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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 927-938
Effect of Steroid Hormones and Retinoids on the Formation of
Capillary-Like Tubular Structures of Human Microvascular Endothelial
Cells in Fibrin Matrices Is Related to Urokinase Expression
By
Mirian Lansink,
Pieter Koolwijk,
Victor van Hinsbergh, and
Teake Kooistra
From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands.
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ABSTRACT |
Angiogenesis, the formation of new capillary blood vessels, is a
feature of a variety of pathological processes. To study the effects of
a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor- . This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and
tumor stroma. Our results show that testosterone and dexamethasone are
strong inhibitors and all-trans retinoic acid (at-RA)
and 9-cis retinoic acid (9-cis RA) are potent
stimulators of the formation of capillary-like tubular structures.
These effects are mediated by their respective nuclear hormone
receptors as demonstrated by the use of specific synthetic receptor
agonists and antagonists. 17 -estradiol, progesterone, and
1,25-dihydroxyvitamin D3 did not affect or only weakly affected in
vitro angiogenesis, which may be related to the lack of significant
nuclear receptor expression. Although hMVEC express both thyroid
hormone receptors and , no effect of thyroid hormone on tube
formation was found. The effects of testosterone, dexamethasone,
at-RA, and 9-cis RA on tube formation were accompanied
by parallel changes in urokinase-type plasminogen activator (u-PA)
expression, at both mRNA and antigen levels. Exogenous suppletion of
the medium with single chain u-PA enhances tube formation in our in
vitro model, whereas quenching of u-PA activity (but not of tissue-type
plasminogen activator activity) or of u-PA binding to u-PA receptor by
specific antibodies suppressed basal and retinoid-stimulated tube
formation. Moreover, addition of scu-PA to testosterone- or
dexamethasone-treated hMVEC restored the suppressed angiogenic activity
for a substantial part. Aprotinin, an inhibitor of plasmin activity,
completely inhibited tube formation, indicating that the proteolytic
properties of the u-PA/u-PA receptor complex are crucial in this
process. Our results show that steroid hormones (testosterone and
dexamethasone) and retinoids have strong, but opposite effects on tube
formation in a human in vitro model reflecting pathological
angiogenesis in the presence of fibrin and inflammatory mediators.
These effects can be explained by hormone-receptor-mediated changes in
u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ANGIOGENESIS is the formation of new
capillary blood vessels by a process of sprouting from existing
microvascular vessels. It has a role during development and in the
normal physiology of reproduction, formation of collaterals, and wound
healing, but is also important under pathological conditions, where it contributes to the pathogenesis of a number of diseases such as diabetic retinopathy, tumor growth, and rheumatoid
arthritis.1-5 In each system, the formation of new
capillaries involves a series of discrete, but overlapping events,
including (1) localized degradation of the endothelial cell basal
lamina; (2) endothelial cell migration and extracellular matrix
invasion; (3) endothelial cell proliferation; and (4) formation of
capillary lumina and reconstitution of the basal lamina.6,7
Given the (patho)physiological importance of angiogenesis, it is of
clinical relevance to identify factors that either stimulate or inhibit
those processes and to elucidate their mode of action.
Several reports have pointed to an effect of steroid hormones and
retinoids (retinoic acid derivatives) on angiogenic activity. However,
results have been conflicting, and both stimulatory and inhibitory
activities of these compounds have been found.8-17 It is
not clear at present, whether the variation in response to hormones
relates to differences in assay systems, species, the parameter
measured, or hormone concentration and metabolism. Many studies have
been performed in nonhuman angiogenesis models and focused on
individual components of the angiogenic process (for instance,
proliferation or migration), rather than on the complete response of
capillary tube formation. To gain more insight into the effect of
hormones on angiogenesis in humans, we have developed an in vitro
model, in which human foreskin microvascular endothelial cells
(hMVEC) can be induced to invade a three-dimensional fibrin
matrix thereby forming capillary-like tubular structures, which
can be quantified by computer-assisted video
analysis.18 In this in vitro model, both a growth factor
(basic fibroblast growth factor [bFGF] or vascular endothelial cell
growth factor) and a factor to induce urokinase-type plasminogen
activator (u-PA), for example tumor necrosis factor- (TNF-), are
necessary to induce capillary-like tubular structures. This model
mimics the in vivo situation where fibrin appears to be a common
component of the matrix present at sites of chronic inflammation and
tumor stroma.19 Electron microscopy of cross sections
showed that the capillary-like tubular structures have a lumen and are
very much like capillary structures in vivo.18
Tube-formation in this model was shown to be dependent on u-PA activity
and plasmin activity, which are thought to play a role in the
regulation of the first steps of angiogenesis.
We have used this in vitro model to examine the angiogenesis-modulating
activity of a group of hormones acting via binding to a specific class
of nuclear receptors, the steroid/retinoid/thyroid hormone receptor
superfamily.20 Our results show both blocking (testosterone
and dexamethasone) and stimulating (all-trans and 9-cis
retinoic acid) activities of the hormones tested. In all cases,
hormone-induced changes in tube formation are paralleled and can be
mimicked by corresponding changes in u-PA levels. Our findings provide
evidence for an important role for hormones and their respective
nuclear receptors in the formation of tube-like structures by
influencing the expression of u-PA and thereby the local proteolytic
capacity of the endothelial cell.
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MATERIALS AND METHODS |
Materials.
17-estradiol, 2-methoxyestradiol, progesterone, testosterone,
dexamethasone, 3,3,5-triiodo-l-thyronine (T3), all-trans
retinoic acid (at-RA), RU486 (mifepristone), dimethyl sulfoxide
(DMSO), and charcoal-stripped, delipidated bovine calf serum were
purchased from Sigma (St Louis, MO). R1881 (methyltrienolone) was
obtained from NEN (Boston, MA). 1,25-Dihydroxyvitamin D3 (Ro 21-5535)
was kindly provided by Dr M.R. Uskokovi (Hoffmann-LaRoche,
Nutley, NJ) and 9-cis retinoic acid (9-cis RA) and the
retinoic acid receptor (RAR) antagonist, Ro 41-5253, were kindly
provided by Drs M. Klaus and C. Apfel (Hoffmann-LaRoche, Basel,
Switzerland). Hydroxyflutamide was a gift from Dr T. Lavecchia
(Schering-Plough, Kenilworth, NJ).
4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid (3-methyl-TTNEB) and
(E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl-1-propenyl] benzoic acid (TTNPB) were kindly provided by Dr S. Karathanasis, American Cyanamid Company (Pearl River, NY) and by Dr M. Issandou, Glaxo Wellcome (Les Ulis, France), respectively. Stock solutions of
hormones (10 mmol/L) were prepared in DMSO and stored at
 20°C. Stock solutions were diluted with incubation medium to
the final test concentrations immediately before the start of an
experiment. All experiments involving retinoids, R1881, and
hydroxyflutamide were performed in subdued light, and the tubes
containing these test compounds were covered with aluminium foil. bFGF
was obtained from Intergen (Purchase, NY), thrombin from Leo
Pharmaceutical Products (Weesp, The Netherlands), and human fibrinogen
from Chromogenix AB (Mölndal, Sweden). Factor XIII was kindly
provided by Dr H. Keuper (Centeon Pharma, Marburg, Germany). Human
recombinant TNF- was a gift from Dr J. Travernier (Biogent, Ghent,
Belgium), and contained 2.45 × 107 U/mg protein and
<40 ng lipopolysaccharide per µg protein. Single chain u-PA was
generously provided by Dr A. Molinari (Farmitalia, Milan, Italy). The
amino terminal fragment of u-PA (ATF, amino acids 1-143) was provided
by Abbott (Abbott Park, IL). Aprotinin was purchased from Pentapharm
Ltd (Basel, Switzerland). Rabbit polyclonal anti-u-PA antibodies and
rabbit polyclonal anti-t-PA antibodies were prepared in our
laboratory. The u-PA receptor (u-PAR) blocking monoclonal antibody H-2
was a gift from Dr U. Weidle (Boehringer-Mannheim, Penzberg, Germany).
Technovit 8100 was obtained from Heraeus Kulzer (Wehrheim, Germany).
Enzyme-linked immunosorbent assay (ELISA) kits for determination of
t-PA antigen (Thrombonostika t-PA) and PAI-1 antigen
(Imulyse) were obtained from Organon Teknika (Boxtel, The
Netherlands) and Biopool (Umeå, Sweden), respectively. Deoxycytidine
5[-32P] triphosphate (dCTP) was from Amersham
(Buckinghamshire, UK). Oligonucleotides used for reverse
transcriptase-polymerase chain reaction (RT-PCR) were purchased from
Isogen Bioscience (Maarssen, The Netherlands). Other materials used
have been specified in the methods described or in the related
references.
Cell culture.
hMVEC were isolated, cultured, and characterized as previously
described.21 The cells were cultured in gelatin-coated
dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20 mmol/L HEPES (pH, 7.3), 10% (vol/vol) human serum, 10% (vol/vol) heat-inactivated newborn calf serum (NBCS), 150 µg/mL crude
endothelial growth factor,22 5 IU/mL heparin, 2 mmol/L
L-glutamine, 100 IU/mL penicillin, and 100 µg/mL
streptomycin at 37°C in a 5% CO2 atmosphere.
Subcultures were obtained by trypsin/EDTA treatment of confluent
monolayers at a split ratio of 1:3.
In vitro angiogenesis model.
Human fibrin matrices were prepared by addition of 0.1 U/mL thrombin to
a mixture of 2.5 U factor XIII, 2 mg fibrinogen, 2 mg Na-citrate, 0.8 mg NaCl, and 3 µg plasminogen per mL DMEM medium without indicator. A
total of 300 µL of this mixture was added to 1 cm2 wells.
After clotting at room temperature, the fibrin matrices were soaked
with 0.5 mL DMEM supplemented with 10% (vol/vol) heat-inactivated human serum, 10% (vol/vol) heat-inactivated NBCS, and
penicillin/streptomycin. Endothelial cells were seeded at high density
to obtain confluent monolayers, and, unless stated otherwise, cultured
in DMEM without indicator supplemented with 10% (vol/vol)
heat-inactivated human serum, 10% (vol/vol) heat-inactivated NBCS, 2 mmol/L L-glutamine, penicillin/streptomycin, 20 ng/mL bFGF, and 20 ng/mL TNF- (referred to as incubation medium). Incubations were for
8 to 12 days, and test compounds were added together with incubation
medium where appropiate. The conditioned medium was collected and
replaced every 2 to 3 days. Invading cells and the formation of
capillary-like tubular structures ("tube formation") of
endothelial cells in the three-dimensional fibrin matrix were analyzed
by phase contrast microscopy and the total number, the total area, and
the total length of capillary-like tubular structures of six randomly
chosen microscopic fields/well (11.6 mm2/field) were
measured using an Olympus microscope equipped with a monochrome CCD
camera (MX5) connected to a computer with Optimas image analysis
software (Tokyo, Japan). Because all three measured parameters correlated well with each other (r > .96), only
data for total length of capillary-like structures are shown. For
determination of t-PA secretion, cells were cultured on gelatin-coated
1-cm2 wells in parallel to cells grown on fibrin matrices.
In experiments with nuclear receptor antagonists, the antagonist
was added to the medium 1 hour before the hormone.
Histochemistry.
Matrices were fixed in 2% (vol/vol) p-formaldehyde in
phosphate-buffered saline (PBS) (0.15 mol/L NaCl; 10 mmol/L
Na2HPO4; 1.5 mmol/L
KH2PO4, pH 7.4), embedded in Technovit 8100, sectioned at 3 µm, and stained with hematoxylin and floxin.
ELISAs.
u-PA antigen was determined by a previously described u-PA ELISA, which
recognizes single-chain u-PA, two-chain u-PA, and u-PA/PAI-1 complex
with the same efficiency.18 t-PA and PAI-1 antigen
determinations were performed by commercially available immunoassay
kits (Thrombonostika t-PA and Imulyse).
Northern blot analysis.
Total RNA from hMVEC (30 cm2) was isolated by the
isothiocyanate/phenol/acid extraction method of Chomcynski et
al.23 The RNA was dissolved in H2O, and the RNA
concentration was determined spectrophotometrically. Equal amounts (7.5 to 10 µg) of RNA were separated on a formaldehyde/agarose gel and
were subsequently capillary transferred to a Hybond N membrane
according to the instructions of the manufacturer (Amersham).
Hybridization was performed in 7% (wt/vol) sodium dodecyl sulfate
(SDS), 1 mmol/L EDTA, 0.5 mol/L
Na2HPO4/NaH2PO4 buffer,
(pH, 7.2) overnight at 63°C with 25 ng of probe labeled with the
random primer method (Mega prime kit, Amersham). The membranes were
subsequently washed three to four times during 20 minutes with 2×
SSC/1% (wt/vol) SDS (1× SSC: 0.15 mol/L NaCl, 0.015 mol/L
Na3 citrate) when using the u-PA and u-PAR probes, twice
with 2× SSC, and twice with 1× SSC when using the other
probes. The filters were exposed to an Amersham Hyper film-MP film with
an intensifying screen at 80°C.
cDNA probes.
The following cDNA fragments were used as probes in the hybridization
experiments: a 1.0-kb fragment of the human u-PA cDNA (a gift from Dr
W-D. Schleuning, Schering AG, Berlin, Germany),24 a 585-bp
BamHI fragment of the human u-PAR cDNA (a gift from Dr F. Blasi, Milano, Italy),25 a 2.7-kb Sma I fragment of
the human androgen receptor cDNA (a gift from Dr A.O. Brinkmann,
Rotterdam, The Netherlands),26 a 2.6-kb Kpn
I/Dra I fragment of the human glucocorticoid receptor (a gift
from Dr S. Wissink, Utrecht, The Netherlands),27 and a
1.2-kb Pst I fragment of hamster actin cDNA.28
Oligonucleotide primers.
The following primer sequences were used in the RT-PCR to detect
receptor mRNA: for the estrogen receptor mRNA: sense
5 -TGATGGGGAGGGCAGGGGTGAAGTG-3 (aa 272-279) and antisense
5-TAGGCGGTGGGCGTCCAGCATCTCC-3 (aa 541-549), as described
by Perrot-Applanat et al.29 For the estrogen receptor mRNA: sense 5-TTGTGCGGAGACAGAGAAGTGC-3 (aa 175-182) and
antisense 5-GGAATTGAGCAGGATCATGGCC-3 (aa
349-355).30 For the progesterone receptor mRNA: sense
5-GTGGGCGTTCCAAATGAAAGCCAAG-3 (aa 660-667) and antisense
5-QAATTCAACACTCAGTGCCCGGGACT-3 (aa 897-905).29 For the thyroid receptor mRNA: sense
5-AGTGGGATCTGATCCACATTGC-3 (aa 164-171) and antisense
5-GATCTTGTCCACACACAGCAGG-3 (aa 332-338).31 For the thyroid receptor : sense
5-GGGAGCTCATCAAAACTGTCAC-3 (aa 214-221) and antisense
5-GGCTACTTCAGTGTCATCCAGG-3 (aa 359-366).32 For the vitamin D3 receptor: sense
5-AGACACACTCCCAGCTTCTCTG-3 (aa 171-180) and antisense
5-ACGTCTGCAGTGTGTTGGACAG-3 (aa 359-366).33
RT-PCR.
RT-PCR was performed under standard conditions following the
specifications recommended by the supplier. In short, cDNAs were synthesized in one reaction mixture containing 1 µg total RNA, 0.45 µg oligo dT primer, and RT-II-superscript (Life Technologies, Paisley, UK); the cDNAs were then heated for 8 minutes at 95°C. Subsequently, the cDNAs were treated with RNAse H (25 U/mL) for 25 minutes at 37°C. Next, the cDNAs (1 µL of a 10× diluted
cDNA reaction mixture) were amplified in the presence of 5% (vol/vol) DMSO and 5% W-1 (vol/vol) (Life Technologies) with the corresponding primers. The amplifications were performed for 30 cycles for all receptors. The denaturation was performed for 60 seconds at 94°C. Primer extension was performed for 60 seconds at 60°C for 4 cycles, then at 58°C for 4 cycles, next at 56°C for 4 cycles, and
finally at 55°C for 18 cycles for the progesterone receptor and the
thyroid receptors, and . For the estrogen receptors and and for the vitamin D receptor, primer extension was performed for 60 seconds at 60°C for 10 cycles and then at 58°C for 20 cycles. The DNA-synthesizing step was performed at 72°C for 1 minute. For
the estrogen receptors and , PCR was also performed for 55 cycles, with a cycling profile of 1 minute at 94°C, 1 minute at
62°C, and 1 minute at 72°C. After 20 cycles with a primer
concentration of 28 nmol/L, additional primers were added to reach a
final concentration of 380 nmol/L. Aliquots of the PCR reaction mixture
were separated on an agarose gel, stained with ethidium bromide, and
visualized with a UV transilluminator.
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RESULTS |
Effect of steroid hormones, retinoids, thyroid hormone, and
1,25-dihydroxyvitamin D3 on tube formation.
To determine the effect of the various hormones on tube formation,
hMVEC were seeded on three-dimensional fibrin matrices and maintained
in standard incubation medium containing 10% (vol/vol) human serum,
10% (vol/vol) NBCS, 20 ng/mL TNF- and 20 ng/mL bFGF, and varying
concentrations of the appropiate hormone. Tube formation required the
presence of both bFGF and TNF-, and none of the hormones tested
showed any angiogenic activity by itself or in combination with either
bFGF or TNF- (data not shown). Tube formation usually became
detectable after 3 to 5 days of cell culture, and the onset of this
process was not or hardly affected by the presence of the hormones.
Effects of hormones on tube formation were most apparent after 8 to 12 days of culture and are illustrated in Fig
1 (A through E) for the most potent compounds, namely
at-RA, testosterone, and dexamethasone. Very similar results
were obtained when the human serum/NBCS was replaced by 20% (vol/vol)
delipidated calf serum (data not shown). Histologic analysis of
cross-sections perpendicular to the surface of the matrix shows that
the tube-like structures are located in the fibrin matrix underneath
the endothelial cell monolayer (Fig 1F). At a hormone concentration of
1 µmol/L, tube formation was strongly suppressed by testosterone and
dexamethasone to 32% ± 9% and 30% ± 17% of control values,
respectively (Fig 2). The synthetic
androgen receptor agonist R1881 also effectively inhibited tube
formation (see Fig 8A). A total of 1 µmol/L 17-estradiol and its
metabolite, 2-methoxyestradiol, only weakly inhibited tube formation to
82% ± 11% and 74% ± 19%, respectively, whereas progesterone
(1 µmol/L) had no significant effect (85% ± 17%) (Fig 2).
at-RA (a ligand for the retinoic acid receptor, RAR) and
9-cis RA (a ligand for both RAR and the retinoid X receptor, RXR) both stimulated tube formation, with 9-cis RA being an
even more potent stimulator (377% ± 80%) than at-RA
(278% ± 40%) (Fig 2B). Comparable results were obtained with the
RAR-specific synthetic ligand TTNPB and the RXR-specific ligand TTNEB
(data not shown), indicating that both RARs and RXRs are able to
mediate the effect of retinoids on tube formation. Neither thyroid
hormone (T3) nor 1,25-dihydroxyvitamin D3 significantly affected tube
formation at a concentration of 1 µmol/L (Fig 2B), but at a
concentration of 10 µmol/L 1,25-dihydroxyvitamin D3 increased tube
formation to 161% ± 35% (data not shown). Effective hormones
influenced tube formation in a concentration-dependent manner with
ED50 values of 20 nmol/L for testosterone, 8 nmol/L for
dexamethasone, 60 nmol/L for at-RA, and 70 nmol/L for
9-cis RA, and maximal effects were reached at hormone
concentrations of 0.1 to 1.0 µmol/L.
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| Fig 1.
Effect of all-trans retinoic acid, testosterone,
and dexamethasone on in vitro angiogenesis. hMVEC were cultured on the
surface of a three-dimensional fibrin matrix in incubation medium
containing 20 ng/mL TNF- and 20 ng/mL bFGF, supplemented with
hormone or vehicle. After 9 days of culture, nonphase contrast
photographs were taken with the plane of focus beneath the endothelial
surface monolayer. (A) Incubation medium from which bFGF and TNF-
had been omitted; (B) incubation medium + vehicle (0.01% [vol/vol] DMSO); (C) incubation medium + all-trans retinoic acid (1 µmol/L); (D) incubation medium + testosterone (1 µmol/L); (E)
incubation medium + dexamethasone (1 µmol/L); (F) incubation medium + vehicle; cross-section through a fibrin matrix perpendicular to the
surface of the matrix, stained with hematoxylin and phloxin. Lumens
surrounded by endothelial cells are indicated by arrows (original
magnification ×40). Bar represents 500 µm (A through E).
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| Fig 2.
Effect of steroid hormones, retinoids, thyroid hormone,
and 1,25-dihydroxyvitamin D3 on tube formation. hMVEC were cultured on
the surface of a three-dimensional fibrin matrix in incubation medium
containing 20 ng/mL bFGF and 20 ng/mL TNF-. (A) Shows the effect of
vehicle, 0.01% (vol/vol) DMSO (con) or 1 µmol/L 17-estradiol
(E2), testosterone (test), progesterone (prog), dexamethasone (dex), or
2-methoxyestradiol (2-ME) and (B) shows the effect of 1 µmol/L of
1,25-dihydroxyvitamin D3 (D3), thyroid hormone (T3), all-trans
retinoic acid (at-RA), or 9-cis retinoic acid (9-cis RA) on
tube formation. The data represent the average ± SD of three to six
experiments with the number of experiments indicated in parentheses for
each condition, with each experiment performed in duplicate, and are
expressed as percentage of control values. Total
tube-length/cm2 under control conditions ranged from 32 to
329 mm/cm2 (average = 130 mm/cm2) between the
different experiments. * P< .05, *** P < .0001.
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Role of u-PA in hormone-modulated tube formation.
Because we had previously demonstrated that tube formation in our in
vitro angiogenesis model depends on u-PA activity,18 we
evaluated the effect of the various hormones on the accumulation of
u-PA in the medium (Fig 3). Under standard
incubation conditions, a continuous increase in u-PA accumulation rate
was observed from day 2 onward over a 9-day period (Fig 3A and B). At a
concentration of 1 µmol/L, testosterone and dexamethasone lowered
u-PA accumulation to 66% ± 4% and 52% ± 6% of control
values, respectively, after 9 days, at-RA and 9-cis RA
enhanced the u-PA accumulation to 236% ± 56% and 284% ± 18%, respectively (Figs 3A and B). The other hormones tested,
17-estradiol, 2-methoxyestradiol, progesterone, T3, and
1,25-dihydroxyvitamin D3, did not change u-PA production significantly
in our in vitro model. The effect of the various hormones on u-PA
accumulation thus highly parallels the effect on tube formation.
Similarly as found for u-PA, PAI-1 accumulation rate increased in time.
Of all the hormones tested, only 9-cis RA and at-RA
slightly increased PAI-1 levels (Fig 3C and D). Because t-PA binds to
fibrin, we performed parallel experiments with hMVEC grown on
gelatin-coated dishes to evaluate the effect of the various experimental conditions on t-PA accumulation. t-PA production proceeded
at a constant rate. Both at-RA and 9-cis RA stimulated t-PA production to 374% ± 91% and 278% ± 25% of control
values, respectively, whereas the other compounds did not affect t-PA production (Fig 3E and F). Omitting bFGF and TNF- from the standard incubation medium resulted in a very low, constant production rate of
u-PA, PAI-1, and t-PA. In all, these findings are consistent with a
role of u-PA (but not of t-PA) in mediating the hormonal effect on tube
formation.
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| Fig 3.
Accumulation of u-PA, PAI-1, and t-PA in hormone-treated
hMVEC-conditioned medium. hMVEC were cultured on three-dimensional fibrin matrices (for determination of u-PA and PAI-1 production) or
gelatin-coated dishes (for determination of t-PA production) in
incubation medium containing 20 ng/mL bFGF and 20 ng/mL TNF- in the
presence of vehicle (con) or the indicated hormones (1 µmol/L). (A
and B) Show the accumulative production in the medium of u-PA
(ng/well), (C and D) show the accumulative production of PAI-1
(ng/well), and (E and F) show the accumulative production of t-PA
(ng/well). u-PA, PAI-1, and t-PA antigen were determined in the
conditioned medium by ELISA as described in Materials and Methods. The
data shown are from one representative experiment (of five performed).
Under control conditions, the cumulative levels of u-PA, PAI-1, and
t-PA after 9 days of culture varied between 20 to 49 ng/well, 2,300 to
2,440 ng/well, and 3.4 to 5.4 ng/well, respectively in the different
experiments. For comparison, u-PA, PAI-1, and t-PA antigen levels were
also determined when cells were cultured in incubation medium from
which bFGF and TNF- had been omitted (BT). E2, 17-estradiol;
test, testosterone; prog, progesterone; dex, dexamethasone; 2-ME,
2-methoxyestradiol; D3, 1,25-dihydroxyvitamin D3; T3, thyroid hormone;
at-RA, all-trans retinoic acid; 9-cis RA, 9-cis
retinoic acid.
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To further substantiate that hormonal modulation of tube formation in
our in vitro model is related to changes in u-PA levels, we performed
experiments in which the amount of u-PA available for angiogenesis was
altered by the addition of specific antibodies or exogenous single
chain u-PA (scu-PA). Figure 4A shows that the addition of antibodies, which neutralize u-PA activity, completely inhibited basal and at-RA-stimulated tube formation, whereas
antibodies, which neutralize t-PA activity, were without effect.
Similarly, antibodies directed against u-PAR strongly inhibited basal
(by 75% ± 26%) and at-RA-stimulated tube formation (by
68% ± 19%) (Fig 4B). The structures, which were formed in the
presence of u-PAR antibody, were very small and resembled single
invading cells rather than capillary-like tubes. The addition of
exogenous scu-PA to control cultures (mimicking retinoid-stimulated
u-PA production) dose-dependently increased tube formation, and
exogenous scu-PA could also restore for a great part the testosterone-
and dexamethasone-inhibited tube formation (Fig 4C). Together these findings indicate that differences in u-PA levels explain the observed
hormone effects on tube formation.
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| Fig 4.
Effects of antibodies against u-PA, t-PA, and u-PAR and
of addition of single chain u-PA on hormone-modulated tube formation. hMVEC were cultured on three-dimensional fibrin matrices in incubation medium containing 20 ng/mL bFGF and 20 ng/mL TNF-, and the
appropriate hormone. Activity blocking antibodies to u-PA, t-PA, and
u-PAR were added at the start of an experiment, whereas single chain u-PA was added from day 2 onward. After 8 to 11 days, the total length
of capillary-like tubular structures was determined as described in
Materials and Methods. (A) Effect of solvent (PBS), antibodies to u-PA
( uPA, 150 µg/mL) or t-PA ( tPA, 150 µg/mL) and rabbit IgG
isolated from pooled normal serum (con-ab, 150 µg/mL) on tube
formation of hMVEC in incubation medium supplemented with vehicle (con,
0.01% (vol/vol) DMSO) or all-trans retinoic acid (RA, 1 µmol/L). (B) Effect of solvent (PBS), antibody to u-PAR ( uPAR, 25 µg/mL) and antibody to FITC (con-ab, 25 µg/mL) on tube formation of
hMVEC in incubation medium supplemented with vehicle (con, 0.01% DMSO)
or all-trans retinoic acid (RA, 1 µmol/L). (C) Effect of
addition of solvent (PBS) and scu-PA (10 or 30 ng scu-PA) on tube
formation of hMVEC in incubation medium supplemented with vehicle (con,
0.01% DMSO), testosterone (test, 1 µmol/L), or dexamethasone (dex, 1 µmol/L). The data represent the average of two experiments performed
in duplicate wells (with ranges given by error bars) and are expressed
as percentages of control values (tube-length/cm2 in the
presence of bFGF and TNF-).
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To examine whether the effect of u-PA involves proteolytic activation
of plasminogen by receptor-bound u-PA, experiments were performed in
the presence of aprotinin (100 KIU/mL), an inhibitor of plasmin
activity. Aprotinin completely inhibited tube formation in the presence
of bFGF and TNF- alone, as well as in the additional presence of
9-cis RA or dexamethasone (Fig 5),
indicating that plasmin activity is critically important for the
formation of tubular structures in the fibrin matrix. To further
demonstrate that receptor-bound u-PA activity is required, we performed
experiments with the amino terminal fragment (ATF) of u-PA. ATF, which
lacks the catalytic domain, binds similar to the u-PA receptor as u-PA, but was found to be inactive in stimulating tube formation (data not
shown).
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| Fig 5.
Effect of aprotinin on hormone-modulated tube formation.
hMVEC were cultured on three-dimensional fibrin matrices in incubation medium containing 20 ng/mL bFGF and 20 ng/mL TNF-, and the
appropriate hormone (1 µmol/L 9-cis RA or 1 µmol/L dexamethasone)
or vehicle (con). For comparison, cells incubated in the absence of
bFGF and TNF- (BT) were included. An inhibitor of plasmin
activity, aprotinin (100 KIU/mL), was added at the start of the
experiment. Total tube-length/cm2 ± standard error of
mean (SEM) was determined of triplicate wells as described in Materials
and Methods.
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u-PA and u-PAR mRNA expression.
Northern blotting studies were conducted to examine whether the effect
of hormones on u-PA protein levels were reflected at the mRNA level.
After a 96-hour exposure of hMVEC to the various hormones, testosterone
and dexamethasone were found to decrease u-PA mRNA levels by
approximately twofold, whereas at-RA and 9-cis RA
increased u-PA mRNA levels twofold and threefold, respectively (Fig 6). The other hormones did not
significantly influence u-PA mRNA levels. Hybridization with the cDNA
for u-PAR showed a very similar induction pattern, with the exception
of dexamethasone, which did not affect u-PAR mRNA levels. Northern
blotting analysis of u-PA and u-PAR mRNA expression after 8 hours of
incubation very much resembled that of the 96-hour hormone exposure,
except for testosterone, which did not cause any early change in u-PA and u-PAR mRNA expression. Omitting bFGF and TNF- from the
incubation medium resulted in a twofold to threefold decrease in u-PA
and u-PAR mRNA levels.
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| Fig 6.
Northern blot analysis of u-PA and u-PAR mRNA of hMVEC
cultured in the presence of steroid hormones, retinoids, thyroid
hormone, or 1,25-dihydroxyvitamin D3. hMVEC were cultured on
gelatin-coated dishes in incubation medium containing 20 ng/mL bFGF and
20 ng/mL TNF- and 0.01% (vol/vol) DMSO (con) or 1 µmol/L of
17-estradiol (E2), progesterone (prog), testosterone (test),
dexamethasone (dex), 1,25-dihydroxyvitamin D3 (D3), thyroid hormone
(T3), all-trans retinoic acid (at-RA), or 9-cis
retinoic acid (9-cis RA). After 2 days, the media were
refreshed. After 4 days, RNA was isolated and analyzed (7.5 µg/lane)
by Northern blot analysis using 32-[dCTP]-labeled probes
for u-PA, u-PAR, and actin mRNA. For comparison, RNA was also isolated
and analyzed from cells before the addition of hormones or vehicle (t
= 0). Signals for u-PA and u-PAR were quantified by densitometry and
adjusted for the corresponding actin mRNA signals. Data are expressed
relative to that found in control (con) cells. Results are means of two
independent experiments.
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Nuclear hormone receptors.
Northern analysis was performed to demonstrate the expression of mRNAs
coding for RAR, RXR, androgen receptor (AR), and glucocorticoid
receptor (GR) (Fig 7). Two transcripts for
RAR (3.6 kb and 2.8 kb) and one for RXR (4.8 kb) were evident in cultured hMVEC. The human AR mRNA migrated as two species of
approximately 10 kb and 7 kb and one band for GR mRNA (7 kb) was found.
To determine whether the effect of testosterone, dexamethasone, and
at-RA on u-PA production and tube formation was mediated by
their respective nuclear receptors, experiments in the presence of
receptor-specific antagonists were performed. As shown in
Fig 8A, the specific androgen receptor
antagonist, hydroxyflutamide,34,35 counteracted the inhibitory effect of the synthetic androgen, R1881. For blocking glucocorticoid receptor-mediated transcriptional activity, we used
RU486 (mifepristone). RU486 is a synthetic progestin and glucocorticoid
antagonist that binds with high affinity to progesterone and
glucocorticoid receptors.36 RU486 prevented the suppressing effect of dexamethasone on tube formation (Fig 8B). The RAR antagonist Ro41-5253 completely inhibited the at-RA-induced increase in
tube formation (Fig 8C). Ro41-5253 is a retinoid that specifically antagonizes the transactivation of RARs by retinoids, having a preference for RAR.37,38 These effects of the
receptor-specific antagonists on tube formation were paralleled by
similar changes in u-PA production (data not shown).
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| Fig 7.
Nothern blot analysis of RAR, RXR, androgen
receptor (AR), and glucocorticoid receptor (GR) mRNA in hMVEC. hMVEC
were cultured on gelatin-coated dishes in incubation medium without
bFGF and TNF-. RNA was isolated and analyzed (10 µg/lane) by
Northern blot analysis using 32-[dCTP]-labeled probes
for RAR, RXR, AR, and GR mRNA. The sizes of the various
transcripts were calculated by comparison to the migration of an RNA
ladder.
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| Fig 8.
Effects of nuclear receptor antagonists on R1881-,
dexamethasone- and at-RA-modulated tube formation. hMVEC were
cultured for 8 to 12 days on three-dimensional fibrin matrices in
incubation medium containing 20 ng/mL bFGF and 20 ng/mL TNF-, and to
which was added 1 nmol/L R1881 (R1881) (A), 10 nmol/L dexamethasone (dex) (B), or 10 nmol/L at-RA (RA) (C). Receptor antagonists
(hydroxyflutamide, RU486 or Ro41-5253; final concentration 1 µmol/L
for hydroxyflutamide and RU486 and 10 µmol/L for RO41-5253) or
vehicle (0.1% or 0.01% DMSO) were added 1 hour before the hormones.
(A) Effect of solvent (DMSO, 0.01% [vol/vol]) or hydroxyflutamide
(OH-flu, 1 µmol/L) on tube formation of hMVEC in incubation medium
alone (con) or supplemented with R1881 (R1881, 1 nmol/L). (B) Effect of
solvent (DMSO, 0.01% [vol/vol]) or RU486 (RU486, 1 µmol/L) on tube
formation of hMVEC in incubation medium alone (con) or supplemented
with dexamethasone (dex, 10 nmol/L). (C) Effect of solvent (DMSO; 0.1 % [vol/vol]) or Ro41-5253 (Ro41-5253, 10 µmol/L) on tube formation of hMVEC in incubation medium alone (con) or supplemented with all-trans retinoic aicd (at-RA, 10 nmol/L). The data
represent the average ± standard deviation of three experiments
performed in duplicate and are expressed as percentages of control
values (tube-length/cm2 in the presence of bFGF and
TNF-).
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To determine whether the weak effects of some of the hormones tested
are related to the low expression levels of the corresponding nuclear
receptors, we performed RT-PCR analysis on RNA isolated from hMVEC
cultured in incubation medium for 4 days. Indeed, the levels of
progesterone receptor mRNA, estrogen receptor mRNA, and estrogen
receptor mRNA in hMVEC were below the detection limit of our assay
(Fig 9A through C). Also, in cells cultured in the absence of bFGF and TNF-, mRNA for these receptors could not
be detected. Only a very weak band for the vitamin D3 receptor could be
demonstrated (Fig 9D). Unexpectedly, both thyroid hormone receptor and were clearly expressed (Fig 9E).
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| Fig 9.
Presence of estrogen receptors and , progesterone
receptor, thyroid hormone receptors and , and vitamin D receptor
mRNA in hMVEC as determined by RT-PCR. hMVEC were cultured for 4 days on gelatin-coated dishes in incubation medium containing 20 ng/mL bFGF
and 20 ng/mL TNF- or in incubation medium from which bFGF and
TNF- was omitted. RNA was isolated from these cells and cDNAs were
synthesized using 1 µg total RNA and oligo dT primer as described in
Materials and Methods. The cDNAs were amplified with primers for
estrogen receptor (A), estrogen receptor (B), progesterone receptor (C), vitamin D receptor (D), and thyroid hormone receptors and (E) as described in Materials and Methods. The expected length
of the amplified DNA fragment of the estrogen receptor is 832 nt,
of the estrogen receptor 541 nt, of the progesterone receptor 737 nt, of the thyroid hormone receptor 523 nt, of the thyroid hormone
receptor 458 nt, and of the vitamin D receptor 579 nt. As a
positive control for the expression of estrogen receptor ,
progesterone receptor, thyroid hormone receptors and , and vitamin D receptor mRNA, RNA isolated from MCF7 cells was used. As a
positive control for the expression of the estrogen receptor mRNA,
RNA isolated from the SV-HFO osteoblast cell line was used. Lane 1, incubation medium from which bFGF and TNF- had been omitted; lane 2, incubation medium containing bFGF and TNF-; lane 3, appropiate
positive control; M, molecular weight marker.
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DISCUSSION |
The present study demonstrates that steroid hormones and retinoids can
have strong, but different effects on the formation of capillary-like
tubular structures by human microvascular endothelial cells cultured on
top of a human fibrin matrix in the presence of bFGF and TNF-. We
found that testosterone and dexamethasone almost completely block tube
formation in this in vitro model, while at-RA and 9-cis
RA strongly potentiate the effect of bFGF and TNF-. These compounds
are likely to exert their action via their respective nuclear
receptors, as demonstrated by the use of receptor-specific antagonists.
17-Estradiol, progesterone, thyroid hormone (T3), and
1,25-dihydroxyvitamin D3 showed no or only minor effects on in vitro
angiogenic activity, which, with the exception of T3, could be related
to the absence of significant nuclear receptor expression.
We found that testosterone and dexamethasone decreased, and
at-RA and 9-cis RA increased u-PA mRNA and antigen
levels. These alterations in u-PA expression not only parallel, but are
also likely to be responsible for the observed changes in angiogenic activity for the following reasons. First, exogenous suppletion of the
medium with scu-PA enhances tube formation in our in vitro model,
whereas quenching of u-PA activity (but not t-PA activity) or u-PA
binding to u-PAR by specific antibodies suppresses basal and
retinoid-stimulated tube formation. Second, addition of scu-PA to
testosterone- and dexamethasone-treated hMVEC restores the suppressed
angiogenic activity for a substantial part.
It is likely that the effect of u-PA on tube formation involves
proteolytic activation of plasminogen by receptor-bound u-PA, because
inhibition of plasmin by aprotinin decreases the formation of
capillaries. This does not exclude the possibility, however, that the
u-PA/u-PA receptor system is also relevant for other aspects of the
angiogenic process. After proteolytic disruption of the cell-matrix
interaction, the cell has to create simultaneously new attachment sites
by which it "pulls" itself into the matrix. Experiments by Yebra
et al39 indicate that the u-PA/u-PA receptor complex has a
cooperative effect with the integrin v5 in promoting cell
migration by providing an additional receptor for attachment to matrix
proteins. Consequently, enhancement of the number of occupied u-PA
receptors not only provides the endothelial cell with an enhanced local
proteolytic capacity, but also provides the cell the increased capacity
to form new attachment sites.
Our results exclude a major contribution of u-PA in the angiogenic
process through signalling via the u-PA receptor.40,41 This
cellular activation by u-PA appears to be mediated through the ATF of
u-PA, which lacks the catalytic domain, but binds similar to the u-PA
receptor as u-PA. However, ATF was unable to replace u-PA in our in
vitro angiogenesis model in stimulating tube formation (see also
Koolwijk et al18).
The small effect of 17-estradiol on tube formation, despite the lack
of an effect on u-PA production, may be related to its conversion to 2 methoxyestradiol (2-ME). 2-ME has been shown to inhibit in vitro
angiogenesis, probably by interfering with tubulin polymerization.15,42 We found 2-ME to inhibit tube
formation in our model without affecting u-PA expression. Inasmuch as
the absence of a direct effect of 17-estradiol on angiogenesis is related to the male origin of the endothelial cells (viz. human foreskin) is not clear at present. For a proper assessment of the role
of sex steroids in angiogenesis, it may be relevant to extend our
present experiments to endothelial cells of female origin.
The mechanism by which the steroid hormones and retinoids alter the
TNF--stimulated u-PA expression in hMVEC is not known, but may be
related to activation of the NF B/Rel system by
TNF-.43,44 In unstimulated endothelial cells, NFB/Rel
family complexes are retained in the cytoplasm by the binding of
inhibitory proteins, including IB. Endothelial activation evokes
dissociation of IB and allows translocation of the transcription
factors to the nucleus. Two functional NFB elements at 1580
and 1865 bp have been identified in the human u-PA
promoter.45 We have found, using a recombinant adenovirus
expressing IB, that overexpressed IB inhibits
cytokine-stimulated NFB activation and u-PA expression in human
umbilical vein endothelial cells (Kooistra and De Martin, unpublished
data, 1997). The dexamethasone-binding glucocorticoid receptor can directly or indirectly bind and inactivate the NFB transcription factor, analogous to the mechanism responsible for the
negative interaction between the glucocorticoid receptor and the AP-1
transcription complex.46,47 More recent studies have also
demonstrated transcriptional activation of the IB gene by
dexamethasone.48,49 The resulting increase in IB
protein synthesis then inhibits NFB translocation to the nucleus.
Testosterone, whose androgen receptor belongs to the same subclass of
nuclear receptors as the glucocorticoid receptor, might also act by
maintaining IB levels, as suggested by Keller et al50
for the repressive effect of androgens on the expression of
interleukin-6. Alternatively or additionally, formation of androgen
receptor/NFB complexes may be responsible for the inhibiting effects
of androgens on NFB-mediated transcription activation.
Our finding that at-RA and 9-cis RA by themselves only
slightly increase u-PA expression, but strongly upregulate the
TNF--induced u-PA synthesis, resembles the results described by
Harant et al51 for the activation of IL-8 gene
transcription in the human melanoma cell line A3. It was shown that
stimulation with at-RA and TNF- resulted in enhanced NFB
binding compared with that induced by TNF- alone and also resulted
in changes in the composition of the NFB complexes bound to the IL-8
NF< |
Abstract
Introduction
Abstract