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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Gaubius Laboratory TNO-PG, Leiden, The
Netherlands, and the Institute for Cardiovascular Research, Vrije
Universiteit, Amsterdam, The Netherlands.
Hypoxia stimulates angiogenesis, the formation of new blood
vessels. This study evaluates the direct effect of hypoxia (1% oxygen)
on the angiogenic response of human microvascular endothelial cells
(hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs
stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor- In pathologic disorders, low oxygen tension or
hypoxia often occurs. Especially in the course of tissue damage, a loss
of adequate blood supply causes hypoxia.1 Angiogenesis or
the formation of new vessels is strongly induced by hypoxic
conditions.2,3 A number of cell types respond to hypoxia
and produce angiogenic factors such as vascular endothelial growth
factor-A (VEGF-A),4 platelet-derived growth factor-B
(PDGF-B),5,6 and fibroblast growth factor-2
(FGF-2).7,8 Macrophages in particular are extremely
sensitive to hypoxia. They may act as oxygen sensors and initiate the
process of vessel renewal by secreting a number of angiogenic
factors.9-11 On the other hand, endothelial cells, which
are the dominant cell type in microvessels, play a key role in the
formation of new vessel networks. Endothelial cells are able to survive
severe hypoxic conditions12,13 and are potential vectors of
hypoxia-induced angiogenesis.
During the onset of angiogenesis, endothelial cells degrade their
basement membrane, migrate into the interstitial matrix, proliferate,
and form new microvascular structures. Matrix remodeling proteases of the plasminogen activator/plasmin- and matrix-degrading metalloproteinase (MMP) cascades, together with their receptors and
inhibitors, play pivotal roles in several of these
steps.14 Depending on the composition of the matrix
proteins in the area of angiogenesis, different groups of proteases are
involved. In pathologic angiogenesis of the adult, angiogenesis is
often accompanied by vascular leakage and by the formation of a
fibrinous exudate.15,16 The fibrin matrix in this exudate
facilitates cell migration by providing additional scaffolding for
invading leukocytes and endothelial cells. Endothelial invasion into a
fibrin matrix and the subsequent formation of capillary structures
require cell-bound urokinase-type plasminogen activator (uPA) and
plasmin activities.17-19 The uPA is secreted as a
single-chain inactive pro-enzyme, which binds to its cellular receptor
uPAR (CD87) and is proteolytically activated. Active uPA converts
plasminogen into plasmin, a broadly acting protease that degrades
several matrix proteins and can activate latent MMPs.16
Both plasmin and uPA are rapidly inactivated, the latter by
PA-inhibitor type-1 (PAI-1). The uPA:PAI-1 complex is internalized
together with uPAR and degraded, while the unoccupied uPAR returns
to the plasma membrane.20
It has been shown that in a number of in vitro as well as in vivo
studies, the binding of uPA to uPAR is essential for its action in
angiogenesis.19,21,22 Besides a role in localizing uPA
activity, the uPAR can also play a role in cell adhesion by interacting
with vitronectin and integrins.23-25 This cell-matrix interaction is modified by the binding of uPA and
PAI-1.26,27 In addition, the occupied uPAR is also
involved in cellular signal transduction.28,29
Hypoxia may stimulate angiogenesis by several mechanisms. It increases
the transcription of VEGF in a number of cells.4,10,30 In
endothelial cells hypoxia-induced VEGF production is
described,31 although the resistance of endothelial cells
to hypoxic stimulation with regard to VEGF production can also be
observed.32,33 Similarly, conflicting data have been
reported regarding the effect of hypoxia on the expression of VEGF
receptors in endothelial cells.33-35 In addition to this,
hypoxia also induced an increase in the expression of
In the present study we evaluated the effect of hypoxia on the
formation of capillary-like tubular structures by human microvascular endothelial cells (hMVECs) in a 3-dimensional fibrin matrix.
In particular the regulation and involvement of the
plasminogen activator system, VEGF, and
Materials
A crude preparation of endothelial cell growth factor (ECGF) was
prepared from bovine brain as described by Maciag et al.40 Human serum (HS) was obtained from a local blood bank, prepared with freshly obtained blood from 10-20 healthy donors, pooled, and
stored at 4°C. Human recombinant tumor necrosis factor (TNF)- Complementary DNA probes
The human Cell culture Foreskin hMVECs were isolated, cultured, and characterized as previously described.46,47 The hMVECs were cultured on gelatin-coated dishes in M199 supplemented with 20 mmol/L HEPES (pH 7.3), 10% HS, 10% heat-inactivated NBCS, 150 µg/mL crude ECGF, 2 mmol/L L-glutamine, 5 U/mL heparin, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5% carbon dioxide (CO2)/95% air atmosphere, unless mentioned otherwise. The experiments were performed with confluent cells (0.7 × 105 cells per cm2) that were cultured without growth factor for at least 24 hours.Establishment of hypoxic culture conditions For culturing in hypoxic conditions, hMVECs were placed in a NAPCO incubator (serial number 7101-C1; Precision Scientific, Chicago, IL) that controls the oxygen concentration by flushing with nitrogen (N2). Oxygen levels in the incubator were monitored by an internal oxygen sensor as well as by external calibration using Dräger Tubes 6728081 (Drägerwerk Ag, Lübeck, Germany). The hypoxic condition is defined as culturing at 37°C under a 1% oxygen (O2)/5% CO2 atmosphere.In vitro angiogenesis model Human fibrin matrices were prepared by the addition of 0.1 U/mL thrombin to a mixture of 2.5 U/mL Factor XIII (final concentrations), 2 mg/mL fibrinogen, 2 mg/mL sodium citrate, 0.8 mg/mL sodium chloride (NaCl), and 3 µg/mL plasminogen in M199 medium (mixture, pH 7.4). We added 300-µL aliquots of this mixture to the wells of 48-well plates. After clotting at room temperature, the fibrin matrices were soaked with M199 supplemented with 10% (vol/vol) HS and 10% (vol/vol) NBCS for 2 hours at 37°C to inactivate the thrombin. Confluent endothelial cells (0.7 × 105 cells per cm2) were detached and seeded in a 1.25:1 split ratio on the fibrin matrices to form a highly confluent monolayer. After a 24-hour culture in M199 medium supplemented with 10% HS, 10% NBCS, and penicillin/streptomycin, the endothelial cells were stimulated with the mediators for the time indicated. At the end of the culturing period, the media were collected, and the formation of tubular structures of endothelial cells in the 3-dimensional fibrin matrix was analyzed by phase contrast microscopy. The total length of tube-like structures of 6 randomly chosen microscopic fields (7.3 mm2 per field) was measured and expressed as mm/cm2 using a Nikon FXA microscope (Nikon, Japan) equipped with a monochrome CCD camera (MX5) connected to a computer with Optimas image analysis software.Enzyme-linked immunosorbent assay We performed uPA, tissue plasminogen activator (tPA), PAI-1, and VEGF antigen determinations using the following commercially available immunoassay kits: uPA EIA HS Taurus (Gaubius Laboratory, Leiden, The Netherlands); Thrombonostika tPA (Organon-Teknika, Turnhout, Belgium); IMULYSE PAI-1 (Biopool, Umea, Sweden); and VEGF ELISA (R&D Systems, Minneapolis, MN). Antibodies used in the uPA ELISA recognize single-chain uPA, 2-chain uPA, and the uPA:PAI-1 complex with the same efficiency. The PAI-1 enzyme-linked immunosorbent assay (ELISA) detects active and latent forms of PAI-1, whereas the tPA:PAI-1 and uPA:PAI-1 complexes are not recovered. The tPA ELISA recognizes both tPA and tPA:PAI-1 complexes.Determination of specific uPA binding Diisopropylfluorophosphate-treated uPA (Ukidan; Pierce Chemical, Rockford, IL) (DIP-uPA) was radiolabeled using Na iodine 125 (125I) and the Iodogen procedure (Pierce). The binding of 125I-DIP-uPA to hMVECs was determined at 0°C. The cells were placed on melting ice and incubated for 10 minutes with 50 mmol/L glycine-hydrochloride (HCl) buffer (pH 3.0) to remove receptor-bound endogenous uPA. Subsequently, the cells were washed twice with ice-cold M199 medium and incubated with 8 nmol/L 125I-DIP-uPA in endothelial cell-conditioned medium (M199 medium supplemented with 1% HS albumin and conditioned for 24 hours) for 3 hours. Incubation was performed in endothelial cell-conditioned medium to exclude the residual binding of uPA to cell-associated PAI-1. In parallel incubations, a 50-fold excess of DIP-uPA was included to assess nonspecific binding. After the incubation period, unbound ligand was removed by extensive washing with ice-cold M199 medium. Cell-bound ligand was solubilized with 0.3 mol/L NaOH, and the radioactivity was determined in a -counter (Cobra Auto Gamma, Packard, Meriden, CT).
Specific binding was calculated by the subtraction of nonspecific
binding from the total binding.
RNA isolation and Northern blot analysis Total RNA was isolated as described by Chomczynski and Sacchi48 and electrophoresed in a 1.2% (wt/vol) agarose gel under denaturing conditions using 1 mol/L formaldehyde. The RNA was transferred to Hybond-N filter by blotting, and the filters were hybridized overnight at 63°C in 7% (wt/vol) sodium dodecyl sulfate (SDS), 0.5 mol/L Na2HPO4/NaH2PO4 buffer (pH 7.2), and 1 mmol/L ethylenediamine tetraacetic acid (EDTA) containing a 3 ng/mL -32P (phosphorous 32) CTP-labeled
probe. The probes were labeled by a Megaprime kit (Amersham
International, Little Chalfont, England), yielding an average activity
of 0.0074 MBq/ng (0.2 µCi/ng) DNA. After hybridization of
the filters, they were washed twice with 2 times sodium chloride/sodium
citrate (SSC) (one times SSC equal to 0.15 mol/L NaCl and
0.015 mol/L sodium citrate-dihydrate [pH 7.0]) and 1% SDS and then
washed twice with 1 times SSC and 1% SDS for 20-minute time periods at
63°C. The filters were exposed to a Fuji imaging plate type BAS-MP
(Fuji Photo Film, Tokyo, Japan), and the quantification of relative
amounts of transcribed mRNA was performed using a Phosphorimager
BAS-reader (Fuji Fujix Bas 1000, Fuji).
Cell attachment assay Cell attachment assays were performed in bacteriological 96-well plates (ELISA plates; Greiner, Frickenhausen, Germany) coated with 10 µg/mL vitronectin as previously described.19Assay of cell-associated plasmin formation We cultured hMVECs until confluency in 96-well culture plates and stimulated them for 72 hours with the factors indicated in the text. The cells were washed 3 times with ice-cold 0.05 mol/L Tris-HCl (tris[hydroxymethyl] aminomethane-HCl) buffer (pH 7.4) supplemented with 0.03% HS albumin. We added 40 µL substrate mix containing plasminogen at a final concentration of 200 nmol/L and chromogenic substrate S-2251 at a final concentration of 0.3 mmol/L in 0.05 mol/L Tris-HCl plus 0.03% HS albumin (pH 7.4). The culture plates were placed at 37°C, and absorbance was monitored at 405 nm using a multichannel spectrophotometer (Titertek multiscan; Flowlabs, McLean, VA), thereby producing the expected increase for p-nitroanilide from plasmin production.Statistical analysis The data are expressed as the mean plus or minus SD. The unpaired t test was used for comparison of groups with equal variance and normal distribution. P < .05 was considered statistically significant.
Effect of hypoxia on cell viability and capillary-like tube formation The viability of hMVECs cultured in an hypoxic condition (1% oxygen atmosphere) was comparable to that in standard oxygen atmosphere (20% oxygen, further indicated as normoxic), as determined by trypan blue exclusion. When confluent hMVECs had been incubated for 72 hours in normoxic and hypoxic conditions, without the addition of growth factors, their viabilities were 98.3% ± 0.1% and 97.0% ± 1.5%, respectively (the mean plus or minus SD of 3 experiments in duplicate, P = .81). The monolayers of hypoxic hMVECs showed a very regular cobblestone appearance on the gelatin-coated dishes (data not shown) and on a 3-dimensional fibrin matrix in the absence of angiogenesis stimulating factors (Figure 1A-B).
In previous studies we have shown that hMVECs cultured on top of a
3-dimensional fibrin matrix can be induced to form capillary-like structures by simultaneous exposure to FGF-2 and TNF-
FGF-2+TNA-
Hypoxia decreases uPA levels in the conditioned media of hMVECs To determine the effect of hypoxia on the fibrinolytic activity of hMVECs, we determined the uPA, tPA, and PAI-1 antigen levels by ELISA in the conditioned media of normoxic and hypoxic hMVECs. Under hypoxic culture conditions the uPA antigen was significantly decreased by 67% in nonstimulated cells, 74% in TNF--stimulated cells, and
77% in FGF-2+TNF--stimulated cells compared to normoxic culture
conditions (Table 1). The amounts of
PAI-1 and tPA produced by the cells were comparable in hypoxic and
normoxic cells (Table 1). The effect of hypoxia on uPA levels in the
conditioned media of hMVECs could not be mimicked by incubation with
cobalt chloride, nickel chloride, or deferoxamine (data not shown).
This decrease in the fibrinolytic potential in response to hypoxia was
not expected because the formation of capillary-like structures
requires the presence of cell-bound uPA (previous paragraph).
uPAR expression is upregulated in hypoxic conditions The uPA level in the conditioned medium of cells is the result of its production and internalization by the cellular receptor uPAR. The effect of hypoxia on the uPAR expression was assayed. Hypoxic conditions increased uPAR mRNA in nonstimulated cells as well as in TNF-- or FGF-2+TNF--stimulated hMVECs (Figure 4A-B). The uPAR mRNA signal was
normalized for actin mRNA and quantified (Figure 4B). Compared to the
normoxic situation, hypoxia stimulated uPAR mRNA expression by 139%,
144%, and 130% in nonstimulated cells, TNF--stimulated cells, and
FGF-2+TNF--stimulated cells, respectively. The uPA mRNA levels were
also determined (Figure 4A-B). These signals were very weak but clearly
enhanced after hypoxic treatment: 101%, 141%, and 124% in
nonstimulated cells, TNF--stimulated cells, and
FGF-2+TNF--stimulated cells, respectively (Figure 4B).
The increase in uPAR mRNA expression in hypoxic conditions was
accompanied by an enhanced uPAR antigen level compared to normoxic conditions. The increase in specific-bound 125I-labeled
DIP-uPA was evident in nonstimulated hMVECs (138% ± 38%; n = 4,
P = .2) and significantly increased in cells stimulated with FGF-2+TNF-
When the uPA:uPAR binding was prevented by the blocking mAb H-2 against
uPAR, the decrease in the uPA antigen level observed under hypoxic
conditions was completely abolished in nonstimulated as well as in
FGF-2+TNF-
Plasmin generation is enhanced in hypoxic culture conditions Stimulation of hMVECs with FGF-2+TNF- resulted in an increased
plasmin formation (Figure 7) compared to
unstimulated cells. The plasmin generation was completely blocked by
aprotinin. Parallel to the increased uPAR expression, hypoxia further
increased plasmin formation by FGF-2+TNF--stimulated hMVECs
compared to their normoxic counterparts (P < .002). This
plasmin formation was completely inhibited by the addition of the
blocking antibody H-2 against uPAR, indicating that solely
receptor-bound uPA was responsible for plasmin formation (Figure 7).
Antibodies against tPA did not influence plasmin formation (data not
shown), meaning that only uPA was responsible for the activation of
plasminogen.
VEGF165 is not involved in hypoxia-induced tube formation by hMVECs in a fibrin matrix It has been reported that VEGF165 expression is enhanced in hypoxic conditions in a number of cell types, including endothelial cells,31 by an increased transcriptional activation50 as well as by mRNA stabilization.51,52 Furthermore, it has been shown in HUVECs and hMVECs that VEGF165 can up-regulate uPAR expression.18,53 Therefore, it was investigated whether endogenous synthesis of VEGF165 might play a role in the observed increase in tube formation of hMVECs in a fibrin matrix under hypoxic conditions.VEGF165 could not be detected by ELISA (detection limit, 5 pg/mL) in the conditioned media of both normoxic and hypoxic hMVECs. In
addition, VEGF165 mRNA was not detected by Northern blot
analysis at 8, 24, or 72 hours nor by RT-PCR (data not shown). The
addition of 29 nmol/L sVEGFR-1 (a 50-fold excess over the
VEGF165 concentration used in the in vitro angiogenesis
experiments) did not affect the increased tube formation of
FGF-2+TNF-
Involvement of v 3- and v5-integrins in angiogenesis and their
regulation by hypoxia have been shown in a number of
studies.36,54-56
After 16 hours of hypoxic culturing, hMVECs showed an increase of
The involvement of
In this report we have shown that hypoxia strongly enhances the
formation of capillary-like tubular structures by hMVECs in a
3-dimensional fibrin matrix. Our data indicate that enhanced expression
of uPAR by hypoxia, at least in part, explains the increased angiogenic
response of endothelial cells. In our experimental model, which only
contains endothelial cells, the expression of Hypoxia is a potent stimulus for angiogenesis.2,3 In vivo macrophages are highly sensitive to hypoxia and are induced by it to produce angiogenic factors such as VEGF.9-11 VEGF is also induced in other tissue cells such as stroma cells and smooth muscle cells.4,10,30 In these cells hypoxia can act both on gene transcription and mRNA stability.50,57 In this report we have shown that hypoxia increases the formation of capillary-like tubular structures in a model in which endothelial cells invade a fibrin matrix. No accessory cells were present, indicating that hypoxia directly affected endothelial cells. This affect appears independent of an increased VEGF165 production because VEGF165 mRNA was not detectable in the cells, VEGF165 antigen was not detectable by ELISA, and the effect could not be inhibited by sVEGFR-1 or anti-VEGFR-2 immunoglobulin G (IgG). While Namiki et al31 reported the induction of
VEGF165 in human endothelial cells, other
investigators33 could not detect any increase in
VEGF165 by hypoxia, which is similar to our findings. In
line with this latter study, our data does not support an autocrine stimulation of VEGF165-induced angiogenesis by hypoxia and
provides information regarding a mechanism in addition to the presently established paracrine stimulation of VEGF165-induced
angiogenesis by hypoxia. A hypoxia-induced up-regulation of
VEGF165 receptors in endothelial cells may be of interest
in this context.33-35 Such mechanisms may contribute to
the hypoxia-enhanced VEGF165+TNF- Human endothelial cells invade a fibrin matrix after exposure to an
angiogenic growth factor, VEGF165 or FGF-2, and the
cytokine TNF- In line with our previous observations that the uPA:uPAR interaction is required for tube formation in a fibrin matrix and that modulation of the uPAR expression influences the rate of this process,18,19 the higher expression of uPAR and consequently a higher plasmin formation are likely to contribute to the increased angiogenic response of hMVECs under hypoxic conditions. In pericellular proteolysis, the uPA/plasmin and MMP cascades often cooperate. One may expect that hypoxia alters the expression of MMPs. Under our experimental conditions we observed that there was no difference in the expression of the mRNA of MMP-1 and MMP-3 as compared to the normoxic conditions. Similarly the expression of MMP-2 and MMP-9 remained unaltered, as revealed by gelatin zymography (our unpublished data). However, these data do not exclude the possibility that another proteolytic enzyme or an unknown proangiogenic factor is influenced by hypoxia. The mechanism by which hypoxia increases the expression of uPAR is not yet known. Cobalt chloride, nickel chloride, and deferoxamine could not mimic the hypoxic response in our system, indicating that a heme protein was not involved. This finding is in contradiction to the studies of Graham et al,39,59 who suggested the involvement of a heme protein in uPAR induction. The difference in these results is not easy to explain. Probably they are caused by the use of different cell types or the different time scale in which the experiments were performed (24 hours compared to 72 hours in this study). In addition to the involvement of a heme protein, redox-based signaling is indicated as a possible oxygen-sensing mechanism.61,62 The activity of a well-studied hypoxia-induced transcription factor-1 (HIF-1) has been linked to the redox state of the cell.63 Three potential HIF-1 binding sequences can be found in the 5'-flanking region of the uPAR gene,39 indicating that hypoxia can directly influence uPAR transcription. In addition or alternatively to its effect on gene transcription, hypoxia may also increase the stability of the uPAR expression. Because hypoxia also affects the expression of other proteins, such as VEGF16557 and erythropoietin,64 by increasing mRNA stability, this is a plausible option. In addition to matrix degradation, the ability of the endothelial cells
to re-adhere to this matrix is also indispensable for invasion. The
matrix-receptors, In conclusion, this study shows that endothelial cells by themselves are able to increase their angiogenic potential in response to hypoxia. In our experimental conditions, the increase of uPAR expression by hypoxia appears an important determinant in this process. We would like to stress that angiogenesis in different conditions is regulated by a number of regulators and that our findings reflect angiogenesis in the temporary repair matrix fibrin. In other conditions, such as development and bone repair, the role of uPA and uPAR may be less prominent, and other proteins, such as MMPs, play a dominant role. We conclude that cell bound uPA is an important determinant in capillary-like tube formation in fibrin matrices. In particular, it is anticipated to act in particular in the recanalization of fibrin clots and fibrous exudates in addition to the paracrine effects of hypoxia involving the induction of VEGF165 by adjacent tissue cells.
We would like to thank Erna Peters and Mario Vermeer for their excellent technical assistance.
Submitted December 15, 1999; accepted June 9, 2000.
Supported by grant 95.193 from The Netherlands Heart Foundation, The Hague, The Netherlands, and grant TNOP 97-1511 from The Dutch Cancer Society, Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Victor W. M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE Leiden, The Netherlands; e-mail: vwm.vanhinsbergh{at}pg.tno.nl.
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© 2000 by The American Society of Hematology.
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  Y. Ben-Yosef, A. Miller, S. Shapiro, and N. Lahat Hypoxia of endothelial cells leads to MMP-2-dependent survival and death Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1321 - C1331. [Abstract] [Full Text] [PDF]
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M. Scortegagna, M. A. Morris, Y. Oktay, M. Bennett, and J. A. Garcia The HIF family member EPAS1/HIF-2{alpha} is required for normal hematopoiesis in mice Blood, September 1, 2003; 102(5): 1634 - 1640. [Abstract] [Full Text] [PDF]
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 D. A. Vallera, C. Li, N. Jin, A. Panoskaltsis-Mortari, and W. A. Hall Targeting Urokinase-Type Plasminogen Activator Receptor on Human Glioblastoma Tumors With Diphtheria Toxin Fusion Protein DTAT J Natl Cancer Inst, April 17, 2002; 94(8): 597 - 606. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
8
mol/L phorbol myristate acetate (PMA). Subsequently, the cells were
cooled on ice, and the specific binding of 125I-labeled
DIP-uPA to hMVECs was determined and expressed as fmol
125I-uPA/105 cells. The data represent the mean
plus or minus SEM of 4 independent experiments performed in duplicate
wells. The asterisk indicates P < .05, which is
significantly different from the normoxic counterpart.
