© 1997 American Heart Association, Inc.
Articles |
Basic Fibroblast Growth Factor–Induced Angiogenic Phenotype in Mouse Endothelium
A Study of Aortic and Microvascular Endothelial Cell Lines
From the Unit of General Pathology and Immunology (M.B., E.E.N., P.Dell'E., M.R., M.P.) and Unit of Histology (M.P.M.-T., S.P.), Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy; Laboratory of Developmental Biology (R.A.), Department of Zoology, University of Wisconsin, Madison; Unit of Immunopathology (L.P.R.), Department of Experimental Medicine and Pathology, University La Sapienza, Rome, Italy; and Institute of Biomedical Sciences (L.P.), University of Ancona, Italy.
Correspondence to Marco Presta, General Pathology, Department of Biomedical Sciences and Biotechnology, via Valsabbina 19, 25123 Brescia, Italy. E-mail presta{at}master.cci.unibs.it
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Abstract The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. Yet, to the best of our knowledge, very few reports on the in vitro interaction of the angiogenic basic fibroblast growth factor (bFGF) with mouse endothelial cells are available. Three mouse endothelial cell lines originated from aorta (MAECs), brain capillaries (MBECs), and heart capillaries (MHECs) were characterized for endothelial phenotypic markers, in vivo tumorigenic activity, and the capacity to respond in vitro to bFGF. These cells express angiotensin-converting enzyme, acetylated LDL receptor, constitutive endothelial nitric oxide synthase, and vascular cell adhesion molecule-1 and bind Griffonia simplicifolia-I lectin. When injected subcutaneously in nude mice, MAECs induced the appearance of slow-growing vascular lesions reminiscent of epithelioid hemangioendothelioma, whereas MBEC xenografts grew rapidly, showing Kaposi's sarcoma–like morphological features. No lesions were induced by injection of MHECs. MAECs, MBECs, and MHECs expressed both low-affinity heparan sulfate bFGF-binding sites and high-affinity tyrosine kinase receptors (FGFRs) on their surfaces. In particular, MAECs expressed FGFR-2/bek mRNA, whereas microvascular MBECs and MHECs expressed FGFR-1/flg mRNA. Accordingly, bFGF induced a mitogenic response and the phosphorylation of extracellular signal-regulated kinase-2 in all the cell lines. In contrast, upregulation of urokinase-type plasminogen activator expression was observed in bFGF–treated microvascular MBECs and MHECs but not in MAECs. Also, bFGF–treated MBECs and MHECs but not MAECs invaded a three-dimensional fibrin gel and formed hollow, capillary-like structures. The relevance of the modifications of the fibrinolytic balance of mouse microvascular endothelium in bFGF–induced angiogenesis was validated in vivo by a gelatin-sponge assay in which the plasmin inhibitors tranexamic acid and
-aminocaproic acid given
to mice in the drinking water inhibited neovascularization induced by
the growth factor. In conclusion, differences in response to bFGF exist
between large-vessel MAECs and microvascular MBECs and MHECs. Both in
vitro and in vivo data point to a role of the profibrinolytic phenotype
induced by bFGF in microvascular endothelial cells during mouse
angiogenesis. Our observations make these endothelial cell lines
suitable for further studies on mouse endothelium during angiogenesis
and in angioproliferative diseases.
Key Words: angiogenesis • endothelium • mouse • basic fibroblast growth factor • plasminogen activators
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Introduction |
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Endothelial cell turnover is normally very slow in adults. Physiological exceptions in which angiogenesis occurs under tight regulation are found in the female reproductive system and during wound healing. In contrast, uncontrolled endothelial cell proliferation takes place in several angioproliferative diseases, including diabetic retinopathy, arthritis, and Kaposi's sarcoma, and during tumor neovascularization.1
Angiogenesis is characterized by increased microvessel endothelial cell proliferation, production and/or activation of matrix degradative enzymes, migration in the subendothelial matrix, and differentiation into functional new capillaries.2 The necessity to study in detail the process of angiogenesis has led to the isolation and culture of endothelial cells from various tissues and from several species, including humans. A high degree of heterogeneity has been observed for endothelial cells of different tissue origins and for endothelium isolated from different animal species. In addition, significant differences exist between large-vessel and microvascular endothelium.3 4
Among the first-characterized and extensively studied angiogenesis factors is bFGF, a peptide product of a single-copy gene found in isoforms of molecular weight ranging from 18 to 24 kD.5 bFGF belongs to a family of heparin-binding growth factors that includes nine members of related peptides6 characterized by the capacity to interact with target cells by binding to low-affinity HSPGs and high-affinity FGFRs.7 8 bFGF is involved in different physiological and pathological conditions associated with angiogenesis, such as embryonic development,9 wound healing,10 solid tumor growth,11 and angioproliferative diseases.12 13 When tested in vivo, bFGF induces neovascularization in different experimental models in various animal species, including the rabbit avascular cornea assay,14 the chick embryo chorioallantoic membrane assay,9 the gelatin sponge,15 and Matrigel16 subcutaneous assays in the mouse. Unfortunately, few data are available on the biological effects of bFGF on endothelial cells isolated from these species. In contrast, a considerable amount of work has been performed on cultured endothelial cells of human, bovine, or porcine origin in which bFGF induces an "angiogenic phenotype" manifested as an increase in cell proliferation, migration, and protease production,17 formation of capillary-like structures in three-dimensional gels,18 invasion of the amniotic membrane,19 modulation of integrin expression,20 urokinase-type receptor upregulation,21 and induction of gap-junction intercellular communication.22 Thus, for a better comparison between in vitro and in vivo observations on the angiogenic activity of bFGF, it is necessary to establish endothelial cell cultures from key species used in biomedical research and to assess their modality of response to the growth factor.
The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. For instance, recent observations have shown that bFGF–transfected tumor cells form highly vascularized tumors that grow faster than parental cells when injected subcutaneously in nude mice.11 23 In the present study, we have characterized the response to bFGF of three mouse endothelial cell lines derived from aorta (MAECs), heart capillaries (MHECs), and brain capillaries (MBECs) to allow for a comparison between the capacity of large-vessel endothelium and of microvascular mouse endothelium to respond to a prototypical angiogenic factor. Cultured cells derived from rodents spontaneously escape senescence with significant frequency, originating cell lines with indefinite life spans that may resemble normal cells in most respects.24 25 26 Accordingly, MAEC, MBEC, and MHEC lines were established from BALB/c mice and retained, at least in part, their endothelial features in long-term culture (see below and Reference 2727 ). The results of the present study demonstrate significant differences among MAECs, MBECs, and MHECs concerning their capacity to respond to bFGF. Indeed, a complete profibrinolytic, angiogenic phenotype was expressed only by microvascular endothelial cells. Also, the cell lines investigated showed a different tumorigenic potential when injected in nude mice and gave rise to vascular lesions with different morphological features. Mouse endothelial cell lines appear to be suitable for additional in vitro and in vivo studies on the biological functions and properties of mouse endothelium during angiogenesis and in endothelium-derived lesions.
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Cell Cultures
BALB/c mouse aortic endothelial 22106 cells (MAECs), brain microvascular endothelial 10027 cells (MBECs), and heart microvascular endothelial 14077 cells (MHECs) were grown in DMEM supplemented with 10% FCS. These spontaneously immortalized cell lines were identified as endothelial on the basis of different phenotypic markers (see Reference 2727 and text for further details). They were maintained in culture at the University of Brescia for more than 200 cell-population doublings with no signs of cell senescence after they were received from the University of Wisconsin. The term "immortalized cell line" is used in the present report solely to define the fact that these cells have escaped senescence and divide indefinitely. As observed for mouse NIH 3T3 cells, immortalization does not necessarily imply the expression of other transformation features, such as in vitro focus formation and in vivo tumorigenicity.28
Immunocytochemical Analysis and AcLDL Uptake
Cells were plated on 12-mm glass coverslips coated with 100
µg/mL poly-L-lysine. After 16 hours, medium was removed
and cell cultures were fixed in PBS containing 4% paraformaldehyde and
0.1 mol/L sucrose for 5 minutes at 37°C and for an additional 10
minutes at room temperature. Cells were washed twice with 0.1 mol/L
sucrose in PBS, then twice with PBS, and were incubated for 15 minutes
at room temperature with goat serum diluted 1:100 with PBS containing
0.3% Triton X-100 (GS/PBS/Triton). Cells were then incubated with
factor VIII–related antigen antiserum provided by G. Fontanini
(University of Pisa, Italy). For immunocytochemical studies of
cell-surface markers, cells were stained with TRITC-conjugated
Griffonia (Bandeiraea) simplicifolia-I lectin (Sigma
Chemical Co) or incubated with anti-CD34 antiserum or with a rat
monoclonal anti-murine CD31 antibody (kindly provided by A. Vecchi,
Mario Negri Institute, Milan, Italy). Immunocomplexes were visualized
by exposure of the cells to FITC-conjugated secondary antibody. For
AcLDL uptake, cells were incubated with 10 µg/mL
1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine
perchlorate–labeled LDL (Biomedical Technologies Inc) for 4 hours at
37°C and fixed in 3% formaldehyde. Where relevant, human umbilical
vein endothelial cells were used as positive controls.
Injection in Nude Mice
MAECs, MBECs, and MHECs were dispersed with trypsin-EDTA, washed
twice in cell-culture medium supplemented with 10% FCS, and
resuspended in PBS. Three-week-old nude mice were inoculated
subcutaneously with 4x106 cells (seven to eight animals
per group). Mice were monitored daily, and the size of the lesion was
measured with calipers. When the animals were killed, lesions were
removed, formalin-fixed, paraffin-embedded, and stained with
hematoxylin and eosin. For factor VIII–related antigen immunostaining,
tumors were fixed in 4% paraformaldehyde. After fixation, specimens
were sequentially washed with 10%, 20%, and 30% sucrose in PBS,
embedded in ornithine carbamyl transferase compound, and frozen, and
5-µm serial sections were obtained with a cryostat microtome. Tissue
sections were rinsed in PBS and incubated for 20 minutes with 0.3%
H2O2 in absolute methanol to block endogenous
peroxidase and for 30 minutes with 1% Triton X-100/PBS. Then, a
30-minute preincubation with diluted normal serum was followed by
incubation at 4°C with factor VIII–related antigen antiserum in a
humidified chamber. Sections were then exposed to biotinylated
secondary antibody (Vector Laboratories) and to
avidin-biotin-peroxidase complex (Dako ABComplex HRP) for 30 minutes.
Peroxidase color reaction was developed with 3-amino-9-ethylcarbazole
(Sigma), and the sections were lightly counterstained with Mayer's
hematoxylin.
125I-bFGF Binding Assay
Human recombinant bFGF was purified from transformed
Escherichia coli cell extract and labeled with
125I (37 GBq/mL; Amersham International) using Iodogen
(Pierce Chemical Co) as described previously14 at a
specific radioactivity equal to 600 cpm/fmol. Cells were seeded in
24-well dishes at a density of 80 000/cm2. After 24 hours,
cells were washed three times with cold PBS and incubated for 2 hours
at 4°C in binding medium (serum-free medium containing 0.15%
gelatin, 20 mmol/L HEPES buffer, pH 7.5) in the presence of
increasing concentrations of 125I-bFGF. Then, after a PBS
wash, cells were washed twice with 2 mol/L NaCl in 20 mmol/L HEPES
buffer (pH 7.5) to remove 125I-bFGF bound to low-affinity
HSPGs and twice with 2 mol/L NaCl in 20 mmol/L sodium acetate (pH
4.0) to remove 125I-bFGF bound to high-affinity
FGFRs.29 Nonspecific binding was measured in the presence
of a 100-fold molar excess of unlabeled bFGF and subtracted from all
the values. Data were analyzed by use of the Scatchard plot
procedure.30
Cell Proliferation Assays
Long-term Assay
Cells were seeded in 24-well plates at 5000 per cm2.
The next day, cells were incubated in fresh medium with 10% FCS in the
absence or presence of bFGF. Then, medium with or without the growth
factor was changed every other day. After 3 more days, cells were
trypsinized and counted.
DNA Synthesis Assay
bFGF was tested for the ability to stimulate
3H-thymidine incorporation into the DNA of mouse
endothelial cells. For this purpose, MAEC, MBEC, and MHEC cultures were
incubated for 2 days with 0.4% FCS. Quiescent cells were then
supplemented with 20 ng/mL bFGF. After 16 hours, cells were pulse
labeled with 3H-thymidine (1 µCi/mL) for 6 hours. The
amount of radioactivity incorporated into the trichloroacetic
acid–precipitable material was then measured.
Western Blot Analysis of ERK-2
Cells were grown at subconfluence in 60-mm dishes in DMEM
containing 10% FCS. Cells were then treated for 20 minutes with fresh
medium with or without 20 ng/mL bFGF. Western blot analysis of the cell
extracts was performed exactly as described previously31
using anti–ERK-2 antibodies (kindly provided by Dr Y. Nagamine,
Friedrich Miescher Institute, Basel, Switzerland). Phosphorylation of
ERK-2 was evidenced as a mobility shift on the gel.31
Plasminogen Activator Assay and SDS-PAGE Zymography
Confluent cultures of MAECs, MBECs, and MHECs were incubated in
fresh medium containing 0.4% FCS in the absence or presence of bFGF.
After incubation at 37°C for 18 to 20 hours, cell layers were washed
twice with PBS and PA activity was measured in the cell
extracts14 by using the plasmin chromogenic substrate
H-D-norleucyl-hexahydrotyrosil-lysine-p-nitroanilide-acetate
(American Diagnostica) according to the manufacturer's instructions.
Human uPA (60 000 IU/mg protein, Calbiochem) was used as a standard.
One international unit corresponds to 0.7 Ploug units. In addition,
20-µg aliquots of cell extracts were run on 10% SDS–polyacrylamide
gel under nonreducing conditions. Zymography for the detection of PA
activity was then performed on a casein/agarose gel as described
previously.11
Preparation of Three-dimensional Gels
Fibrin gels were prepared as described
previously.32 Briefly, fibrinogen (2.5 mg/mL) was
dissolved in calcium-free medium. Clotting was then started by addition
of thrombin (250 mU/mL), and the mixture was immediately transferred
into 24 well-plates and allowed to gel for 5 minutes at 37°C.
Endothelial cells were then seeded at 75 000/cm2 onto
fibrin-coated dishes. Culture medium with or without 30 ng/mL bFGF was
renewed every 48 hours.
Processing for Light Microscopy
Fibrin gels were fixed for 2 hours with 2.5% glutaraldehyde/1%
tannic acid in 0.1 mol/L sodium cacodylate buffer, pH 7.4. After
extensive washing, gels were postfixed in 1% osmium tetroxide,
dehydrated through a graded series of ethanol, stained en bloc with
0.3% uranyl acetate in 100% ethanol, treated with propylene oxide,
and embedded in epoxy resin 812.33 Samples were then
trimmed to 3x3-mm block faces. Semithin (1 µm) sections were
cut perpendicularly to the culture plane and stained with 1% toluidine
blue.
Northern Blot Analysis
Northern blot analysis of total RNA (20 µg/sample) was
performed according to standard procedures.34 Uniform
loading of the gels was assessed by methylene blue staining of the
filter. FGFR-1 and FGFR-2 probes were kindly provided by A. Mansukhani
(New York University, NY), FGFR-3 and FGFR-4 probes by J. Partanen
(University of Helsinki, Finland), uPA probe by P. Mignatti (University
of Pavia, Italy), and constitutive endothelial nitric oxide synthase
probe by I. Suzuki (University of Verona, Italy).
Angiogenesis Assay
The angiogenic activity of bFGF was evaluated in a mouse
gelatin-sponge assay.15 Gelatin sponges (Gelfoam, Upjohn)
were adsorbed with 300 µL of PBS containing 30 µg of the growth
factor. Each sponge was then implanted subcutaneously in the dorsal
region of a female C3H mouse (Charles River, Italy). Animals were then
grouped at random in three cages (three to four animals per group) and
were given tranexamic acid or -aminocaproic acid (both at 50 mg/mL)
or vehicle in the drinking water. Animals were killed after 14 days.
Sponges were dissected free of adherent tissue, fixed in formalin, and
processed for immunohistochemical analysis using anti-laminin antibody
(HEYL) for the detection of blood vessels. Sections were then exposed
to biotinylated secondary antibody and to avidin-biotin-peroxidase
complex for 30 minutes. Peroxidase color reaction was developed with
3-amino-9-ethylcarbazole, and the sections were lightly counterstained
with Mayer's hematoxylin.
To assess microvessel density, the whole surface area of each section was examined at x400 magnification, and the number of laminin-positive blood microvessels per field was counted. Vascular counts for individual sections were then produced using the mean of all the field counts. In addition, the Magiscan Image Analysis System (Joyce Loebl) was used to perform a quantitative evaluation of the laminin-positive areas in each section. Briefly, the section image was input via a TV camera mounted on a Nikon light microscope, digitized on the high-resolution monitor, and stored within the Magiscan's image memory. The ratio between the integrated density values of the laminin-positive area and of the selected surface was determined for each section, and the angiogenesis response was then expressed as relative units.
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General Characteristics of MAECs, MBECs, and MHECs
MAECs, MBECs, and MHECs were isolated from aorta and from microvessels from brain and heart of BALB/c mice, respectively, and maintained in long-term culture.27 They had been identified as endothelial cells on the basis of different criteria, including cobblestone morphology, expression of AcLDL receptor and angiotensin-converting enzyme, and lack of expression of Mac-1 and Ly-5.27
Several years after isolation, these cells retain cobblestone
morphology (Fig 1
) and the capacity to take up AcLDL
(Fig 2A). We have also characterized these cell lines
for the expression of other phenotypic markers by immunocytochemistry
and Northern blot analysis. Immunocytochemical analysis demonstrates
that MAECs, MBECs, and MHECs do not express significant amounts of
factor VIII–related antigen or the cell-surface adhesion molecules
CD31/PECAM and CD34 (data not shown). However, they retain the capacity
to bind Griffnia simplicifolia-I agglutinin, a mouse
endothelial cell marker35 (Fig 2B), and to express
constitutive endothelial nitric oxide synthase mRNA36 (Fig 2C). They also constitutively express vascular cell adhesion molecule-1
on the cell surface (data not shown).
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When grown on tissue-culture plastic in the presence of 10% FCS
without any growth supplement, MAECs proliferate more rapidly than
MHECs and MBECs. Doubling time was 
24 hours for MAECs compared with
48 hours for MHECs and MBECs. In addition, MAECs reach a density at
confluence (1.7x105 cells/cm2) higher than
MBECs and MHECs that are growth arrested at 0.9x105 and
0.6x105 cells/cm2, respectively (Fig 3A). It should be noted that MAECs, MBECs, and MHECs do
not express significant amounts of bFGF as assessed by Northern
blotting, Western blot analysis of the cell extracts, and
immunocytochemistry (data not shown), thus ruling out the possibility
of an autocrine mitogenic role for endogenous bFGF in these cell
lines.
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, MAECs; 
,
MBECs; and 
, MHECs.
MAECs, MBECs, and MHECs were evaluated for their behavior when injected
subcutaneously in nude mice (Fig 3B
). Within 1 week after injection,
all mice inoculated with MBECs (n=8) developed lesions that reached an
average size of 4.0 cm3 at 8 weeks (range, 0.7 to 9.0
cm3). In contrast, lesions induced by MAECs developed much
more slowly in four of seven animals, being just palpable 1 week after
injection and reaching an average size of 0.5 cm3 at 25
weeks (range, 0.2 to 1.5 cm3). Inoculation of MHECs did not
result in the formation of lesions.
The histology of MAEC and MBEC xenografts was markedly different.
MBEC-induced lesions were highly monomorphic and consisted of
spindle-shaped cells arranged in a storiform pattern (Fig 4A). Numerous slitlike spaces reminiscent of Kaposi's
sarcoma were present (Fig 4B). Tumor cells showed oval nuclei with
multiple nucleoli (Fig 4C) and were highly proliferative, as
demonstrated by the presence of mitotic figures. In contrast, the
histology of MAEC xenografts was similar to that of an epithelioid
hemangioendothelioma, a vascular tumor of low-grade malignancy. Indeed,
MAEC-induced lesions were made of short strands or solid nests of
rounded epithelioid cells, and clear evidence of vascular
differentiation was provided by the presence of canalized vascular
channels (Fig 4D). Neoplastic cells showed round nuclei with frequent
lobations and indentations and a prominent nucleolus (Fig 4E). Mitotic
figures were less numerous than in MBEC lesions.
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Immunostaining of the tissue slides for factor VIII–related antigen
showed positivity of newly formed blood vessels in both MBEC- and
MAEC-induced lesions (Fig 4F). Because this antibody does not react
with MBECs and MAECs in vitro (see above), immunostaining reflects the
presence of endothelial cells of the host within the lesion, thus
indicating the capacity of MBECs and MAECs to recruit endothelial cells
of the host.
bFGF-Receptor Expression
bFGF interacts with the endothelial cell surface by binding to two
distinct classes of receptors: low-affinity sites, represented by
cell-surface HSPGs, and high-affinity tyrosine-kinase
FGFRs.7 8 We evaluated MAECs, MBECs, and MHECs for their
ability to bind 125I-bFGF (Fig 5A and 5B).
Scatchard plot analysis of the binding data revealed the presence of
both low- and high-affinity sites in all three endothelial cell lines.
MHECs bound 125I-bFGF on HSPGs less efficiently than did
MAECs and MBECs. This was due to differences in the number of
low-affinity sites, which was three to four times lower for MHECs than
for MAECs and MBECs, rather than a difference in affinity for the
growth factor (Table). In contrast, the three cell types
bound 125I-bFGF to FGFRs with similar capacity. Indeed,
when compared with MAECs and MBECs, the reduced affinity of FGFRs for
125I-bFGF in MHECs was balanced by a higher number of
receptors.
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) were plated at 80 000 cells/cm2. After 24 hours,
cells were incubated for 2 hours at 4°C with increasing
concentrations of 125I-bFGF, and binding to high-affinity
sites (A) and low-affinity sites (B) was evaluated. Scatchard plot
analysis of the binding data is shown in the insets. C, Total RNA
isolated from murine endothelial cells was probed with FGFR-1 and
FGFR-2 cDNAs in Northern blot. Uniform loading of the samples was
checked by ethidium bromide staining of the gel. A digitized image of
stained 18S RNA is shown in the lower panel.
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FGFRs represent a family of structurally related tyrosine-kinase
receptors. The four members of the FGFR family are encoded by distinct
genes and differ in terms of ligand specificity.8 On this
basis, total RNA extracted from MAECs, MBECs, and MHECs was hybridized
with specific probes for FGFR-1/flg, FGFR-2/bek,
FGFR-3, and FGFR-4 in a Northern blot. The results indicated that MBECs
and MHECs expressed significant amounts of FGFR-1 mRNA, whereas MAECs
expressed FGFR-2 mRNA only (Fig 5C). A faint band corresponding to
FGFR-2 mRNA was also detectable in MHECs. No FGFR-3 or FGFR-4 mRNAs
were detectable in any cell type (data not shown).
Mitogenic Response to bFGF
bFGF induces the proliferation of endothelial cells from different
species.6 Accordingly, human recombinant bFGF stimulated
the proliferation of MBECs and MHECs grown on tissue-culture plastic in
the presence of 10% FCS in a long-term assay. The effect was dose
dependent with an ED50 of 2 and 5 ng/mL for MBECs and
MHECs, respectively. At 10 to 30 ng/mL, bFGF caused twofold and
threefold increases in MHEC and MBEC number, respectively. In contrast,
no significant increase in cell number was observed for bFGF–treated
MAECs compared with untreated cells (Fig 6A). The
apparent lack of response of MAECs to the mitogenic stimulation of bFGF
was probably due to the high rate of proliferation of these cells under
basal growth conditions in 10% FCS (Fig 3A), cell density in control
bFGF–untreated cultures being equal to 90 000 cells/cm2
for MAECs versus 12 000 cells/cm2 and 30 000
cells/cm2 for MBECs and MHECs, respectively. Indeed, when
cells were starved for 48 hours in medium containing 0.4% FCS, a
significant increase in 3H-thymidine incorporation was
observed in all cell types 16 hours after addition of 20 ng/mL bFGF to
the culture medium, even though the stimulation of DNA synthesis
induced by the growth factor was limited compared with that induced by
treatment with 10% FCS (Fig 6B).
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To confirm these data, we evaluated the capacity of bFGF to activate an
FGFR-dependent intracellular signaling pathway in MAECs, MBECs, and
MHECs. For this purpose, we assessed the capacity of bFGF to cause the
phosphorylation of ERK-2, a key molecule in the signal transduction
Ras/Raf-1/MEK/ERK-2 pathway switched on by the growth factor in
responsive cells.31 As shown in Fig 6B (inset), bFGF
caused the rapid phosphorylation of ERK-2 in all cell lines, detected
by Western blot analysis as a mobility shift in
SDS-PAGE.31
uPA Upregulation Induced by bFGF
bFGF upregulates uPA in endothelial cells of bovine
origin14 17 but not of human origin (M. Presta,
unpublished observations, 1995). Mouse endothelial cells were therefore
evaluated for the capacity to increase uPA production in response to
bFGF. Under standard culture conditions, cell-associated PA activity
was equal to 42 mU, 11 mU, and 350 mU per milligram of protein for
MAECs, MBECs, and MHECs, respectively. As shown in Fig 7A, bFGF induced an increase in cell-associated PA
activity in MBECs and MHECs but not in MAECs. The ED50 was
lower for MBECs than for MHECs (2 versus 5 ng/mL, respectively). PA
activity was identified as uPA by SDS-PAGE of the cell extracts
followed by casein-zymography of the gel in which a clear induction of
50 kD uPA was observed after a 24-hour treatment with 30 ng/mL of bFGF
in MHECs and MBECs but not in MAECs (Fig 7B). Also, an increase in the
steady-state levels of uPA mRNA was observed for bFGF–treated MBECs
and MHECs but not for bFGF–treated MAECs (Fig 7A, inset). Similar
results were obtained when mouse endothelial cells were treated with
two other members of the bFGF family. Indeed, 10 ng/mL acidic
bFGF/bFGF-1 or 30 ng/mL K-FGF/FGF-4, both in the presence of 10 µg/mL
heparin, induced uPA activity in MBECs and MHECs similar to bFGF but
were ineffective in MAECs. Interestingly, the treatment with 100 ng/mL
of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, a
typical uPA-inducer in various cell types including bovine endothelial
cells,37 also did not elicit a significant increase in uPA
activity in MAECs, MBECs, or MHECs (data not shown).
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Induction of Three-dimensional Gel Invasion by bFGF
Invasion of endothelial cells in the pericellular tissue is one of
the first steps during angiogenesis.2 Accordingly,
endothelial cells invade fibrin matrix when stimulated in vitro by
angiogenic factors.32 uPA is involved in fibrin
degradation by converting plasminogen to plasmin, a key enzyme involved
in extracellular matrix degradation.38 The differential
effect of bFGF on the three mouse endothelial cell lines regarding uPA
upregulation prompted us to examine the capacity of bFGF–treated
MAECs, MBECs, and MHECs to invade a three-dimensional fibrin gel.
Phase-contrast microscopy (Fig 8) and semithin sections
(Fig 9) demonstrated that all cell lines formed a
confluent monolayer of flattened endothelial cells when cultivated on
fibrin gels under basal conditions. In the presence of 30 ng/mL bFGF,
MHECs and MBECs invaded fibrin and extended long processes under the
surface of the monolayer, penetrating in the three-dimensional mesh
(Fig 8). Interestingly, capillary-like structures containing a lumen
were observed after long-term culture in the presence of bFGF (Fig 9).
As anticipated, bFGF–treated MAECs did not invade the fibrin gel (Fig 8). Thus, only endothelial cells of microvascular origin invaded and
underwent morphogenesis within three-dimensional fibrin gel when
stimulated by bFGF.
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Inhibition of bFGF–Induced Angiogenesis by Plasmin
Inhibitors
Our data indicate that bFGF is able to induce an angiogenic
phenotype based on enhanced uPA activity and invasive capacity in
cultured mouse microvessel endothelial cells. To assess the
contribution of the profibrinolytic phenotype acquired by microvascular
endothelium to the angiogenesis process, the plasmin inhibitors
tranexamic acid and -aminocaproic acid39 were tested
for their capacity to affect bFGF–induced neovascularization in a
mouse gelatin-sponge assay.15 For this purpose, bFGF was
adsorbed in sponges that were implanted subcutaneously in the dorsal
region of female mice. Animals were then treated with 50 mg/mL
tranexamic acid, -aminocaproic acid, or vehicle added to the
drinking water, according to previously described
protocols.40 Previous experiments15 have
shown that an intense blood vessel infiltration of the bFGF–treated
sponges could be detected 2 weeks after implantation under the
experimental conditions adopted in the present study. Thus, animals
were killed after 14 days, and angiogenesis was visualized by
immunostaining of sponge sections with an anti-laminin antibody to
highlight newly formed blood vessels (Fig 10A). For all
sections, neovascularization was quantified both by counting the number
of blood vessels per microscopic field and by computerized image
analysis of the laminin-positive areas. As shown in Fig 10B, bFGF
caused a significant angiogenic response in all the animals.
bFGF-2–induced neovascularization was reduced by 50% in mice
treated with the plasmin inhibitors tranexamic acid or -aminocaproic
acid, as evaluated both by blood vessel counting and computerized image
analysis.
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Mice are the most commonly used experimental animals for a variety of studies in biomedical research, including tumor growth and angiogenesis. Yet few reports on the biological features of cultured murine endothelium and its interaction with angiogenesis factors are available. This appears to be a serious limitation for studies aimed at elucidating the mechanisms of angiogenesis, in which in vivo data are frequently compared with in vitro observations obtained with endothelium originating from different animal species and of large-vessel origin. A typical example is the comparison of the results obtained in chick embryo chorioallantoic membrane and/or rabbit cornea angiogenesis assays with observations on cultured endothelium isolated from bovine aorta and/or human umbilical vein. These limitations are emphasized by the high degree of heterogeneity observed in endothelial cells originating from different tissues, vessels of different caliber, or different animal species.3 4
In the present study, we investigated three murine endothelial cell lines derived from large blood vessel (MAECs) or capillaries (MBECs and MHECs). These cells were originally established as long-term cultures and identified as endothelial cells on the basis of various phenotypic markers.27 We have confirmed the previous phenotypic characterization and observed that these cells also express other endothelial markers, including endothelial nitric oxide synthase, vascular cell adhesion molecule-1, and binding to Griffonia simplicifolia-I lectin, even though they have lost expression of factor VIII–related antigen, CD31, and CD34. It is also interesting to note that MBECs and MHECs seeded on three-dimensional gels, like endothelial cells of different origins,18 retained the capacity to form hollow, capillary-like structures when stimulated by bFGF. Thus, we believe that these cells can be described as bona fide immortalized cells of endothelial origin. The main purpose of the present study was to determine whether they can provide a useful tool for investigating some aspects of angiogenesis, in particular the response of mouse endothelium to bFGF.
The results indicate that cultured macrovascular and microvascular mouse endothelial cells respond differently to bFGF. bFGF induces the activation of ERK-2, a key step in the signal-transduction pathway activated by bFGF in target cells,31 and exerts a mitogenic response in all the cell lines. However, bFGF induces uPA upregulation only in microvascular MBECs and MHECs. Also, MBECs and MHECs invade fibrin and organize themselves in capillary-like structures when exposed to bFGF, whereas MAECs are unable to invade or to undergo morphogenesis within a three-dimensional fibrin gel. The different angiogenic responses of murine microvascular versus macrovascular endothelial cells to bFGF is consistent with the observation that new blood vessels arise primarily from the microvasculature, which suggests that endothelial cells of microvascular origin react to angiogenic factors with a full complement of responses. This is consistent with previous observations indicating that microvascular endothelium is more prone than large-vessel endothelium to form tubelike structures when seeded on extracellular matrix preparations and to respond to certain cytokines (discussed in Reference 44 ). Also, angiogenic stimuli have been shown to increase collagenase production in cultured bovine endothelial cells isolated from microvessels but not in bovine endothelial cells of large-vessel origin, even though uPA upregulation was observed in both endothelial cell types.41 However, at present we cannot rule out the possibility that the different response of the three mouse endothelial cell lines to bFGF may reflect, at least in part, alterations consequent to their in vitro immortalization.
In accordance with our in vitro data, the results obtained in vivo
using the mouse gelatin-sponge assay confirm the central role that the
fibrinolytic system plays during angiogenesis, as underlined by the
ability of the plasmin inhibitors tranexamic acid and -aminocaproic
acid to inhibit bFGF–induced neovascularization. Previous observations
had shown that plasmin and uPA inhibitors affect angiogenesis in vitro
and in vivo in different experimental models, including bFGF–induced
bovine endothelial cell invasion of the human amniotic
membrane,19 bovine endothelial cell organization on
collagen gel,42 and neovascularization of rabbit
cornea.43 44 Taken together, the data support the
hypothesis that the reduced rate of growth of experimental tumors in
mice caused by antifibrinolytic drug treatment may depend on a reduced
vascularization of the tumor.45 46 47
The inability of bFGF, bFGF-1, and bFGF-4 to induce uPA production in MAECs is of interest. MAECs express FGFR-2, whereas MBECs and MHECs express FGFR-1. The differential expression of FGFR types contributes to the heterogeneity observed between large-vessel and microvascular mouse endothelium. However, it may not be sufficient per se to explain the lack of response of MAECs to bFGF regarding uPA upregulation. Indeed, both FGFR-1 and FGFR-2 are able to transduce a uPA-inducing signal in CHO transfectants treated with bFGF,48 indicating that the lack of response of MAECs is not due to an intrinsic incapacity of FGFR-2 to activate the intracellular machinery required for uPA upregulation. MAECs respond instead to bFGF by increasing DNA synthesis. Accordingly, ERK-2 activation is observed after short-term treatment with bFGF. Recent observations have shown that the activation of the signal-transduction Ras/Raf-1/MEK/ERK-2 pathway is responsible for the uPA-inducing activity of bFGF in mouse NIH 3T3 fibroblasts.31 The results in MAECs indicate that ERK-2 activation is not sufficient per se to upregulate uPA in bFGF–responsive cells. It is also possible that the signal-transduction pathway responsible for uPA induction in mouse endothelial cells differs from that characterized in mouse fibroblasts. This is suggested by the observation that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, a potent uPA inducer in 3T3 cells,31 is ineffective in all mouse endothelial cell lines investigated. Further experiments are required to clarify this point.
The interaction of bFGF with FGFRs is modulated by low-affinity
HSPGs.49 50 When the binding of bFGF to low-affinity sites
is prevented by treating the cells with heparinase or chlorate, the
binding of bFGF to FGFRs is reduced together with its capacity to
stimulate cell proliferation.51 In addition, heparin
facilitates the formation of the bFGF/FGFR complex52 and
induces oligomerization of the growth factor, thus facilitating FGFR
dimerization.53 MHECs express a lesser number of
low-affinity sites on their surface than MBECs. In agreement with the
role of HSPGs in receptor binding and biological activity of bFGF, the
affinity of FGFR-1 for 125I-bFGF is lower and the minimal
effective dose and potency of bFGF in inducing cell proliferation and
uPA upregulation are higher in MHECs than in MBECs. Thus, the
heterogeneity of the expression of endothelial cell–surface HSPGs may
play an important role in modulating the angiogenic activity of bFGF as
well as of other heparin-binding angiogenic factors such as vascular
endothelial growth factor,54 hepatocyte growth
factor/scatter factor,55 and the HIV-1 transactivating
factor Tat.56 This, in conjunction with differences in
FGFR expression and signal-transduction machinery, may reflect a
possible tissue-dependent specificity or sensitivity of microvascular
endothelial cells to growth factor action. This hypothesis is in
agreement with previous observations indicating that the microvascular
response to different cytokines, including epidermal growth factor,
interleukin 2, platelet-derived growth factor, and transforming growth
factor-
, strictly depends on the tissue origin of the endothelium
(see Reference 44 and references therein).
Finally, we have observed that MAECs and MBECs but not MHECs induce angioproliferative lesions when injected in nude mice. MAECs form slow-growing lesions resembling epithelioid hemangioendothelioma, whereas MBECs give rise to fast-growing lesions with histological features similar to Kaposi's sarcoma. Both xenografts are able to recruit the endothelium of the host, a capacity shared by murine endothelioma cells expressing the polyoma middle T oncogene.57 58 Interestingly, we have found that MAECs transfected with bFGF cDNA induce lesions in nude mice characteristic of early-stage Kaposi's sarcoma and composed largely of recruited host cells, including endothelial cells.59 These lesions, unlike those originated by parental MAECs, resemble those induced by MBECs. Because MBECs do not express significant levels of bFGF,60 our results indicate that bFGF expression is not an absolute requirement for the induction of angioproliferative Kaposi's sarcoma–like lesions in nude mice.
Stable murine endothelial cell lines may represent a useful tool in angiogenesis research to achieve reproducibility and to avoid the variability of endothelial cell characteristics usually observed in primary cultures. MAECs, MBECs, and MHECs appear suitable for further studies on mouse endothelium during angiogenesis and in angioproliferative diseases.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from C.N.R. (Progetto Finalizzato Biotecnologie e Biostrumentazioni, Sottoprogetto Biofarmaci, and grants No. 94,00316,CT14 and No. 95,02925,CT14 to Dr Presta; No. 95,02983,CT14 to Dr Rusnati; No. 95,02880,CT14 to Dr Dell'Era), from Associazione Italiana per la Ricerca sul Cancro and from the AIDS Project to Dr Presta, and from European Communities (Human Capital Mobility Project "Mechanisms for the Regulation of Angiogenesis") to Drs Presta and Bastaki.
Received April 16, 1996; accepted July 15, 1996.
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References |
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D. Qiao, K. Meyer, C. Mundhenke, S. A. Drew, and A. Friedl Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Signaling in Brain Endothelial Cells. SPECIFIC ROLE FOR GLYPICAN-1 IN GLIOMA ANGIOGENESIS J. Biol. Chem., April 25, 2003; 278(18): 16045 - 16053. [Abstract] [Full Text] [PDF]
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 S. Zhang and G. Magnusson Cellular Mobile Genetic Elements in the Regulatory Region of the Pneumotropic Mouse Polyomavirus Genome: Structure and Function in Viral Gene Expression and DNA Replication J. Virol., March 15, 2003; 77(6): 3477 - 3486. [Abstract] [Full Text] [PDF]
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 B. Troyanovsky, T. Levchenko, G. Mansson, O. Matvijenko, and L. Holmgren Angiomotin: An Angiostatin Binding Protein That Regulates Endothelial Cell Migration and Tube Formation J. Cell Biol., March 19, 2001; 152(6): 1247 - 1254. [Abstract] [Full Text] [PDF]
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L. Moldovan, N. I. Moldovan, R. H. Sohn, S. A. Parikh, and P. J. Goldschmidt-Clermont Redox Changes of Cultured Endothelial Cells and Actin Dynamics Circ. Res., March 17, 2000; 86(5): 549 - 557. [Abstract] [Full Text] [PDF]
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S. Liekens, D. Leali, J. Neyts, R. Esnouf, M. Rusnati, P. Dell'Era, P. C. Maudgal, E. De Clercq, and M. Presta Modulation of Fibroblast Growth Factor-2 Receptor Binding, Signaling, and Mitogenic Activity by Heparin-Mimicking Polysulfonated Compounds Mol. Pharmacol., July 1, 1999; 56(1): 204 - 213. [Abstract] [Full Text]
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M. Presta, M. Rusnati, M. Belleri, L. Morbidelli, M. Ziche, and D. Ribatti Purine Analogue 6-Methylmercaptopurine Riboside Inhibits Early and Late Phases of the Angiogenesis Process Cancer Res., May 1, 1999; 59(10): 2417 - 2424. [Abstract] [Full Text] [PDF]
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M. Rusnati, E. Tanghetti, C. Urbinati, G. Tulipano, S. Marchesini, M. Ziche, and M. Presta Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures Mol. Biol. Cell, February 1, 1999; 10(2): 313 - 327. [Abstract] [Full Text]
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R Giuliani, M Bastaki, D Coltrini, and M Presta Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2 J. Cell Sci., January 8, 1999; 112(15): 2597 - 2606. [Abstract] [PDF]
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P. Dell'Era, M. Mohammadi, and M. Presta Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis Mol. Biol. Cell, January 1, 1999; 10(1): 23 - 33. [Abstract] [Full Text]
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M. Presta, M. Belleri, A. Vecchi, J. Hesselgesser, A. Mantovani, and R. Horuk Noncompetitive, Chemokine-mediated Inhibition of Basic Fibroblast Growth Factor-induced Endothelial Cell Proliferation J. Biol. Chem., April 3, 1998; 273(14): 7911 - 7919. [Abstract] [Full Text] [PDF]
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L. Goretzki, C. R. Lombardo, and W. B. Stallcup Binding of the NG2 Proteoglycan to Kringle Domains Modulates the Functional Properties of Angiostatin and Plasmin(ogen) J. Biol. Chem., September 8, 2000; 275(37): 28625 - 28633. [Abstract] [Full Text] [PDF]
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B. L. Allen, M. S. Filla, and A. C. Rapraeger Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition J. Cell Biol., November 26, 2001; 155(5): 845 - 858. [Abstract] [Full Text] [PDF]
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R. J. Tomanek, A. Sandra, W. Zheng, T. Brock, R. J. Bjercke, and J. S. Holifield Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Differentially Modulate Early Postnatal Coronary Angiogenesis Circ. Res., June 8, 2001; 88(11): 1135 - 1141. [Abstract] [Full Text] [PDF]
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