrigel surface and
formed branching, anastomosing tubes with multicentric junctions,
originating a meshwork of capillary-like structures (Fig 4D).
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| Fig 4.
Morphogenic activity of rHuEpo. EA.hy926 cells were
seeded on Matrigel and incubated in DMEM added with 0.1% BSA (A) or
with 0.5 U/mL (B), 1.0 U/mL (C), or 2.0 U/mL (D) rHuEpo. After 6 hours,
cells were photographed using an inverted phase contrast
photomicroscope. A dose-dependent morphogenetic effect of rHuEpo was
observed.
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rHuEpo induces an angiogenic response in the chick embryo CAM.
The ability of rHuEpo to stimulate both early and late responses of the
angiogenic cascade in EA.hy926 cells in vitro prompted us to assess its
angiogenic capacity in vivo. To this purpose, chick embryo CAMs at day
8 of incubation were implanted with gelatin sponges adsorbed with
rHuEpo dissolved in PBS. Sponges adsorbed with vehicle alone or with
FGF2 were used as negative and positive controls, respectively.
At day 12 of incubation, macroscopic observation of the CAMs showed
that rHuEpo induced an angiogenic response characterized by the
presence of allantoic vessels spreading radially towards the sponge in
a spoked wheel pattern
(Fig
5A). The effect of rHuEpo was dose-dependent, with the number of
positive implants (of a total of 30 embryos per group) being equal to
0, 6, 9, 27, and 27 implants for 1.0 U, 2.0 U, 5.0 U, 10.0 U, and 20.0 U of rHuEpo per sponge, respectively. A similar macroscopic angiogenic
response was observed in 16 of 20 implants treated with 1 µg of FGF2
(data not shown), whereas no vascular reaction was detectable around the sponge in the 20 specimens treated with PBS alone (Fig 5B).
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| Fig 5.
rHuEpo stimulates angiogenesis in the chick embryo
CAM. CAM of 12-day-old chick embryo incubated for 4 days with a gelatin
sponge adsorbed with 10 U of rHuEpo. Note the presence of an increased
number of blood vessels with a radially arranged spoked wheel pattern
around the implant. (B) CAM of 12-day-old chick embryo incubated for 4 days with a sponge adsorbed with vehicle alone (PBS) used as negative
control. No vascular response is detectable around the sponge. (C)
Histological section of a gelatin sponge treated with 10 U of rHuEpo.
Note the collagenous matrix containing numerous capillaries among the
sponge trabeculae and a cellular infiltrate prevalently formed by
fibroblasts. (D) Histological section of a sponge treated with PBS. No
collagenous matrix, blood vessels, or fibroblasts are detectable. Bar
for (A) and (B) is 25 µm and for (C) and (D) is 12.5 µm.
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Microscopically, the sponges adsorbed with rHuEpo showed a collagenous
matrix containing numerous small blood vessels and fibroblasts
localized among the sponge trabeculae (Fig 5C). Numerous host
capillaries piercing the sponge in some points were also recognizable
at the boundary between the sponge and the CAM mesenchyme. A very
scarce mononuclear cell infiltrate was present among the sponge
trabeculae. Similar findings had been reported previously for
FGF2-treated sponges.37 No collagenous matrix, blood
vessels, or fibroblasts were instead present among the sponge
trabeculae in the samples treated with PBS (Fig 5D). In keeping with
the strong angiogenic response elicited by rHuEpo, numerous mitotic figures of the endothelial cells were recognizable at ultrastructural level in CAMs treated with rHuEpo but not in control, PBS-treated CAMs
(Fig 6).
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| Fig 6.
Ultrastructural features of rHuEpo-treated CAM
vasculature. An ultrathin section showing a mitotic endothelial cell
(EC) in rHuEpo-treated CAM. Bar is 0.3 µm.
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In agreement with the observations listed above, evaluation of the
microvessel density of the CAM at day 12 of incubation demonstrated
that rHuEpo exerted an angiogenic response in the chick embryo
quantitatively similar to that elicited by FGF2
(Table 1). It must be pointed out that no
correlation was observed between microvessel density and the number of
infiltrating mononuclear cells in histological sections of
rHuEpo-treated CAMs from 8 independent experiments (Pearson's
r = .53; P = .17).
In vitro studies had shown that rHuEpo stimulates ET-1 production in
endothelial cells and that rHuEpo-induced endothelial cell sprouting
from rat aorta rings is partially blocked by neutralizing anti-ET-1
antibodies, thus suggesting rHuEpo activity can be partially modulated
in vitro by an autocrine action of ET-1.25 However, no
angiogenic response was observed in the CAM when sponges were adsorbed
with ET-1 at 106 to 108 mol/L per
implant, ruling out the possibility that ET-1 may play a significant
role in mediating the angiogenic activity exerted by rHuEpo in this in
vivo model (data not shown).
The capacity of rHuEpo to exert an angiogenic response in the chick CAM
prompted us to assess this tissue for the presence of EpoRs. As shown
in Fig 7B, a marked immunoreactivity to anti-EpoR antibody was observed in the microvessels of the CAM of untreated embryos starting from day 8 of incubation. EpoR immunostaining colocalized with factor VIII positivity (Fig 7A), thus confirming that
EpoR expression is limited to endothelial cells of the CAM.
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| Fig 7.
Immunohistochemical localization of EpoR in CAM.
Immunoperoxidase staining of an 8-day-old chick embryo CAM using
polyclonal antibodies to factor VIII (A) and anti-EpoR (B). Note the
coexpression of factor VIII and EpoR on vascular endothelial cells. The
negative control (preimmune serum replacing the primary antibody) is
shown in (C). Bar for (A) through (C) is 5 µm.
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| |
DISCUSSION |
Previous observations have shown the capacity of endothelial cells to
express EpoR and to respond to this cytokine with an increase in cell
proliferation and chemotaxis.22,23 rHuEpo has also been
shown to stimulate endothelial cell sprouting in the in vitro rat aorta
ring assay.25 These findings were taken as a suggestion
that Epo might be endowed with an angiogenic activity. However, the
ability of rHuEpo to stimulate neovascularization in vivo was not assessed.
We have demonstrated here that rHuEpo induces a pro-angiogenic
phenotype in human endothelial EA.hy926 cells. This phenotype includes
both early (ie, increase in cell proliferation and MMP-2 production)
and late angiogenic events (differentiation into vascular tubes).
Accordingly, EA.hy926 cells express EpoR that binds to JAK-2 and
induces its transient activation after rHuEpo exposure. An in vivo
chick embryo CAM assay showed that rHuEpo elicits an angiogenic
response quantitatively and qualitatively similar to that exerted by
the prototypic angiogenic factor FGF2 in the absence of a significant
mononuclear cell infiltrate. Accordingly, endothelial cells of the CAM
express EpoRs that colocalize with factor VIII positivity. Taken
together, the data demonstrate that Epo can act as a bona fide direct
angiogenic factor.
Previous observations had shown that blood vessel outgrowth induced in
vitro in the rat aorta ring assay by rHuEpo is partially dependent on
ET-1 production by endothelial cells.25 Our data show that
ET-1 is unable to stimulate new blood vessel growth in the CAM and
agree with other in vivo studies demonstrating that ET-1 is not
angiogenic.49 This suggests that the role of ET-1
upregulation in mediating Epo activity on endothelial cells may be
restricted to some experimental systems.
EpoR is a member of the cytokine receptor superfamily and lacks a
kinase domain. Epo induces tyrosine phosphorylation in EpoR-expressing cells and this is correlated with gene transcription and
mitogenesis.41 Experimental evidence indicates that JAK-2
serves as a signaling molecule for EpoR. Indeed, JAK-2 is tyrosine
phosphorylated and associates to EpoR after stimulation with
rHuEpo.41 EpoR intracellular signaling has been poorly
investigated in endothelial cells. rHuEpo induces an increase in
cytosolic free calcium concentration26 and causes tyrosine
phosphorylation of various proteins, including the transcription factor
STAT-5, in endothelial cell cultures.50 We have shown here
that rHuEpo causes a rapid and transient phosphorylation of JAK-2 and
its association with EpoR in endothelial EA.hy926 cells. It is
interesting to note that JAK-2 is involved in the intracellular
signaling of receptors for various cytokines, including the angiogenic
G-CSF and GM-CSF (see Witthuhn et al41 for discussion of
this issue). Recent observations have shown that GM-CSF induces JAK-2
activation in EA.hy926 cells51 and in HUVECs.52
Taken together, these results suggest a possible role for JAK-2/STAT-5 signaling pathway in cytokine-mediated angiogenesis. Further
experiments are required to elucidate this point.
MMP-2 is a major extracellular matrix proteolytic enzyme that degrades
various constituents of the interstitial stroma and basement membrane,
including type IV, type V, type VII, and type X collagens; fibronectin;
laminin; and elastin.42 It is secreted when endothelial
sprouting takes place, thus enhancing endothelial cell migration across
the matrix.42 Indeed, MMP inhibitors prevent endothelial
cell invasion and angiogenesis.53 The ability of rHuEpo to
induce a significant increase of MMP-2 activity released by EA.hy926
cells may thus represent an important step in Epo-mediated neovascularization. Accordingly, rHuEpo has been demonstrated to
increase vessel outgrowth from rat aortic rings embedded in a
reconstituted basement matrix.25
The culture of certain populations of endothelial cells on Matrigel
results in the formation of vascular tubes and cord-like structures.43-47 EA.hy926 cells exposed to rHuEpo migrate
throughout the Matrigel surface and align to form branching and
anastomosing tubes. This originates a meshwork of capillary-like
structures, indicating that Epo is able to stimulate morphogenesis in
cultured endothelium. Thus, as demonstrated for FGF2 and
VEGF,29,30 Epo is able to stimulate the early invasive
phase of the angiogenic process that leads to endothelial sprouting
(characterized by an increase in cell motility, matrix degradation, and
cell proliferation) and the late differentiation phase required for the
formation of hollow vascular structures.
Overall, our observations suggest a role of Epo in vasoproliferative
processes and emphasize its direct interaction with endothelial cells.
These data are in agreement with increasing experimental evidence on
the role of various hematopoietic cytokines in angiogenesis. As stated
above, receptors for G-CSF and GM-CSF have been detected on the surface
of endothelial cells.1,6,8 These cytokines induce
endothelial cells to migrate and proliferate and are angiogenic in vivo
in the rabbit cornea.1,2 GM-CSF also induces angiogenesis in rat connective tissue by a direct effect on endothelial cells and/or
by the recruitment and activation of macrophages that release their own
angiogenic factors.4 Human endothelial cells express receptors for IL-3.3,8 IL-4, a lymphocyte growth and
differentiation factor, stimulates the growth of microvascular
endothelial cells5 and exerts an anti-angiogenic effect in
vivo.31 IL-6, a B-cell differentiation factor, regulates
the growth of vascular cells and its overexpression in the central
nervous system of transgenic mice correlates with
neovascularization.7,9 IL-6 mRNA is expressed in
endothelial cells during the angiogenesis that accompanies folliculogenesis and formation of decidua.54 Furthermore,
IL-6 participates in an autocrine manner to the growth of Kaposi's sarcoma55 and exerts an autocrine activity in middle
T-antigen-transformed endothelial cells.56 IL-6 is also
secreted by non-Hodgkin's lymphoma, lymphoblastic leukemia, and
multiple myeloma,57 tumors in which the extent of
angiogenesis is related to neoplastic progression.40,58,59 Finally, IL-8, which regulates the functions of mature myeloid cells,
is an angiogenic factor that induces proliferation and chemotaxis of
human endothelial cells.60-63
Differentiation of vascular endothelium is closely linked to the
appearance of primitive hematopoietic cells, suggesting that both cell
lineages share a common progenitor, the hemangioblast.10,11 Experimental studies of the angiogenic growth factor receptor VEGFR2,
which was initially thought to be expressed specifically in cells of
endothelial lineage,64 supported this hypothesis. Endothelial and hematopoietic cells are, in fact, absent in homozygous VEGFR2 knock-out mice,16 both cell types share a
VEGFR2+ common precursor in the chick embryo,18
and VEGFR2 expression defines a population of early embryonic
hematopoietic precursors.17 In addition, putative
endothelial cell progenitors have been isolated from human peripheral
blood.19 Epo functions as the primary humoral regulator of
erythropoiesis. Our data suggest that its full action and hence the
production of erythrocytes and their release into the blood are
rendered possible by the convergence of two phenomena, namely (1)
proliferation and differentiation of progenitor erythroid cells and (2)
bone marrow angiogenesis.
| |
ACKNOWLEDGMENT |
The authors thank F. Bussolino (University of Torino, Torino, Italy)
for critical reading of the manuscript and Janssen-Cilag (Cologno
Monzese, Milan, Italy) for the generous supply of rHuEpo.
| |
FOOTNOTES |
Submitted June 25, 1998; accepted December 1, 1998.
Supported in part by a grant from Ministero dell'Università e
della Ricerca Scientifica e Tecnologica (MURST, 60%), Rome, Italy, to
D.R.; by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan,
Italy, to D.R, F.D., and M.P.; and by Istituto Superiore di
Sanità (AIDS Project), Rome, Italy, to M.P.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Domenico Ribatti, MD, Institute of Human
Anatomy, Histology and Embryology, University of Bari, School of
Medicine, Piazza G. Cesare, 11, Policlinico, I-70124 Bari, Italy;
e-mail: ribatti{at}anatomia.uniba.it.
| |
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X. Yu, C.-S. Lin, F. Costantini, and C. T. Noguchi
The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse
Blood,
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[Abstract]
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G. Acs, P. Acs, S. M. Beckwith, R. L. Pitts, E. Clements, K. Wong, and A. Verma
Erythropoietin and Erythropoietin Receptor Expression in Human Cancer
Cancer Res.,
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[Abstract]
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A.-L. Sirén, M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, et al.
Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress
PNAS,
March 16, 2001;
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51606598.
[Abstract]
[Full Text]
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V. H. Haase, J. N. Glickman, M. Socolovsky, and R. Jaenisch
Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor
PNAS,
February 13, 2001;
98(4):
1583 - 1588.
[Abstract]
[Full Text]
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V. Divoky, Z. Liu, T. M. Ryan, J. F. Prchal, T. M. Townes, and J. T. Prchal
Mouse model of congenital polycythemia: Homologous replacement of murine gene by mutant human erythropoietin receptor gene
PNAS,
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[Abstract]
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G. Bernardini, G. Spinetti, D. Ribatti, G. Camarda, L. Morbidelli, M. Ziche, A. Santoni, M. C. Capogrossi, and M. Napolitano
I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo
Blood,
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96(13):
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[Abstract]
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D. Salani, G. Taraboletti, L. Rosano, V. Di Castro, P. Borsotti, R. Giavazzi, and A. Bagnato
Endothelin-1 Induces an Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo
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[Abstract]
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H. H. Marti, M. Bernaudin, E. Petit, and C. Bauer
Neuroprotection and Angiogenesis: Dual Role of Erythropoietin in Brain Ischemia
Physiology,
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[Abstract]
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J. Y. Pan, W. E. Fieles, A. M. White, M. M. Egerton, and D. S. Silberstein
Ges, a Human Gtpase of the Rad/Gem/Kir Family, Promotes Endothelial Cell Sprouting and Cytoskeleton Reorganization
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[Abstract]
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A. Vacca, M. Iurlaro, D. Ribatti, M. Minischetti, B. Nico, R. Ria, A. Pellegrino, and F. Dammacco
Antiangiogenesis Is Produced by Nontoxic Doses of Vinblastine
Blood,
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[Abstract]
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A. Rivard, L. Berthou-Soulie, N. Principe, M. Kearney, C. Curry, D. Branellec, G. L. Semenza, and J. M. Isner
Age-dependent Defect in Vascular Endothelial Growth Factor Expression Is Associated with Reduced Hypoxia-inducible Factor 1 Activity
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M. Ogilvie, X. Yu, V. Nicolas-Metral, S. M. Pulido, C. Liu, U. T. Ruegg, and C. T. Noguchi
Erythropoietin Stimulates Proliferation and Interferes with Differentiation of Myoblasts
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[Abstract]
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A.-L. Siren, M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, et al.
Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress
PNAS,
March 27, 2001;
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION