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VASCULAR BIOLOGY

EMMPRIN promotes angiogenesis through hypoxia-inducible

factor-2␣–mediated regulation of soluble VEGF isoforms and their receptor
VEGFR-2
*Faten Bougatef,1-3 *Cathy Quemener,1-3 Sabrina Kellouche,2,4 Benyoussef Naïmi,5 Marie-Pierre Podgorniak,1-3 Guy Millot,1,2
Eric E. Gabison,5,6 Fabien Calvo,1-3 Christine Dosquet,2,4 Céleste Lebbé,1,2,7 Suzanne Menashi,5,6 and Samia Mourah1-3
1Inserm Unité 940, Paris; 2Université Paris 7-Institut Universitaire d’Hematologie, Paris; 3Laboratory of Pharmacology, Assistance Publique-Hôpitaux de Paris,

Hôpital Saint-Louis, Paris; 4Inserm Unité 553, Paris; 5Université Paris 12, Créteil; 6Centre National de la Recherche Scientifique-Unité Mixte de Recherche 7149,
Laboratoire Croissance, Réparation et Régénération Tissulaires, Créteil; and 7Département de Dermatologie, Hôpital Saint Louis, Paris, France

Extracellular matrix metalloproteinase in- dothelial cells, we show that EMMPRIN ily through hypoxia-inducible factor-2␣
ducer (EMMPRIN/CD147) is thought to selectively increased the soluble VEGF expression, also up-regulated by EMMPRIN.
promote tumor angiogenesis mostly isoforms (121 and 165), but not the matrix- VEGFR-2 increase was also observed in
through its protease-inducing function bound VEGF 189 form. In addition, vivo in a mouse model of xenograph tumors
and more recently by its ability to in- EMMPRIN up-regulated the expression of overexpressing EMMPRIN. These results
crease tumor cell expression of vascular VEGFR-2 without an effect on VEGFR-1. suggest that in addition to increasing
endothelial growth factor (VEGF). In this This increase in VEGFR-2 was respon- protease production, EMMPRIN may contrib-
study, we present evidence that EMMPRIN sible for the observed EMMPRIN stimula- ute to the formation of a reactive stroma
can promote angiogenesis by a direct tion of the migratory and tube formation also through the up-regulation of hypoxia-
effect on endothelial cells through a capacity of endothelial cells. EMMPRINⴕs inducible factor-2␣, VEGFR-2, and the
paracrine regulation of the VEGF/VEGF- effects, which were matrix metalloprotein- soluble forms of VEGF in endothelial cells,
receptor (VEGFR) system. Using human ase and urokinase-type plasminogen acti- thus directly regulating the angiogenic pro-
microvascular endothelial cell line–1 en- vator independent, were mediated primar- cess. (Blood. 2009;114:5547-5556)

Introduction
Angiogenesis is essential for the primary and metastatic growth of 165, 183, 189, and 206 amino acids that have been described.9
tumors, and has become a target for anticancer therapy, although VEGF121, VEGF165, and VEGF189 are the predominant isoforms
the mechanisms by which the tumor regulates angiogenesis are not secreted by a wide range of normal and transformed cells.10
fully known. The ability of tumors to recruit endothelial cells and Whereas VEGF121 diffuses relatively freely in tissues, VEGF189 has
stimulate their proliferation, migration, or survival is thought to be high affinity for heparin and is almost completely retained in the
central in tumor-induced angiogenesis. Much attention has been extracellular matrix (ECM); VEGF165 exists in both a soluble and
focused on the vascular endothelial growth factor (VEGF) family an ECM-bound form.11 Thus, the ECM represents an important
of growth factors and the receptor tyrosine kinases that mediate source of VEGF that can be released in a diffusible and bioactive
their angiogenic effects.1,2 The implication of VEGF as a mediator form by proteolysis.
of tumor angiogenesis is supported by multiple evidence. Up- VEGF acts through 2 high-affinity receptor tyrosine kinases,
regulation of VEGF has been observed in many human tumors, and VEGF receptor (VEGFR)-1/flt-1 and VEGFR-2/kinase domain
VEGF expression is closely correlated with tumor progression and receptor (KDR)/flk-1; both are expressed on normal vascular
less favorable prognosis.3-6 Furthermore, monoclonal antibodies to endothelial cells.12 VEGF signals mainly through VEGFR-2, which
VEGF and other anti-VEGF treatments have been shown to inhibit upon ligand binding becomes tyrosine phosphorylated and acti-
growth of several tumor cell lines in nude mice.7,8 VEGF expres- vates multiple signaling networks that lead to increase prolifera-
sion can be induced by numerous environmental factors. Several tion, sprouting, migration, and tube formation of endothelial
growth factors, oncogenic proteins, or transcription factors were cells.13-15 VEGFR-2 is expressed at elevated levels by endothelial
shown to up-regulate VEGF mRNA expression. VEGF gene cells engaged in angiogenesis. VEGFR-1 is expressed not only in
expression is also induced by exposure to low oxygen tension. endothelial cells, but also in macrophage-lineage cells, and was
The biologic significance of VEGF depends on the content and shown to promote tumor growth and inflammation at least partly in
ratio of the different isoforms of this growth factor and on the a macrophage-dependent manner.16 The role of VEGFR-1 in
expression of their receptors. VEGF is expressed through alterna- neoangiogenesis is much less clear, as it binds VEGF with
tive splicing as 6 different isoforms (VEGF121, VEGF145, VEGF165, approximately 10 times the affinity of VEGFR-2 binding, but its
VEGF183, VEGF189, and VEGF206) having, respectively, 121, 145, signal-transducing properties are extremely weak.17 As was already

Submitted April 19, 2009; accepted September 3, 2009. Prepublished online as The online version of this article contains a data supplement.
Blood First Edition paper, October 16, 2009; DOI 10.1182/blood-2009-
The publication costs of this article were defrayed in part by page charge
04-217380.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*F.B. and C.Q. contributed equally to this work. © 2009 by The American Society of Hematology

BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27 5547

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5548 BOUGATEF et al BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27

shown in embryogenesis, VEGFR-1 may function as a negative containing EMMPRIN full-lengh cDNA, as previously described,24 and
regulator of angiogenesis, by binding and trapping VEGF and stably transfected cells were designated as CHO-Emp cells.
preventing its binding to VEGFR-2.18 In the different experiments, HMEC-1 cells were incubated with
It is generally held that in tumor angiogenesis, VEGF action is 20 ␮g/mL membrane extract from CHO-Emp or CHO control cells,
designated Emp and Ctl, respectively. To determine how many CHO-Emp,
attributable to a paracrine mechanism by tumor cells, which
or, by comparison, tumor cells (mammary and melanoma MDA-MB231
produce VEGF, but cannot respond to it directly because they do and WM278 obtained from ATCC and Wistar Institute, respectively) are
not have cell surface VEGFRs. In contrast, endothelial cells needed to deliver 20 ␮g of EMMPRIN-enriched membranes, membrane
engaged in angiogenesis express numerous VEGFRs, but they fractions were immunoblotted for EMMPRIN alongside extracts obtained
produce only low levels of endogenous VEGF.19 However, a recent from increasing number of the above cells. The results show that 20 ␮g of
study has shown that this low expression of endogenous autocrine- EMMPRIN-enriched membranes corresponds to between 25 000 (CHO-
acting VEGF is nevertheless crucial for endothelial cell survival Emp and WM278) and 50 000 (MDA-MB231) cells. Coculture experi-
and is needed to regulate vascular homeostasis by signaling ments were performed by incubating endothelial cells (HMEC-1 or
through intracellular VEGFR-2.20 It seems, therefore, that even HUVEC) with either EMMPRIN- or mock-transfected CHO cells at a 1:1
though VEGF can originate from a variety of cells and be present in ratio for 24 hours.
In some experiments, cells were incubated with either EMMPRIN
sufficient amount in the tumor environment, its expression by the
blocking antibody (20 ␮g/mL, UM-8D6; Ancell), uPA inhibitor amiloride
endothelial cells themselves may present an essential role in (25nM; Sigma-Aldrich), MMP inhibitor marimastat (20␮M; British Biotech-
driving the angiogenesis process. nology), anti-uPA blocking antibody (50 ␮g/mL; American Diagnostica),
To form new blood vessels, activated endothelial cells must recombinant tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2
degrade the basement membrane of the original vessel and remodel (20 ␮g/mL), or the iron chelator deferoxamine (Sigma-Aldrich) to mimic
the ECM around neovasculature sites, and proteases, such as the hypoxia (100␮M).
matrix metalloproteinase (MMPs) and the urokinase-type plasmin-
ogen activator (uPA), have been largely implicated in this process. Membrane preparation
CD147/extracellular MMP inducer (EMMPRIN), a membrane
Cell membranes were isolated, as previously described.32 Briefly, CHO
glycoprotein greatly enriched on the surface of tumor cells, was
cells were scrapped in serum-free medium containing 1/100 protease
shown to stimulate neighboring stromal cells, such as fibroblasts inhibitor mixture Set V (AEBSF [4-(2-aminoethyl)benzene sulfonyl hydro-
and endothelial cells, to increase their synthesis of several choloride], aprotinin, E-64, leupeptin, and 1mM EDTA [ethylenediaminetet-
MMPs.21-23 EMMPRIN was also shown to increase expression of raacetic acid]), sonicated on ice, and centrifuged at 1000g for 10 minutes at
the plasminogen activation system, including uPA and its receptor, 4°C to remove unbroken cells. After removal of the organelles by 19 000g
both in tumor and endothelial cells, further increasing its proteo- centrifugation for 30 minutes, the membranes were pelleted at 100 000g for
lytic potential in the stroma.24 Elevated EMMPRIN levels were detected 1 hour, washed, and resuspended in serum-free Dulbecco modified Eagle
in numerous malignant tumors and have been correlated with tumor medium. The bioactivity of EMMPRIN-containing membranes was veri-
progression in experimental and clinical conditions.25,26 In some cases, it fied by its activity in stimulating uPA expression in HMEC-1 cells.24
was also associated with poor prognosis.25,27-29 Experimentally increas-
ing EMMPRIN by cDNA transfection into human breast cancer cells Enzyme-linked immunosorbent assay measurement of VEGF
greatly enhanced tumor growth in nude mice.30 HMEC-1 cells were seeded in 12-well plates (105 cells/well). After
The role of EMMPRIN in tumor progression has been attributed 24 hours, the cells were incubated with serum-free medium for 6 hours and
mostly to its protease-inducing function in both tumor cells and the then treated for 1 hour with 20 ␮g/mL anti-EMMPRIN blocking antibody
surrounding stromal cells. However, Tang et al31 have recently (Covalab) or with an immunoglobulin G (IgG) control antibody before the
reported that the up-regulation of EMMPRIN in MDA-MB231 addition of Emp for 24- and 48-hour incubation. The concentration of
tumor cells can also increase VEGF expression in these cells, VEGF protein in the conditioned media (CM) of treated HMEC-1 cells was
evoking a potential role of EMMPRIN in tumor angiogenesis. This determined using human VEGF enzyme-linked immunosorbent assay
(ELISA) quantikine kit (R&D Systems). This kit recognizes the different
prompted us to explore whether EMMPRIN can promote angiogen-
VEGF isoforms, and the detection limit was 5 pg/mL. Results were
esis through a direct effect on endothelial cells in a paracrine
normalized to cell number in the corresponding well at the time of sample
manner. Therefore, the effect of EMMPRIN on the expression of collection.
VEGF as well as that of its receptors in endothelial cells was
examined. RNA extraction, reverse transcription, and real-time
quantitative polymerase chain reaction

Total RNA was isolated using TRIzol (Invitrogen). cDNA was synthesized
Methods using random hexamers and Moloney murine leukemia virus (Invitrogen),
Cell culture according to the manufacturer’s protocol. VEGF isoforms (121, 165, and
189), VEGFRs (R1 and R2), hypoxia-inducible factor (HIF)-1␣, and
Human microvascular endothelial cell line (HMEC-1) derived from dermal HIF-2␣ mRNA expression levels were measured by real-time quantitative
microvasculature (T. Lawley, Emory University, Atlanta, GA) was main- polymerase chain reaction (qRT-PCR) using Perfect MasterMix-Probe
tained in MCDB-131 medium (Sigma-Aldrich) supplemented with 10% (AnyGenes) on LightCycler 2.0 (Roche), according to the manufacturer’s
fetal bovine serum (FBS), 2 mL glutamine (Invitrogen), 10 ng/mL endothe- protocol. The expression levels of interest transcripts were normalized to
lial growth factor (Upstate Biotechnology/Millipore), and 1 ␮g/mL hydro- the housekeeping TATA-box binding protein (TBP) gene transcripts.
cortisone (Sigma-Aldrich). Primary human umbilical vein endothelial cells
(HUVECs) obtained from PromoCell (Promo Cell) were cultured in
Western blotting analysis
endothelial cell growth medium (Promo Cell) following the instructions.
HUVECs at early passages (passages 2-4) were used in all of the Cells were lysed in TBS-Nonidet P-40 solution comprising 50mM Tris
experiments. Chinese hamster ovary (CHO) cells (ATCC) were cultured in buffer, pH 7.5, 150mM NaCl, 1% Nonidet P-40, 2mM EDTA (ethylenedia-
Dulbecco modified Eagle medium/F12 (Invitrogen) supplemented with minetetraacetic acid), and protease inhibitor mixture Set V. Cell lysates
10% FBS and 2 mL glutamine. CHO cells were transfected with a plasmid (30 ␮g) and the concentrated CM (10 ␮g) were analyzed by being subjected
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BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27 EMMPRIN PROMOTES ANGIOGENESIS THROUGH HIF-2␣ 5549

to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immuno-

blotted with anti-VEGF (Tebu-bio), anti–VEGFR-2 (R&D Systems),
anti–HIF-1␣ (BD Biosciences), or anti–HIF-2␣ antibodies (Interchim). The
Results
proteins were visualized with enhanced chemiluminescence reagent, and EMMPRIN up-regulates VEGF121, VEGF165, and VEGFR-2, but
their relative expression was determined by densitometry using ImageJ not VEGF189 and VEGFR-1 expression in endothelial cells
software program and normalized relative to the total protein concentration
of cell lysates or to ␤-actin. As EMMPRIN is an intrinsic membrane protein and primarily
functions by direct cell-cell contact mechanism, we used
Small interfering RNA transfection EMMPRIN-enriched membranes to study EMMPRIN⬘s effect
VEGF, VEGFR-1, VEGFR-2, HIF-1␣, HIF-2␣, and EMMPRIN small on endothelial cells. To determine whether EMMPRIN could
interfering RNA (siRNA) oligos (Ambion/Applied Biosystems) or scrambled induce VEGF production in a paracrine manner, we analyzed by
siRNA oligos (25 nmol/L) were transfected into HMEC-1 cells using the ELISA the level of VEGF in the conditioned media of HMEC-1
BLOCK-iT transfection kit and Lipofectamine-2000 (Invitrogen), accord- cells treated with 20 ␮g/mL membranes issue from EMMPRIN-
ing to the manufacturer’s protocol. Cells were then incubated for 24 hours transfected CHO cells (designated Emp) and compared with
before qRT-PCR and Western blotting, or trypsinized for endothelial
membranes prepared in parallel from mock-transfected CHO
capillary-like structure formation or migration assays.
cells. Western blot calibration using purified EMMPRIN has
shown that 20 ␮g of Emp membrane proteins contained 0.5 ␮g
Endothelial capillary-like structure formation
of EMMPRIN, whereas none could be detected in the mock
Human fibrinogen (Biogenic) suspended in phosphate-buffered saline was control membranes. By similar calibration alongside cell ex-
added to human thrombin (Calbiochem). Plates coated with this suspension tracts from CHO-Emp and 2 different tumor cell lines, 20 ␮g of
(500 ␮L/well) were placed at 37°C for 1 hour. The fibrin gel was Emp was estimated to be derived from between 25 000 (CHO-
equilibrated overnight with MCDB-131 medium supplemented with 2%
Emp and WM278) and 50 000 (MDA-MB231) cells.
FBS. HMEC-1 previously transfected with siRNA directed against VEGF,
VEGFR-2, or control siRNA were seeded (2.5 ⫻ 105 cells/well) on top of
As shown in Figure 1A, treatment with 20 ␮g/mL Emp resulted
these gels in 1 mL of serum-free medium, and 20 ␮g/mL Emp was then in a marked increase in VEGF secretion, from 21 pg/106 cells in
added. A mix of human recombinant VEGF (25 ng/mL) and basic fibroblast control cells to 154 pg/106 cells in Emp-treated cells after 24 hours
growth factor (bFGF; 25 ng/mL; R&D Systems) was used as a positive and from 207 to 287 pg/106 cells after 48-hour incubation.
control, and serum-free medium was used as a negative control. After To determine whether this increased VEGF in the conditioned
24 hours, the formation of capillary-like structures was photographed, and medium is due to a transcriptional up-regulation of the specific
the measure of the capillary tube length was carried out using the ImageJ isoforms of VEGF, we conducted qRT-PCR assay using isoform-
software program. specific VEGF primers and probes. Figure 1B shows that only the
soluble VEGF isoforms were significantly induced by EMMPRIN
In vitro migration assay
(1.8- and 1.6-fold for VEGF121 and VEGF165, respectively). Analy-
In vitro migration was assessed using a modified Boyden chamber assay.33 sis of VEGFRs mRNA revealed that EMMPRIN also up-regulates
HMEC-1 previously transfected with siRNA directed against VEGF, VEGFR-2 transcript (⬃ 1.7-fold) without an effect on VEGFR-1
VEGFR-1, VEGFR-2, or control siRNA were seeded (105cells/well) on the (Figure 1B). To examine the specificity of this pattern of regulation
upper chamber of the insert, and 20 ␮g/mL Emp was then added. Human by EMMPRIN, the results were compared with those obtained after
recombinant VEGF (200 ng/mL; R&D Systems) was used as a positive
treatment of HMEC-1 with bFGF, a known potent angiogenic
control. After 48 hours of incubation, cells were fixed, stained with Diff
factor. Unlike EMMPRIN, bFGF increased the transcription of all
Quik (Dade Behring), and counted under a microscope.
VEGF isoforms, including VEGF189 (1.7-, 1.5-, and 1.35-fold for
Animal experiments VEGF121, VEGF165, and VEGF189, respectively), although its effect
on the VEGFRs was similar to that of EMMPRIN (VEGFR-2,
All in vivo experiments were carried out with local ethical committee 1.25-fold).
approval of University Paris 12 and according to United Kingdom This up-regulation was observed also at the protein level, as
Co-ordinating Committee on Cancer Research guidelines. Nu/nu mice
shown by the Western blot analysis. VEGF121 and VEGF165 protein
(Janvier) were purchased at 4 weeks of age. Transformed mammary tumor
cells NS2T2A24 were transfected with EMMPRIN cDNA, as described
levels were increased by approximately 45% and 80%, respec-
above for CHO cells. Suspensions of NS2T2A-Emp or NS2T2A-mock cells tively, in the conditioned media, and by 55% and 60% in the cell
(4 ⫻ 106 in 100 ␮L of phosphate-buffered saline) were subcutaneously lysates (Figure 1C) after treatment with Emp. VEGFR-2 protein
injected into the left side of 6 nude mice per group.24 Tumors were resected level was increased by 65%.
5 weeks after injection and stored in liquid nitrogen before RNA extraction We next performed coculture experiments using EMMPRIN- or
and immunohistochemistry. mock-transfected CHO cells with HMEC-1 cells to confirm that the
same effect could be observed using intact cells. Figure 1D shows
Immunohistochemistry that both VEGF and VEGFR-2 mRNA are up-regulated in HMEC-1
Immunohistochemical analyses of tissue sections of tumors obtained as cells when cultured with CHO-Emp cells, as was found with the
above were carried out using antibody directed against VEGFR-2 (R&D membrane fractions. Similar results were obtained with primary
Systems). Sections were incubated overnight with the primary antibody and umbilical endothelial cells (HUVECs, Figure 1D), showing that
then incubated with the biotinylated secondary antibody. Peroxidase this effect is not specific for the HMEC-1 cell line. Western blot
reactivity was visualized using 3-amino-9-ethylcarbazole (DakoCytomation). analysis confirmed this regulation at the protein level in both cell
type cocultures (Figure 1D).
Statistical analysis In vivo analysis of VEGFR-2 expression, by both qRT-PCR and
Data are presented as the mean values plus or minus SD. Mann-Whitney immunohistochemistry in sections obtained from tumors grown in
test was used to evaluate differences between groups. P value less than .05 nude mice (as we previously described24), suggests that a similar
was accepted as significant. regulation of VEGFR-2 by EMMPRIN occurs in vivo (Figure 1E).
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5550 BOUGATEF et al BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27

A VEGF total protein B 4 VEGF isoforms/ VEGFR-2 transcripts

NS

Relative m RNA level transcript

3,5

NS
Control
*

copies/TBP
2,5

Emp 2

1,5
*
350 Control
* 1
300
* Emp
VEGF (pg/10 cells)

0,5
250
0
6

200
* 4 VEGF121 VEGF165 VEGF189 VEGFR-2 VEGFR-1

Relative mRNA level transcript

150 3,5
100 3

copies/TBP
50 2,5

0 2
1,5
24h 48h Control
1
10ng/mL bFGF
0,5
0
C VEGF121 VEGF165 VEGF189 VEGFR-2 VEGFR-1

Cell lysates Conditionned D HMEC-1+ CHO-Mock HUVEC + CHO-Mock *

14
media 1,8 HMEC-1+ CHO-Emp HUVEC + CHO-Emp
1,6
* 12
NS
1,4 10
Emp - + - + 1,2
8
1 *
VEGF189 0,8 6 * * NS

0,6 * 4
VEGF165 0,4
0,2 2
VEGF121 0 0
VEGF121 VEGF165 VEGFR-2 VEGFR-1 VEGF121 VEGF165 VEGFR-2 VEGFR-1
VEGFR-2
HMEC-1+ HMEC-1+ HUVEC + HUVEC +
CHO-Mock CHO-Emp CHO-Mock CHO-Emp

VEGFR-2 VEGFR-2
β-actin
VEGF165 VEGF165
β-actin β-actin
E
3
transcript copies/TBP

*
Mouse VEGF-R2

2
VEGFR-2 mRNA

0
NS2T2A-MOCK NS2T2A-EMMPRIN

VEGFR-2 protein

NS2T2A-MOCK NS2T2A-EMMPRIN
Figure 1. EMMPRIN up-regulates VEGF121, VEGF165, and VEGFR-2, but not VEGF189 and VEGFR-1 expression in endothelial cells. HMEC-1 cells were incubated with 20 ␮g/mL Emp
in serum-free medium. (A) After 24 and 48 hours, CM were collected and soluble VEGF production was measured by ELISA. Results were normalized to the cell number and expressed as
pg/106 cells. Columns indicate means of 3 independent experiments; and bars, SD. *P ⬍ .05. (B) Total RNA was extracted from HMEC-1 cells treated with Emp or with 10 ng/mL bFGF for
4 hours. mRNAlevels for VEGF121, VEGF165, VEGF189, VEGFR-2, and VEGFR-1 were quantified using qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene
of at least 3 independent experiments; and bars, SD. *P ⬍ .05. (C) Cell lysates were immunoblotted for VEGF isoforms and VEGFR-2 (␤-actin was used as loading control). Concentrated CM
were immunoblotted with anti-VEGF antibody. Band densities were expressed relative to total protein concentration of the corresponding cell lysates. Representative blots of 3 independent
experiments. (D) HMEC-1 and HUVECs cocultured with EMMPRIN- or mock-transfected CHO cells. Total RNA was extracted from cocultured cells. mRNA levels for VEGF121, VEGF165,
VEGFR-2, and VEGFR-1 were quantified using qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD.
*P ⬍ .05. Cocultured cell lysates were immunoblotted for VEGF isoforms and VEGFR-2 (␤-actin was used as loading control). Representative blots of 3 independent experiments.
(E) EMMPRIN regulates mouse VEGFR-2 in xenograph tumors in vivo. Tumors grown in nude mice obtained from EMMPRIN- or mock-transfected mammary tumor sections (NS2T2A) were
analyzed for mouse VEGFR-2 expression by both qRT-PCR and immunohistochemistry. Mouse VEGFR-2 transcripts were quantified using qRT-PCR. Columns indicate means of relative
expression to mouse TBP housekeeping gene of at least 3 independent experiments. Bars indicate SD. *P ⬍ .05. Mouse flk-1/VEGFR-2 protein was analyzed by immunohistochemistry.
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BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27 EMMPRIN PROMOTES ANGIOGENESIS THROUGH HIF-2␣ 5551

A B
VEGF total protein VEGF isoforms/ VEGFR-2 transcripts

NS 3,0

VEGF121 mRNA
2,0

400 * * 1,0
* VEGF121
VEGF pg/106 cellules

350 0,0
ct 1h 2h

300
0,4
250

VEGF165 mRNA
0,3
200
150 0,2 *
100 0,1
VEGF165
50 0
ct 1h 2h
0
cho Emp Emp + IgG ct Emp + ac bloq Emp 3
NS

VEGF189 mRNA
2
Anti-EMMPRIN blocki
king
Antibody 1
VEGF189
IgG ct Antibody 0
ct 1h 2h

3,0

VEGF-R2 mRNA
2,0

1,0 * VEGFR-2
0,0
ct 1h 2h
Emp + IgG ct Antibody

Emp + Anti-EMMPRIN
blocking Antibody
Figure 2. Anti-EMMPRIN antibody inhibits VEGF isoforms and VEGFR-2 expression induced by EMMPRIN. (A) HMEC-1 cells were incubated with anti-EMMPRIN
blocking antibody or with an IgG control antibody in serum-free medium before the addition of Emp for 48 hours, and VEGF in the CM was quantified by ELISA. Results were
normalized to the cell number and expressed as pg/106 cells. Columns indicate means of 3 independent experiments; and bars, SD. *P ⬍ .05. (B) HMEC-1 cells were
incubated for 2 hours with 20 ␮g/mL anti-EMMPRIN blocking antibody or with an IgG control antibody in serum-free medium. VEGF121, VEGF165,VEGF189, and VEGFR-2
transcripts were quantified by qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD.
*P ⬍ .05.

Mouse VEGFR-2 transcripts were more expressed (⬃ 2.4-fold) in the VEGF-siRNA–transfected cells with EMMPRIN partially
the tumors obtained from EMMPRIN-transfected mammary tumor restored tube formation. This increased tube formation by
cells (NS2T2A) compared with mock-transfected cells. This was EMMPRIN, which did not involve a parallel increase in VEGF
associated with a more intense staining of VEGFR-2 protein shown levels, may be explained by the increased VEGFR-2 transcription
by immunostaining. by EMMPRIN (Figure 3A), which could then be activated by the
To evaluate the specificity of EMMPRIN⬘s effects, we then remaining uninhibited VEGF protein.
examined the effect of blocking anti-EMMPRIN antibody added On the contrary, the inhibition of tube formation observed with
with the EMMPRIN-containing membranes. As shown in Figure VEGFR-2 siRNA-transfected cells could not be restored by the
2A, HMEC-1 cells treated for 48 hours with 20 ␮g/mL blocking addition of EMMPRIN (Figure 3B) in spite of the presence of
anti-EMMPRIN antibody resulted in a significant decrease in VEGFR-1 and the induction of VEGF by EMMPRIN. In addition,
VEGF level in the culture supernatants, whereas IgG control the measured level of VEGFR-2 transcript mirrored that of tube
antibody had almost no effect. This down-regulation was also formation (Figure 3B), further suggesting that the induction of this
observed at the transcript level (Figure 2B), where the incubation biologic function by EMMPRIN passes primarily through
with the anti-EMMPRIN antibody showed a maximal decrease at VEGFR-2.
2-hour incubation for VEGF121, VEGF165, and VEGFR-2 (⬃ 25%,
35%, and 32%, respectively) with no effect on VEGF189. EMMPRIN induces endothelial cell migration through VEGFR-2

EMMPRIN induces endothelial cell tube formation through VEGF has been shown to regulate endothelial cell migration, an
VEGF/VEGFR-2 essential step in the angiogenesis process, and both VEGFR-1 and
VEGFR-2 have been implicated. To determine whether EMMPRIN
To determine the biologic implications of the regulation of VEGF can regulate endothelial cell migration through the up-regulation of
by EMMPRIN, we studied tube formation by HMEC-1 cells grown VEGFR-2, the expression of both VEGFR-1 and VEGFR-2 was
on a fibrin gel (Figure 3). EMMPRIN was able to significantly inhibited by siRNA transfection and cell migration was followed
induce tube formation of HMEC-1 cells (3.6-fold compared with by a modified Boyden chamber. Migration induced by VEGF
control), reaching 60% of the maximal value obtained with the mix (⬃ 2.3-fold increase) or inhibited by VEGF siRNA (70% inhibi-
of human recombinants VEGF and bFGF used as a positive control. tion) was used as control (Figure 4).
VEGF siRNA transfection of HMEC-1 cells significantly reduced Transfection of HMEC-1 cells with VEGFR-2 siRNA inhibited
the VEGF expression both at the protein and mRNA levels (60% cell migration to a much greater extent (63%) than with VEGFR-1
reduction of RNA levels) as well as their capillary-like formation siRNA (24%). EMMPRIN enhanced cell migration (25% in
compared with scrambled control siRNA transfection. Treatment of comparison with control cells) and was able, in addition, to restore
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5552 BOUGATEF et al BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27

VEGF + bFGF Scrambled siRNA siRNA VEGF

A 120

Cumulative tube
100

length (%)
80 *
60

40 *
20

*
30
*

mRNA level
25

20
VEGF121
15
VEGF165
10
VEGFR-2
5
VEGFR-1
0
Emp - + - +
B

Cumulative tube
70 Scrambled siRNA siRNA VEGFR-2
*

length (%)
50
NS
20

0
45

*
VEGFR-2
mRNA

30

NS
15

0
Emp - + - +
Figure 3. EMMPRIN induces capillary-like structure formation by endothelial cells via VEGF and VEGFR-2. HMEC-1 transfected with VEGF, VEGFR-2 siRNA, or
scrambled siRNA were seeded on top of a fibrin gel and treated with Emp for 24 hours in serum-free medium. (A) Capillary-like structure formation by HMEC-1 cells transfected
with siRNA-targeting common sequence of VEGF isoforms. A mix of VEGF and bFGF and serum-free medium was used as positive and negative controls, respectively.
VEGF121, VEGF165,VEGF189, VEGFR-1, and VEGFR-2 transcript quantification by qRT-PCR. (B) Capillary-like structure formation by HMEC-1 cells transfected with VEGFR-2
siRNA. VEGFR-2 transcript quantification by qRT-PCR. Results are presented as the mean of 3 independent experiments and expressed as a percentage of the cumulative
tube length obtained for the positive control set at 100%. Columns indicate means of relative expression to TBP housekeeping gene of 3 independent experiments; and bars,
SD. *P ⬍ .05.

cell migration in VEGFR-1, but not VEGFR-2 siRNA-inhibited EMMPRIN up-regulates VEGF and VEGFR-2 in a MMP- and
cells. This may be explained by the fact that EMMPRIN, by uPA-independent manner
up-regulating both VEGF and VEGFR-2, can compensate for the
inhibited VEGFR-1, but because it does not regulate VEGFR-1, the As EMMPRIN is best known as a MMP inducer, and MMPs, such as
addition of EMMPRIN to the cells treated by VEGFR-2 siRNA had membrane type 1 (MT1)–MMP,34 were shown to transcriptionally
no significant effect. This was confirmed by the RNA measure- regulate VEGF, we sought to determine whether the induction of VEGF
ments showing that EMMPRIN increased both VEGF and VEGFR-2 by EMMPRIN may involve a MMP-dependent mechanism. To this end,
transcripts in the VEGFR-1–inhibited cells, but only the VEGF we examined the effect of marimastat, large spectrum inhibitor of
transcript in VEGFR-2 siRNA-transfected cells (data not shown). MMPs, or recombinant physiologic MMP inhibitors (TIMP-1 and

VEGF Scrambled VEGFR-2 VEGFR-1

4000 siRNA siRNA siRNA siRNA
Migrated cell

3000
number

*
2000 *
NS
1000

VEGF + - - - - - - -
Emp - - - + - + - +
Figure 4. EMMPRIN induces migration in HMEC-1 endothelial cells via VEGFR-2. HMEC-1 cells transfected with VEGF, VEGFR-1, VEGFR-2 siRNA, or scrambled siRNA were
seeded in a 12-well insert of Boyden chambers and then treated or not with Emp for 24 hours. VEGF (200 ng/mL) was used as a positive control. After 48 hours of incubation, cells were
fixed, stained with Diff-Quik, and counted under a microscope. Columns indicate means of 3 independent experiments carried out in triplicate; and bars, SD. *P ⬍ .05.
 From [Link] by guest on September 11, 2016. For personal use only.

BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27 EMMPRIN PROMOTES ANGIOGENESIS THROUGH HIF-2␣ 5553

A B
MMP pathway uPA pathway
i

transcript copies/TBP
1,4

Relative mRNA level

transcript copies/TBP

2
Relative mRNA level

1,2
1
1,5 0,8
0,6
1 0,4
0,2
0,5 0
VEGF121 VEGF165 VEGF R2
0
Ct 10h
VEGF121 VEGF165 VEGF189 VEGF R2
10h 25nM amiloride
HMEC-1 HMEC-1 + Emp 24h 25nM amiloride
+ Emp + 20µM marimastat
ii

transcript copies/TBP
1,4

Relative mRNA level

1,2
1
0,8
0,6
0,4
0,2
0

VEGF121 VEGF165 VEGF R2

10h IgG ct Antibody (50µg/mL)

10h Anti-uPA blocking Antibody (50µg/mL)
24h Anti-uPA blocking Antibody (50µg/mL)
Figure 5. EMMPRIN up-regulates VEGF and VEGFR-2 in a MMP- or uPA-independent manner. (A) HMEC-1 cells were incubated for 12 hours with 20␮M marimastat in
serum-free medium before treatment with Emp for 2 hours. VEGF121, VEGF165, VEGF189, and VEGFR-2 transcripts were quantified by qRT-PCR. (B) HMEC-1 cells were
incubated with 25nM amiloride (i) or 50 ␮g/mL anti-uPA blocking antibody (ii) for 10 hours and 24 hours in serum-free medium. VEGF121, VEGF165,VEGF189, and VEGFR-2
transcripts were quantified by qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD.

TIMP-2) on VEGF and VEGFR-2 mRNA expression in HMEC-1 cells treatment may result from a stimulation of these transcription
treated by EMMPRIN. factors. The data in Figure 6 show that EMMPRIN up-regulated
As shown in Figure 5A, the levels of either VEGF isoforms or HIF-1␣ and HIF-2␣ both at the RNA and protein level. This
VEGFR-2 expression induced by EMMRPIN were not affected by increase, already noted after 30 minutes at the RNA and 1 hour at
marimastat (20␮M), suggesting a MMP-independent mechanism. the protein level, preceded that of VEGF and VEGFR-2 (data not
The implication of MT1-MMP, known to be inhibited only by shown). The increase in HIF-1␣ and HIF-2␣ was confirmed with
TIMP-2 and not by TIMP-1, was also excluded by demonstrating both HMEC-1s and HUVECs when cultured in a coculture system
that EMMPRIN-mediated VEGF induction was similarly unaf- with intact CHO-Emp cells (Figure 6C).
fected by either TIMPs (data not shown). We next examined the effect of HIF silencing, by using both
We next examined the possible implication of uPA, which we HIF-1␣ and HIF-2␣ siRNA, on EMMPRIN regulation of VEGF
have previously shown to also be induced by EMMPRIN in both system. Specific inhibition of either HIF-1␣ or HIF-2␣ was
epithelial and endothelial cells.24 The treatment of the cells with achieved, as shown by HIF-1␣ and HIF-2␣ RNA analysis
25nM amiloride, a synthetic uPA inhibitor (Figure 5Bi) or 50 ␮g/ (Figure 6A). When silencing HIF-1␣, EMMPRIN only in-
mL anti-uPA blocking antibody (Figure 5Bii) for 10 hours and creased HIF-2␣ with no effect on HIF-1␣; similarly, silencing
24 hours in serum-free medium did not affect EMMPRIN-induced HIF-2␣ did not affect EMMPRIN induction of HIF-1␣. Figure
VEGF or VEGFR-2 mRNA expression. 6B shows that VEGFR-2 up-regulation by EMMPRIN was
significantly inhibited by siRNA of HIF-2␣, but not of HIF-1␣.
EMMPRIN up-regulates VEGF and VEGFR-2 through
However, the induction of VEGF121 (and VEGF165; data not shown)
stimulation of HIF
appears to involve both HIF-1␣ and HIF-2␣, although HIF-2␣ silencing
As HIF-1␣ and HIF-2␣ are considered as key regulators of VEGF had a much greater inhibitory effect than that of HIF-1␣. These results
and VEGF-R gene expression, we sought to determine whether the suggest that HIF-2␣ plays a central role in EMMPRIN regulation of
increased expression of VEGF and VEGFR-2 after EMMPRIN VEGFR-2 as well as VEGF.
 From [Link] by guest on September 11, 2016. For personal use only.

5554 BOUGATEF et al BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27

A
i ii
HIF-1α ctl Emp HIF-2α ctl Emp

7
Ctl Emp Ctl DFO
6 * 14
*
HIF-1α mRNA

12

HIF-2α mRNA
5
HIF-1α
* 10
*
4
8 HIF-2α
3 6 β−actin
2 NS 4
NS
1 2

0 0
Scrambled Scrambled HIF-1 siRNA HIF-2 siRNA
HIF-1 siRNA HIF-2 siRNA
siRNA siRNA

B C HMEC-1+ CHO-Mock HUVEC+ CHO-Mock

3,5
HMEC-1+ CHO-Emp HUVEC+ CHO-Emp
3 * 8
7
*

HIF mRNA

HIF mRNA
2,5 6
2 5
VEGF121 VEGFR-2
4
*
ctl ctl Emp 1,5
Emp
1
* 3
2
30 * *
VEGF-R2 mRNA

0,5
*
VEGF121 mRNA

60 1
25
50 0 0
20 HIF-1α HIF-2α
40 * NS
HIF-1α HIF-2α

30
15
NS + 1+ + +
-1 k C ck
10 EC Moc C- mp C p
VE Mo UVE Em
20
HM O- HME -E HU O- H -
O O
10 5 CH CH CH CH
0 0 HIF-2α HIF-2α
Scrambled HIF-1 siRNA HIF-2 siRNA Scrambled HIF-1 siRNA HIF-2 siRNA
siRNA siRNA β-actin β-actin

Figure 6. EMMPRIN up-regulates VEGF and VEGFR-2 through HIF stimulation. (A) EMMPRIN up-regulates HIF-1␣ and HIF-2␣ under normoxic conditions. (i) HMEC-1
cells were transfected with HIF-1␣, HIF-2␣ siRNA, or scrambled siRNA before treatment with 20 ␮g/mL Emp for 1 hour. HIF-1␣ and HIF-2␣ transcripts were quantified by
qRT-PCR. (ii) The cells were incubated with Emp or desferrioxamine (DFO). The HIF-1␣ and HIF-2␣ levels were detected by Western blotting. (B) EMMPRIN up-regulates
VEGFR-2 and soluble VEGF isoforms via HIF-2␣. HMEC-1 cells were transfected with HIF-1␣, HIF-2␣ siRNA, or scrambled siRNA before treatment with Emp. VEGFR-2 and
VEGF121 transcripts were quantified by qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars,
SD. *P ⬍ .05. (C) HMEC-1 and HUVECs cocultured with EMMPRIN- or mock-transfected CHO cells. Total RNA was extracted from cocultured cells. mRNA levels for HIF-1␣
and HIF-2␣ were quantified using qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD.
*P ⬍ .05. Cocultured cell lysates were immunoblotted for HIF-2␣ (␤-actin was used as loading control). Representative blots of 3 independent experiments.

EMMPRIN can enhance tumor angiogenesis by a paracrine effect

Discussion on endothelial cells’ expression of VEGFR-2 and may account, at
least in part, for the elevated VEGFR-2 expression observed in
EMMPRIN is highly expressed on the surface of tumor cells and tumor vasculature.39 As VEGF can be expressed by a variety of
stimulates surrounding fibroblasts and endothelial cells to produce cells, the effect of EMMPRIN on VEGF production in tumor cells31
MMPs and uPA in a paracrine fashion.24,25,35 Certain MMPs can and in endothelial cells, as shown in this study, would be expected
release bound VEGF, thus involving EMMPRIN in the regulation to increase the pool of VEGF in the tumoral tissue. It is interesting
of VEGF bioavailability.36 UPA was also shown to cleave the in this respect that endogenous endothelial VEGF was shown,
matrix-bound VEGF189, releasing a diffusible fragment having the albeit under nonpathologic conditions, to serve a particular role in
ability to activate VEGFR-2 and induce proliferation.37 However, cell survival that cannot be provided by VEGF secreted from
EMMPRIN was also shown to stimulate the transcriptional expres- adjacent cell types.20 It remains to be seen whether this endothelial
sion of VEGF in tumor cells via the phosphatidylinositol 3-kinase– autocrine production induced by EMMPRIN may also have a more
Akt signaling pathway.31,38 In the present study, we further show specific function in tumor angiogenesis, such as increasing viabil-
that EMMPRIN can have a direct effect on endothelial cells, ity and stability of the newly formed blood vessels.
specifically up-regulating VEGFR-2 and its soluble ligands 121 EMMPRIN has already been suggested to play a role in
and 165, and as a consequence, increasing both migration and tube angiogenesis through the induction of MMPs and, hence, tissue
formation. Neither the expression of VEGFR-1 nor that of the remodeling, endothelial cell migration, and invasion. MT1-MMP, a
larger matrix-bound VEGF isoforms was affected by EMMPRIN. transmembrane metalloproteinase whose role as an activator of
This may have an important biologic significance, as although pro-MMP-2 has been extensively studied, was also shown to
VEGF can bind to both receptors, VEGFR-2 appears to play a more promote angiogenesis independently of MMP-2 by enhancing
important role in initiating signal transduction pathways within VEGF production in tumor cells.40 This effect of MT1-MMP,
endothelial cells due to its greater kinase activity and, thus, in which was shown to involve the Src tyrosine kinase signaling
promoting endothelial cell tube formation.9 These results further pathway, was nevertheless dependent on the catalytic domain of the
expand on the function of EMMPRIN in tumorogenesis and molecule.34 As EMMPRIN was shown to increase expression of
metastasis, as in addition to its known effect on tumor invasion MT1-MMP,41 we investigated the possibility that its effect on
through the induction of proteinases in the surrounding tissue, VEGF production may be mediated through this proteinase. Our
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BLOOD, 24 DECEMBER 2009 䡠 VOLUME 114, NUMBER 27 EMMPRIN PROMOTES ANGIOGENESIS THROUGH HIF-2␣ 5555

results show, however, that neither marimastat, a broad spectrum in agreement with the previously reported specific regulation of
MMP inhibitor, nor TIMP-1 and TIMP-2, which differentially VEGFR-2 by HIF-2␣, but not by HIF-1␣.47
inhibit MT1-MMP, nor MT1-MMP siRNA (data not shown) had By inducing HIF-2␣ under normal oxygen levels, EMMRPIN
any effect on the synthesis of VEGF induced by EMMPRIN. may contribute to the development of tumor aggressiveness by
Furthermore, the inhibition of uPA, the serine protease also induced inducing the program for a hypoxic phenotype also at near
by EMMPRIN24 and which was likewise shown to up-regulate physiologic oxygen tensions, thus promoting angiogenesis, through
VEGF, was without an effect, further confirming that VEGF VEGF and VEGFR-2 production, in nonhypoxic conditions. This
regulation by EMMPRIN is independent of either the MMP or the view is supported by the experimental evidence from different
uPA systems. cellular models showing that HIF-2␣ is more stable than HIF-1␣ at
It was previously suggested that the MMP-inducing function of normoxia and is transcriptionally active at such conditions.48 It is
EMMPRIN within tumor cells involves homophilic interactions, with interesting that HIF-2␣ was also shown to regulate other gene
EMMPRIN acting as a counterreceptor for itself.42 Such interactions targets such as uPA receptor49 and MT1-MMP,50 which are both
may exist in our system, as the inhibition of endogenous EMMPRIN increased by EMMPRIN. It remains to be seen, however, whether
expression in endothelial cells with siRNA inhibited cell migration as the increase in HIF-2␣ represents a central pathway for the increase
well as the mRNA levels of VEGR-2 and HIF-2␣, although it did not of these EMMPRIN target genes.
completely abrogate EMMPRIN’s effects (see supplemental File 1, Our results presented in this study strengthen the emerging view
available on the Blood website; see the Supplemental Materials link at that the role of EMMPRIN in tumor progression is not strictly
the top of the online article). This may be due to the noninhibited restrained to its widely demonstrated role in MMP induction and
fraction of EMMPRIN that still remains after the siRNA treatment, matrix turnover, but also through VEGF/VEGFR-2–mediated
although the possibility that EMMPRIN can also exert its effects on control of the angiogenesis process by a paracrine effect on the
endothelial cell migration through other nonhomophilic cell-cell interac- endothelial cells. Such findings have direct impact in cancer
tions may not be excluded. therapy by providing a possibility, through the inhibition of
EMMPRIN regulated differently the various VEGF isoforms; EMMPRIN, to inhibit simultaneously VEGF signaling and tissue
those are known to be generated by different processes. These may remodeling associated with tumoral angiogenesis.
involve the alternative 5⬘ splicing, giving rise to either VEGF189 or
VEGF206, the alternative 3⬘ splicing to form either VEGF165 or the
antiangiogenic peptide VEGF165b,43 and the cassette exons forming Acknowledgments
VEGF165 or VEGF121.15 Little is known at present on the mecha-
We thank Dr E. Huet for stimulating discussions.
nism of the regulation of VEGF splicing, although 2 factors, the
This work was supported by the Fondation Charles Oberling
Sam68-like mammalian protein 244 and coactivator of activator
(C.Q.), the Société Française de Dermatologie, and the Fondation
protein 1 and estrogen receptor alpha (CAPER␣),45 have both been
de L’Avenir (study ET8-489).
shown to be able to alter splicing of the VEGF gene. It is hard to
speculate at this point on the mechanism by which EMMPRIN
modifies the ratio of the different VEGF isoforms, but it is Authorship
interesting to note that CAPER␣ inhibition resulted in an increase
in VEGF121/189 ratio similar to EMMPRIN⬘s effect. Contribution: F.B., C.Q. and E.E.G. performed research and
Our results also show that the regulation of VEGF and analyzed data; S.K. performed research and analyzed data; B.N.
VEGFR-2 by EMMRPIN is mediated through the regulation of performed research, provided analytic tools, and analyzed data;
HIF␣, transcription factors that have a central role in angiogen- M.-P.P. performed research; G.M. performed microscopic imaging;
esis.46 Even though these factors are known to be highly regulated F.C. designed the research; C.D. analyzed data and conducted
by cellular oxygen levels, other elements of the inflammatory and research discussions; C.L. analyzed data and conducted research
tumor microenvironment were shown to influence their accumula- discussions; and S. Menashi and S. Mourah designed the research,
tion and activity under normal oxygen concentration. Whereas both analyzed data, and wrote the paper.
HIF-1␣ and HIF-2␣ were up-regulated by EMMPRIN, our results Conflict-of-interest disclosure: The authors declare no compet-
show that HIF-2␣ has a more prominent role in EMMPRIN ing financial interests.
regulation of the VEGF system and more particularly of VEGFR-2, Correspondence: Samia Mourah, Laboratoire de Pharmacologie
as the inhibition of HIF-2␣, but not that of HIF-1␣, almost and Inserm Unité 940, Hôpital Saint-Louis, 27, rue Juliette Dodu,
completely blocked EMMPRIN⬘s induction of VEGFR-2. This is 75010 Paris, France; e-mail: [Link]@[Link].

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 From [Link] by guest on September 11, 2016. For personal use only.

2009 114: 5547-5556

doi:10.1182/blood-2009-04-217380 originally published
online October 16, 2009

EMMPRIN promotes angiogenesis through hypoxia-inducible factor-2α−

mediated regulation of soluble VEGF isoforms and their receptor
VEGFR-2
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