From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Endothelial intercellular adhesion molecule (ICAM)–2 regulates angiogenesis
Miao-Tzu Huang, Justin C. Mason, Graeme M. Birdsey, Valerie Amsellem, Nicole Gerwin, Dorian O. Haskard,
Anne J. Ridley, and Anna M. Randi
Endothelial junctions maintain endothelial integrity and vascular homeostasis.
They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis.
Junctional adhesion molecules such as
vascular endothelial (VE)–cadherin and
CD31/platelet endothelial cell adhesion
molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule
intercellular adhesion molecule 2 (ICAM-2)
is expressed at the endothelial junctions.
In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2–deficient mice and
ICAM-2–deficient endothelial cells, we
show that the lack of ICAM-2 expression
results in impaired angiogenesis both in
vitro and in vivo. We show that ICAM-2
supports homophilic interaction, and that
this may be involved in tube formation.
ICAM-2–deficient cells show defective in
vitro migration, as well as increased
apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine.
ICAM-2 signaling in human umbilical vein
endothelial cells (HUVECs) was found to
activate the small guanosine triphosphatase (GTPase) Rac, which is required
for endothelial tube formation and migration. These data indicate that ICAM-2 may
regulate angiogenesis via several mechanisms including survival, cell migration,
and Rac activation. Our findings identify
a novel pathway regulating angiogenesis
through ICAM-2 and a novel mechanism
for Rac activation during angiogenesis.
(Blood. 2005;106:1636-1643)
© 2005 by The American Society of Hematology
Introduction
Angiogenesis involves a cascade of events that requires the
disassembly of endothelial junctions, followed by detachment,
proliferation, and migration of endothelial cells (ECs), and finally
the re-establishment of cell-cell and cell-matrix contacts.1 Adhesion molecules at the endothelial junctions, such as CD31/platelet
endothelial cell adhesion molecule (PECAM) and junctional adhesion molecule (JAM)–A, support endothelial cell-cell contact through
homophilic binding, and are involved in regulating endothelial homeostasis and angiogenesis via different mechanisms. Junctional molecules
also mediate leukocyte transendothelial migration through homophilic
or heterophilic interactions with leukocytes.2
Intercellular adhesion molecule 2 (ICAM-2) is a member of the
immunoglobulin (Ig) superfamily, constitutively expressed at the
endothelial junctions.3 Its structure includes 2 extracellular Ig
domains, a transmembrane region, and a short intracellular tail.4
ICAM-2, originally identified as a ligand for leukocyte integrin
lymphocyte function–associated antigen-1 (LFA-1),4 also interacts
with the integrin Mac-15 and the dendritic cell receptor DC-SIGN
(dendritic cell–specific ICAM-3–grabbing nonintegrin).6 Through these
interactions, ICAM-2 mediates leukocyte trafficking.4,6,7 Recent data
indicate that ICAM-2 involvement in leukocyte transmigration may be
stimulus specific.8 However its role in inflammation is still unclear:
inflammatory cytokines that up-regulate the expression of leukocyte
adhesion molecules such as ICAM-1 or vascular cell adhesion molecule
1 (VCAM-1), down-regulate ICAM-2 expression and induce its redistribution away from the junctions.3 A similar pattern is observed for other
junctional adhesion molecules such as CD31/PECAM9 and JAM-A.10
On the basis of the similarities in function, structure and
localization between ICAM-2 and other junctional adhesion molecules involved in vascular homeostasis, we postulated that
ICAM-2 also may play a role in angiogenesis. Data on the
regulation of ICAM-2 expression further support this hypothesis.
In endothelial cells, ICAM-2 expression can be regulated by
cell-cell contact and growth factors (M.-T.H. and A.M.R., manuscript in preparation). ICAM-2 endothelial expression is driven by
the transcription factor Erg,11 which is required for endothelial cell
(EC) tube formation on Matrigel12 and also regulates the expression of angiogenesis mediators including thrombospondin, SPARC
(secreted protein acidic and rich in cysteine), RhoA, and vascular
endothelial (VE)–cadherin.12,13
From the British Heart Foundation (BHF) Cardiovascular Sciences Unit, Eric
Bywaters Centre for Vascular Inflammation, Hammersmith Hospital, Imperial
College, London, United Kingdom; Ludwig Institute for Cancer Research,
Royal Free and University College School of Medicine, London, United
Kingdom; and The Center for Blood Research, Harvard Medical School,
Boston, MA.
V.A. performed research, contributed to data analysis and interpretation; N.G.
provided key tools; D.O.H. contributed to design of research, data analysis and
interpretation, and key tools; A.J.R. contributed to design of research, data
analysis and interpretation, and key tools; and A.M.R. provided the original
idea, designed research, contributed key tools and reagents, analyzed and
interpreted data, and wrote the paper.
Submitted December 13, 2004; accepted May 7, 2005. Prepublished online as
Blood First Edition Paper, May 26, 2005; DOI 10.1182/blood-2004-12-4716.
An Inside Blood analysis of this article appears in the front of this issue.
Supported by the British Heart Foundation (A.M.R., D.O.H., M.-T.H., G.M.B.),
Ludwig Institute for Cancer Research (A.J.R.), and European Union Network of
Excellence MAIN (FP6-502935) (V.A.)
M.-T.H. performed research, generated reagents, data analysis and
interpretation, contributed to design of research and writing the paper; J.C.M.
performed research, contributed to data analysis and interpretation; G.M.B.
performed research, contributed to data analysis and interpretation;
1636
Reprints: Anna M. Randi, Imperial College, BHF Cardiovascular Sciences
Unit, Hammersmith Hospital, Du Cane Road, London W12 0NN United
Kingdom; e-mail: [email protected].
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.
© 2005 by The American Society of Hematology
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
In this paper we demonstrate that ICAM-2 is required for in
vitro and in vivo angiogenesis. We also demonstrate for the first
time that ICAM-2 can engage in homophilic interaction, and is
involved in endothelial survival and migration. Finally, we show
that ICAM-2 signaling activates the small guanosine triphosphatase (GTPase) Rac, a key regulator of signal transduction
pathways to the cytoskeleton. In conclusion, we have identified a
novel angiogenesis pathway, involving the adhesion molecule
ICAM-2 and the activation of the small GTPase Rac.
Materials and methods
Animals
ICAM-2⫺/⫺ mice were generated as described.7 129SV ⫻ C57BL/6 wildtype (WT) mice were generated at the Biological Service Unit, Hammersmith Hospital (London, United Kingdom). Experiments were performed
according to the Animals Scientific Procedures Act of 1986.
ENDOTHELIAL ICAM-2 REGULATES ANGIOGENESIS
1637
formation was studied over 8 hours and serial photographs of representative 100 ⫻ fields were taken hourly. Endothelial tubes were quantified by
counting branches from each EC. Where indicated, cells were preincubated
with anti–ICAM-2 or control Ab for 15 minutes at room temperature (RT)
before plating onto Matrigel.
In vivo Matrigel plug assay
Matrigel (300 ␮L) containing 40 ng/mL vascular endothelial growth factor
(Peprotech EC, London, United Kingdom) and 10 U/mL heparin (Roche,
Lewes, East Sussex, United Kingdom) was injected subcutaneously into the
abdominal area of the mouse. Seven days later, Matrigel plugs were
carefully excised, fixed with 10% formalin, and processed for hematoxylineosin (H&E) staining. The area of vascular lumen within the plugs was
measured by Image ProPlus software (Media Cybernetics, Wokingham,
United Kingdom). Two independent experiments were performed, each
with 5 to 6 mice per group. For each plug, 5 representative 100 ⫻ fields
from each of 3 serial sections were analyzed. The data are shown as
percentage of vessel-occupying area per 100 ⫻ field area.
Cells
Endothelial proliferation assay
Murine cardiac endothelial cells (MCECs) were isolated as previously
described.14 Briefly, mice heart tissue was diced and incubated with 5 mL of
1 mg/mL collagenase A for 30 minutes at 37°C followed by 5 mL
trypsin/EDTA (ethylenediaminetetraacetic acid) for 10 minutes at 37°C.
The released ECs were isolated by positive selection with anti-endoglin
antibody (Ab; MJ7/18, 10 mg/mL) and MiniMac microbeads system
(Miltenyi Biotech, Surrey, United Kingdom). MCECs were cultured on 1%
gelatin in Dulbecco modified Eagle medium (DMEM) supplemented with
10% fetal calf serum (FCS), 30 ␮g/mL endothelial cell growth supplement
(ECGS; Sigma, St. Louis, MO), 10 U/mL heparin, 2 mM L-glutamine, 100
IU/mL penicillin, and 0.1 mg/mL streptomycin. Cells were used for
experiments at passages 1 and 2. To generate the immortalized endothelial
cell lines, passage 1 MCECs from WT or ICAM-2⫺/⫺ mice were transduced
with a retrovirus encoding the temperature-sensitive mutant simian virus 40
(SV40) large T antigen tsTA58, a gift from Prof Yuti Chernajovsky (Barts,
London, United Kingdom), as described.15 After infection, the cells were
left to recover for 48 hours in fresh medium. Cells were then cultured in the
above medium containing G418 (400 ␮g/mL) at 33°C for 2 to 3 weeks, and
resistant colonies were pooled and expanded. Cells were switched to 37°C
to arrest proliferation 24 hours before experiments. The endothelial origin
of the WT (WT-MCEC-SV)– and ICAM-2⫺/⫺ (IC2-MCEC-SV)–derived
endothelioma lines was verified by fluorescence-activated cell sorting
(FACS), using anti-endoglin and anti-CD31 Abs. The phenotype of the
IC2-MCEC-SV endothelioma line was similar to that observed in primary
MCECs, based on ICAM-2 expression, Matrigel tube formation, and cell
proliferation.
HUVECs were isolated as described,16 and cultured in 1% gelatincoated tissue-culture ware in Medium 199 supplemented with 20% FCS, 30
␮g/mL ECGS, 10 U/mL heparin, 2mM L-glutamine, 100 IU/mL penicillin,
and 0.1 mg/mL streptomycin.
Cell proliferation was measured over 5 days using the methyl thiazolyl
tetrazolium (MTT) assay (Promega, Southampton, United Kingdom). The
assay was performed as per manufacturer’s instructions, on cells plated onto
collagen-coated 96-well tissue-culture plates at 2000 cells/well.
Flow cytometry
FACS analysis was performed by standard methods.14 Cell samples were
incubated with primary or isotype-matched control Abs for 30 minutes at
4°C and washed in phosphate-buffered solution (PBS) twice followed by
incubation with fluorescence-conjugated secondary Ab. After 2 more
washes in PBS, samples were resuspended in 4% paraformaldehyde and
analyzed with Epic XL-MCL flow cytometer and System II software
(Beckman Coulter, High Wycombe, United Kingdom). The primary Ab data
were normalized against isotype control and expressed as relative fluorescence
intensity (RFI).
In vitro Matrigel tube formation assay
Matrigel (300 ␮L/well) was plated evenly in a 24-well plate, and incubated
at 37°C for 30 minutes before adding MCECs (0.5 ⫻ 105 cells/well). Tube
Apoptosis assays
Apoptosis was induced by 3 different methods to investigate both the
intrinsic and the extrinsic pathways,17,18 using serum/growth-factor deprivation or staurosporine (20 nM) for the former and anti-Fas Ab for the latter.
For the serum deprivation experiments, cells were maintained in 1% bovine
serum albumin (BSA)/DMEM for the duration of the experiment. For
anti-Fas–induced apoptosis, cells were preincubated with 100 ng/mL
interferon-␥ (IFN-␥) for 16 hours to up-regulate Fas expression, followed
by anti-Fas Ab (10 ␮g/mL, Clone Jo-2; BD Biosciences, Oxford, United
Kingdom) for the indicated time. Apoptosis was evaluated by 2 methods: FACS
analysis using the fluorescein isothiocyanate–conjugated annexin V (AnxV)/
propidium iodide (PI) assay19 and acridine orange staining of pyknotic
nuclei undergoing apoptosis.14
In vitro wound migration assay
The in vitro wound assay was used to measure endothelial unidirectional
migration. WT or IC2-MCEC-SV cells were seeded at a density of
6.5 ⫻ 105/cm2 onto gelatin-coated 10-mm dishes and allowed to form
confluent monolayers. Two separate scratch wounds were generated with a
rubber cell scraper (1-mm width). Images were taken at the time of the
wound and at intervals up to 48 hours after wounding under phase-contrast
microscopy with an Olympus camera (objective, 10 ⫻; Olympus, London,
United Kingdom). Migration was calculated as the distance between the
migration front and the wound edge at each time point, as described.20 For
each time point, 8 measurements from 4 fields from 2 independent wounds
were taken.
Generation of stable ICAM-2 transfectants
Full-length human ICAM-2 in pcDNA3, kindly provided by Dr David
Simmons (Oxford, United Kingdom), was used to generate ICAM-2 stable
lines. Chinese hamster ovary (CHO) cells were transfected with linearized
ICAM-2 plasmid DNA using Lipofectamine 2000 (Invitrogen, Paisley,
United Kingdom) as per manufacturer’s instructions. Surface ICAM-2
expression on each subclone was analyzed by FACS (anti–ICAM-2 Ab,
Clone B-T1; Serotec, Oxford, United Kingdom) and 3 subclones with the
highest ICAM-2 expression were selected for further experiments. Mocktransfected cells were generated and grown in parallel. CHO cells stably
transfected with E-selectin were generated as described.21
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1638
HUANG et al
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
Immunofluorescence
Cells were washed in PBS and fixed in freshly prepared 3% (wt/vol)
paraformaldehyde for 20 minutes at room temperature, followed by 50 mM
NH4Cl for 10 minutes to neutralize paraformaldehyde. The cells were then
processed for indirect immunofluorescence using the anti–human ICAM-2
monoclonal Ab (clone B-T1, 10 ␮g/mL) followed by a goat anti–mouse
Alexa Fluor 555 Ab (Molecular Probes, Eugene, OR) at 10 ␮g/mL. All
incubations were performed at room temperature for 15 minutes in PBS
containing 3% BSA. After each incubation, the cells were washed in PBS.
Slides were mounted onto coverslips using VectorShield (Vector Laboratories, Peterborough, United Kingdom). Cells were visualized with a Zeiss
LSM510-META confocal laser-scanning microscope (Carl Zeiss, Welwyn
Garden City, United Kingdom) and images were processed using Adobe
Photoshop CS (Adobe Systems, San Jose, CA).
Homophilic ICAM-2 binding assay
CHO-IC2 cells were grown to confluence in 96-well tissue culture plates. A
recombinant chimeric molecule with the ICAM-2 extracellular region fused
to the Fc portion of human (h) IgG1 (ICAM-2–Fc; R&D Systems,
Minneapolis, MN), was preincubated with polystyrene beads conjugated
with anti-hFc Ab (HBP-30-5, 1% wt/vol; Spherotech, Libertyville, IL), at
37°C for 1.5 hours. Fifty microliters of the mixture was added to CHO-IC2
cells, to reach a final concentration of 10 ␮g/mL protein-Fc construct and
0.15% wt/vol beads. After 1.5 hours of incubation at 37°C, unbound beads
were carefully removed with 6 washes in warm medium. CHO cells stably
transfected with E-selectin (CHO-Esel), ICAM-1–Fc, and VCAM-1–Fc
(R&D Systems) were used as control. Experiments were carried out in
triplicates. Images from 3 representative 400 ⫻ fields from each replicate
were taken and bound beads counted. In indicated experiments, anti–
ICAM-2 Ab (polyclonal goat anti–ICAM-2 Ab [pAb]; R&D Systems),
monoclonal anti–ICAM-2 Ab (mAb), CBR-IC2/2 (Research Diagnostics,
Concord, MA) or goat IgG isotype control Ab (R&D Systems) was added to
the protein-beads mixture in the final 30 minutes of the preincubation step.
Rac pull-down assay
The ability of Rac-GTP to bind to the p21-binding domain (PBD) of
p21-activated kinase (PAK) (PAK1-PBD) was used to study Rac activation.22 To evaluate Rac activation during MCEC tube formation on
Matrigel, cell extracts were prepared at 40 minutes and 4 hours after plating
on Matrigel. For the ICAM-2 cross-linking experiments, HUVECs were
grown in full medium until 80% to 90% confluent, and subsequently
maintained in 1% BSA/M199 for 16 hours. Cells were then incubated with
pAb (15 ␮g/mL) for 40 minutes at 37°C, followed by incubation with
secondary cross-linking Ab (30 ␮g/mL) for the indicated time. Control cells
were treated with primary or secondary Ab alone. For total cell extracts,
cells were washed twice with Hanks balanced salt solution (HBSS) and
lysed with ice-cold buffer containing phosphatase and protease inhibitors.
After sonication for 30 seconds and centrifugation at 14 000g for 5 minutes
at 4°C, the supernatants were incubated with glutathione S-transferase
(GST)–PBD agarose beads (Upstate Biotechnology, Dundee, United Kingdom) for 1 hour at 4°C. The bound fraction was separated onto a sodium
dodecyl sulfate (SDS)–polyacrylamide gel and the presence of Rac in the
bound fraction was detected by Western blotting using primary anti-Rac Ab
(clone 23A8; Upstate Biotechnology). The amount of activated Rac was
normalized against total Rac in the corresponding lysate. Integrated density
values of the bands were measured using Alpha Innotech Chemilmager
5500 program (Alpha Innotech, San Leandro, CA).
Results
Endothelial ICAM-2 is required for in vitro angiogenesis
To determine whether ICAM-2 is involved in angiogenesis, we
studied the ability of primary ICAM-2–deficient ECs to form tubes
Figure 1. ICAM-2 mediates angiogenesis in vitro: tube formation on Matrigel.
(A) Primary murine cardiac endothelial cells (MCECs) isolated from control (WT) or
ICAM-2⫺/⫺ mice were seeded on Matrigel. In each experiment, 6 age- and
sex-matched mice/group were used. The figure shows 1 representative of 3 separate
experiments, each performed in triplicate. Left panels show WT; right panels,
ICAM-2⫺/⫺. In WT, cells were spreading, elongating, and making contacts with
neighboring cells at 3 hours, and an interconnecting tube network was formed over
4 to 5 hours. At all time points studied, the branches and tubes formed by ICAM-2⫺/⫺
cells were much less developed compared with control. Scale bar equals 100 ␮m.
Images were acquired using a Leitz Labovert inverted microscope (Leica Microsystems, Milton Keynes, United Kingdom) fitted with a 10 ⫻ phase-contrast objective
lens (Leitz-Phaco 10 ⫻/0.25 NA). Images were captured with a digital camera model
DP50-CU (Olympus) using Viewfinder Lite (v. 1) software (Olympus). Image processing was carried out using Adobe Photoshop CS (Adobe Systems). (B) Branches from
each cell were counted from 3 representative 100 ⫻ field/well. Error bars indicate
mean ⫾ SEM. **P ⬍ .01; ***P ⬍ .001 analysis of variance (ANOVA).
in an in vitro tube formation assay on Matrigel. MCECs were
isolated from mouse hearts by positive endoglin selection. The
endothelial origin of MCECs was confirmed by FACS analysis of
endoglin and CD31/PECAM-1 expression; the lack of ICAM-2
expression in the ICAM-2⫺/⫺ cells was also confirmed (not shown).
MCECs from ICAM-2⫺/⫺ or WT mice were seeded on Matrigel
and images of representative fields were taken hourly. In WT
MCEC samples, network formation took place between 3 and 6
hours after seeding. Tube formation was quantified after 3, 4, and 5
hours by counting branches from 3 representative fields per
replicate. As shown in Figure 1A, ICAM-2⫺/⫺ cells showed a
significantly impaired ability to form tubes on Matrigel compared
with WT MCECs. A significant reduction in tube formation in the
ICAM-2⫺/⫺ cells compared with WT was observed at all time
points studied (Figure 1B). The difference was most marked at
3 and 4 hours, when a tubular network of interconnecting branches
was well established in WT samples, while few contacts between
cells with shorter protrusions and few branches were observed in
the ICAM-2⫺/⫺ samples. These data indicate that ICAM-2 is
required for endothelial tube formation on Matrigel.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
Figure 2. ICAM-2 mediates angiogenesis in vivo: Matrigel plug assay. Matrigel
plugs were generated by subcutaneous injection of Matrigel; the plugs were removed
7 days later and processed for H&E staining. (A) In samples from WT mice (top),
formation of vascular structures within the plugs is accompanied by erythrocyte and
leukocyte infiltration. In samples from ICAM-2⫺/⫺ mice (bottom) a significant reduction in vascular structures and cellular infiltration within the plugs was observed.
Arrowheads show blood vessels. Scale bar equals 100 ␮m. Images were acquired as
in Figure 1A. (B) The vascular lumen area was measured using Image ProPlus
software. The average vessel-containing area was calculated from 5 representative
100 ⫻ fields of 3 representative serial sections of each plug and is shown as the
percentage of area occupied by blood vessel (BV)/100 ⫻ field. Compared with WT
mice, a 50% reduction in vessel-containing areas were observed in samples from
ICAM-2⫺/⫺ mice. Data represent 1 of 2 independent experiments; 5 to 6 mice per
group were used in each experiment. Error bars indicate mean ⫾ SEM. **P ⬍ .01,
unpaired t test.
ENDOTHELIAL ICAM-2 REGULATES ANGIOGENESIS
1639
shows that ICAM-2–Fc bound to CHO-IC2 (top) but not CHO-Esel
cells (bottom), indicating that ICAM-2 supports homophilic interaction. The interaction was specific to ICAM-2, since neither
ICAM-1–Fc beads (Figure 3B, middle) nor VCAM-1–Fc beads
(not shown) bound to CHO-IC2 cells. Quantification of bound
protein-coupled beads to CHO-IC2 or CHO-Esel is shown in
Figure 3C. Only ICAM-2–Fc–coupled beads, but not ICAM-1–Fc–
or VCAM-1–Fc–coupled beads, bound to CHO-IC2 cells. No
binding to CHO-Esel was detected with any of the recombinant
chimeric proteins. ICAM-2–Fc also bound to ICAM-2 on resting
HUVECs (data not shown). Therefore ICAM-2, like other endothelial junctional molecules, can engage in homophilic interaction.
To determine whether ICAM-2 homophilic interaction was
required for endothelial tube formation, we first searched for an
antibody that could inhibit this interaction. Two anti–ICAM-2
antibodies were screened in the bead binding assay. ICAM-2–Fc
binding to CHO-IC2 cells was specifically inhibited by a pAb
anti–ICAM-2 antibody but not by the mAb CBR/IC2-2 or control
Ab (Figure 3D). Interestingly, this mAb is known to inhibit
ICAM-2/LFA-1 interaction,26 suggesting that the epitope(s) involved in ICAM-2 homophilic interaction may be different from
those involved in binding to integrins. We then addressed the
Endothelial ICAM-2 is required for in vivo angiogenesis
To validate the defect observed in vitro, we studied angiogenesis in
vivo in using the Matrigel plug assay.23 Matrigel was injected
subcutaneously into the abdominal areas of WT and ICAM-2⫺/⫺
mice; after 7 days, the vascularized plugs were removed and
processed for H&E staining to identify the area covered by vessels.
As shown in Figure 2A, new vessel formation within the plugs,
accompanied by erythrocyte and leukocyte infiltration, was observed in plug samples from WT mice (top). Samples from
ICAM-2–/– mice (bottom) showed significantly less vascular and
cellular infiltration within the plugs. Measurement of the neovascularized area showed a 50% reduction in the vessel-containing area
in the ICAM-2⫺/⫺ mice compared with WT mice (32% ⫾ 5.99% vs
11.8% ⫾ 0.42%; Figure 2B).23 Therefore, both in vitro and in vivo
assays demonstrate defective angiogenesis in the ICAM-2⫺/⫺ mice.
ICAM-2 supports homophilic interaction
Having demonstrated that endothelial ICAM-2 is involved in
angiogenesis, we investigated the underlying mechanism. The
assembly and disassembly of endothelial junctions is critical in the
regulation of angiogenesis. Other junctional molecules, such as
CD31/PECAM and JAM-A, engage in homophilic interaction
between adjacent EC.24,25 Because of its similarities to these
molecules, we speculated that ICAM-2 could also engage in
homophilic binding. To test this hypothesis, stable CHO cell lines
expressing ICAM-2 (CHO-IC2) were generated for ligand-binding
studies. Surface ICAM-2 expression in these clones was verified by
FACS (not shown) and immunofluorescent staining, which showed
that ICAM-2 expression was concentrated mainly at the cell
junctions (Figure 3A). The recombinant soluble chimeric protein
ICAM-2–Fc, coupled to anti-Fc–conjugated polystyrene beads,
was used as ligand. CHO cells stably expressing E-selectin
(CHO-Esel) and bead-coupled recombinant chimeric molecules
ICAM-1–Fc and VCAM-1–Fc were used as controls. Figure 3B
Figure 3. ICAM-2 supports homophilic interaction. (A) ICAM-2 surface expression on CHO cells stably transfected with human ICAM-2 (CHO-IC2), detected by
immunofluorescence using an anti–ICAM-2 mAb (clone B-T1). ICAM-2 expression is
concentrated at the cell junctions, similarly to what is observed in endothelial cells.
Staining of mock-transfected CHO cells with the same antibody is also shown. (B)
ICAM-2 homophilic interaction. Polystyrene beads conjugated with anti–human Fc
Ab, preincubated with soluble ICAM-2–Fc, were added to CHO-IC2 (top) or control
cells (CHO-Esel, bottom) in 96-well plates. ICAM-1–Fc (middle) or VCAM-1–Fc
(image not shown) were used as control proteins. Images were acquired as in Figure
1A. (C) The number of beads retained in the wells after washings was counted per
400 ⫻ field of each replicate. Experiments were performed in triplicate. ICAM-2–Fc
significantly bound to CHO-IC2 cells, compared with ICAM-1–Fc and VCAM-1–Fc.
None of the protein-Fc constructs bound to CHO-Esel. ***P ⬍ .001, compared with
control protein constructs and binding to CHO-Esel, ANOVA. n ⫽ 4. (D) Two
anti–ICAM-2 Abs were tested for their ability to block ICAM-2 homophilic interaction,
by preincubation with the protein-Fc-beads complex before adding to CHO-IC2. The
polyclonal anti–ICAM-2 Ab (pAb), but not the monoclonal anti–ICAM-2 Ab
(CBR-IC2/2) or goat IgG isotype control (Ctrl), significantly inhibited binding of
ICAM-2 Fc to CHO-IC2 by 50%. ***P ⬍ .001, ANOVA. n ⫽ 3. (E) Matrigel tube
formation is inhibited by anti–ICAM-2 mAb. HUVECs were preincubated with pAb
anti–ICAM-2 or goat IgG control for 15 minutes before plating onto Matrigel.
Photos were taken hourly after seeding, and branches counted as described (see
“Materials and methods”). In the presence of anti–ICAM-2 pAb, HUVEC tube
formation was inhibited by approximately 30% compared with control Ab (Ctrl Ab)
or untreated samples (Ctrl) at 3, 4, and 5 hours. ***P ⬍ .001, ANOVA. n ⫽ 3.
(C-E) Error bars indicate mean ⫾ SEM.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1640
HUANG et al
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
Figure 4. ICAM-2 protects endothelial cells from apoptosis. (A) Serum starvation-induced apoptosis. Cells were maintained in DMEM with 1% BSA for 24 or 48 hours.
Apoptosis was quantified by FACS analysis using the annexin V (AnxV) and propidium iodide (PI) method (A, inset) and shown as fold increase of the AnxV (⫹)/PI (⫺) cells
compared with control. (B) Anti-Fas–induced apoptosis. MCECs were treated with 100 ng/mL IFN-␥ for 16 hours followed by 10 ␮g/mL anti-Fas Ab for 24 or 48 hours. Data are
shown as fold increase of the AnxV (⫹)/PI (⫺) cells compared with control. (C) Staurosporine-induced apoptosis. MCECs were incubated with 20 nM staurosporine for 3 or 6 hours.
Apoptosis was measured by acridine orange staining (C, inset) and pyknotic nuclei count. Image was acquired as in Figure 1A. Data are shown as average percentage of apoptotic
cells/200 ⫻ field. Scale bar equals 20 ␮m. With all 3 stimuli, MCECs from ICAM-2⫺/⫺ mice were significantly more susceptible to apoptosis compared with WT MCECs. *Comparison with
WT cells at the same time point. †Comparison with ICAM-2⫺/⫺ cells at 24 hours. *P ⬍ .05; ***P ⬍ .001; †P ⬍ 0.05, ‡P ⬍ .01, ANOVA. n ⫽ 3. (A-C) Error bars indicate mean ⫾ SEM.
question whether ICAM-2 homophilic interaction is required for
angiogenesis, by testing the effect of the anti–ICAM-2 pAb which
inhibits ICAM-2 homophilic interaction (Figure 3D) on in vitro
endothelial tube formation on Matrigel. In the presence of the
ICAM-2 pAb, tube formation on Matrigel was significantly
inhibited by 30% compared with untreated control samples, or with
control Ab (Figure 3E). These results suggest that ICAM-2
homophilic interaction may be involved in endothelial tube formation and angiogenesis.
ICAM-2 protects endothelial cells from apoptosis
Apoptosis is an important regulator of angiogenesis1 and junctional
adhesion molecules can provide survival signals to ECs. ICAM-2
has been shown to protect T cells and ICAM-2–overexpressing
fibroblasts from apoptosis.27 Therefore, we speculated that ICAM-2
may support angiogenesis in part by providing survival signals to
ECs. To test this hypothesis, the ability of WT and ICAM-2⫺/⫺
MCECs to withstand apoptotic stimulation was investigated.
Apoptosis was induced in primary MCECs isolated from ICAM2⫺/⫺ or WT mice by serum and growth-factor deprivation (Figure
4A), anti-Fas Ab (Figure 4B), or staurosporine (Figure 4C). Cell
death was measured by Anx-V/PI staining (Figure 4A, inset) or by
acridine orange staining (Figure 4C, inset). With all stimuli used,
ICAM-2⫺/⫺ cells were significantly more prone to apoptosis than
WT cells. Serum and growth-factor deprivation induced a significant increase in apoptosis in the ICAM-2⫺/⫺ cells after 48 hours,
compared with WT (Figure 4A). A similar pattern was observed
when apoptosis was induced by anti-Fas antibody (Figure 4B), with
significantly increased apoptosis in the ICAM2⫺/⫺ cells after 48
hours of stimulation, compared with WT cells. Staurosporineinduced apoptosis was significantly higher in the ICAM-2⫺/⫺ cells
compared with WT cells at both 3 hours and 6 hours after
stimulation (Figure 4C). These data indicate that the lack of
ICAM-2 expression predisposes endothelial cells to apoptosis and
that ICAM-2 provides protection against both intrinsic and extrinsic pathways of apoptosis. The susceptibility of the ICAM-2⫺/⫺
cells to apoptosis may, at least in part, be responsible for the defect
in angiogenesis observed in the ICAM-2⫺/⫺ mice.
WT cells, suggesting that the defect in angiogenesis exhibited by
the ICAM-2⫺/⫺ mice is independent of endothelial cell proliferation.
ICAM-2 is involved in endothelial-cell migration
Endothelial migration is a key step in angiogenesis, and involves
major reorganization of the actin cytoskeleton. ICAM-2 is linked to
the actin cytoskeleton via the ezrin-radixin-moesin (ERM) family,
which acts as linkers between plasma membrane proteins and the
cytoskeleton,28 and has been shown to regulate cell migration.29 We
speculated that the absence of ICAM-2 could affect endothelial
migration. In order to test this hypothesis, unidirectional migration
of endothelial cells was assessed using an in vitro wound-healing
assay. Confluent monolayers of MCEC-SV from WT or ICAM2⫺/⫺ mice were wounded using a thick rubber scraper. The average
width of the wound was similar in WT-MCEC-SV compared with
IC2-MCEC-SV monolayers (mean ⫾ SD: 897.7 ␮m ⫾ 188.7 ␮m
vs 971.4 ␮m ⫾ 115 ␮m, respectively; P ⫽ .645). Pictures of the
wound were taken over 48 hours. ICAM2⫺/⫺ MCEC-SV migrated
more slowly than WT cells; the difference in migration rate was
significant at 14, 18, 22.5, and 23.5 hours (Figure 6). At 40 hours,
however, both WT and ICAM2⫺/⫺ cells filled the wound area (not
shown). These results indicate that ICAM-2 is involved in EC
migration, and this is likely to contribute to the angiogenesis defect
observed in the ICAM-2⫺/⫺ mice.
ICAM-2 regulates Rac activation during endothelialtube formation
The defect in cell migration observed in the ICAM-2–deficient
cells suggests that ICAM-2 may be required to trigger signal
transduction pathways involved in migration. Rho-like GTPases
are key regulators of the cytoskeletal changes required for cell
migration. We therefore speculated that ICAM-2 may regulate
small GTPases and mediate angiogenesis through this mechanism.
ICAM-2 is not involved in endothelial proliferation
Another possible mechanism by which ICAM-2 may be involved
in angiogenesis is through regulation of endothelial cell proliferation. To test this hypothesis, proliferation of primary MCECs from
ICAM-2⫺/⫺ or WT mice was measured in vitro using the MTT
assay over 5 days. As shown in Figure 5, no difference in
proliferation over 5 days was observed between ICAM-2⫺/⫺ and
Figure 5. ICAM-2 does not affect endothelial cell proliferation. Cell proliferation
was measured over 5 days on WT and ICAM-2⫺/⫺ MCECs plated onto collagencoated 96-well tissue culture using the MTT assay. No difference in the proliferation
rate of MCECs was observed in the ICAM-2–deficient cells. Data are presented as
absorbance at optical density (OD) 490 nm (SEM ⫾ SD of 3 replicate experiments).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
ENDOTHELIAL ICAM-2 REGULATES ANGIOGENESIS
1641
Figure 6. ICAM-2 is involved in endothelial-cell migration. WT-MCEC-SV and IC2-MCEC-SV cells were used in a wound-healing assay. Confluent monolayers were
scratch-wounded and incubated for up to 48 hours. (A) Photographs of wounded monolayers taken at 1 and 23.5 hours. The vertical lines indicate the wound edge and the
migration front (dotted line). The horizontal arrows indicate the migration distance. Scale bar equals 50 ␮m. Images were acquired as in Figure 1A. (B) Migration distance,
measured at 14, 18, 22.5, and 23.5 hours. IC2-MCEC-SVs show decreased migration at all time points, compared with WT. Values represent mean ⫾ SD of 8 measurements.
*P ⬍ .001, unpaired t test.
Of the best-characterized small GTPases, Rac was shown to
regulate capillary tube formation on Matrigel.30 Therefore, we
tested whether ICAM-2 is required for Rac activation during tube
formation on Matrigel. Rac activity in MCECs from WT or
ICAM2⫺/⫺ mice seeded on Matrigel was measured by GST-PBD
pull-down assay.22 As shown in Figure 7A, Rac was activated in
WT cells during spreading (40 minutes), and the activation was
sustained at 4 hours, coinciding with maximal tubular network
formation. In the ICAM-2⫺/⫺ cells, Rac activation at the spreading
stage (40 minutes) was similar to WT. However ICAM-2⫺/⫺ cells
failed to sustain Rac activation at 4 hours and formed significantly
reduced tubular networks. These results show that ICAM-2 is
required to sustain Rac activation during tube formation.
To investigate further the relationship between ICAM-2 signaling and Rac activation, Rac activation in HUVECs following
cross-linking of ICAM-2 with the anti–ICAM-2 pAb, which blocks
ICAM-2 homophilic interaction and HUVEC tube formation on
Matrigel, was investigated. As shown in Figure 7B, ICAM-2
cross-linking resulted in Rac activation, with a peak at 20 minutes
after cross-linking and a return to baseline after 30 minutes. These
results indicate that ICAM-2 homophilic interaction directly acti-
Figure 7. ICAM-2 regulates activation of the small GTPase Rac during endothelial tube formation. (A) Rac activation during Matrigel tube formation. MCECs plated
onto Matrigel were analyzed for Rac activation at spreading (40 minutes) and tube
formation stage (4 hours) by GST-PBD pull-down assays. During spreading (40 minutes), ICAM-2⫺/⫺ and WT cells had comparable levels of Rac activation; however,
during tube formation (4 hours), ICAM-2⫺/⫺ cells were unable to sustain Rac
activation. *P ⬍ .05, unpaired t test. n ⫽ 4. (B) ICAM-2 signaling induces Rac
activation. HUVECs grown to 80% confluence were starved for 16 hours and
incubated with goat anti–ICAM-2 Ab at 15 ␮g/mL (pAb) for 30 minutes, followed by
cross-linking with anti–goat IgG Ab. Rac activation was measured at 10, 20, 30, and
90 minutes following cross-linking. Negative control samples were incubated with
either the cross-linking Ab or primary Ab alone. ICAM-2 cross-linking induced Rac
activation, which peaked at 20 minutes and returned to baseline after 30 minutes.
*P ⬍ .05, unpaired t test. n ⫽ 5. (A-B) Error bars indicate mean ⫾ SEM.
vates Rac and identifies a novel pathway for Rac activation during
endothelial junction formation and angiogenesis.
Discussion
Endothelial junctions govern vascular homeostasis by regulating
leukocyte transmigration, permeability, endothelial cell survival,
and proliferation. Adhesion molecules expressed at the endothelial
junctions mediate contact with leukocytes and with neighboring
endothelial cells. Ligand binding, both homophilic and heterophilic, results in the activation of signal transduction pathways.
Examples are CD31/PECAM, which is involved in regulating both
leukocyte trafficking into tissue and endothelial survival,31
and JAM-A, which has been shown to regulate monocyte migration32 as well as endothelial cell motility.33 Therefore, it appears
that leukocyte adhesion molecules play multiple roles in endothelial cells.
ICAM-2 is concentrated at endothelial cell-cell contacts3 and is
known as a leukocyte adhesion molecule. In this study we
demonstrate that ICAM-2 can also regulate angiogenesis, and
identify several new functions for ICAM-2 which are related to its
role in angiogenesis. Using ICAM-2⫺/⫺ mice and primary endothelial cells derived from these mice, we show that lack of ICAM-2
expression on ECs results in defective angiogenesis, both in vitro
and in vivo. A role for ICAM-2 in angiogenesis in vivo is further
supported by data from Melero et al, which showed that an
anti–ICAM-2 Ab eradicated established murine colon carcinoma
through mechanisms involving cytotoxic T-cell activation and, to a
lesser extent, inhibition of angiogenesis.34 In our study, defective
angiogenesis in the ICAM-2⫺/⫺ mouse in vivo was reflected in the
inability of ICAM-2–deficient endothelial cells to form tubes on
Matrigel in vitro, indicating a primary endothelial defect.
Several studies have shown that ICAM-2 expression on endothelial cells is concentrated at the junctions, although the exact
location of ICAM-2 within junctions has not yet been determined.
Adhesion between adjacent endothelial cells is mediated by
homophilic interactions between junctional molecules, forming a
zipper-like structure.35 We therefore investigated whether ICAM-2
could support homophilic interaction. Using CHO transfectants
stably overexpressing ICAM-2, we showed that ICAM-2 is capable
of supporting homophilic interaction. This is the first evidence of
the presence of an endothelial ligand for ICAM-2, suggesting that
ICAM-2 may contribute to the formation of junctional structures.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1642
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
HUANG et al
Tube formation on Matrigel was inhibited by an anti–ICAM-2
antibody that also blocked homophilic interaction, suggesting that
the homophilic interaction between ICAM-2 on opposing ECs may
be involved in angiogenesis. Further experiments with purified
F(ab)⬘ fragments and recombinant ICAM-2–Fc will be required to
define the role of ICAM-2 homophilic interaction in angiogenesis.
Our results do not rule out the possibility that ICAM-2 may interact
with other endothelial ligands, or that ICAM-2 may engage with
ICAM-2 on leukocytes or platelets.
Several mechanisms could be responsible for the angiogenesis
defect in the ICAM-2⫺/⫺ mice. Cross-linking of ICAM-2 has been
shown to protect a variety of cells, including T/B lymphocytic cell
lines, primary human B cells, and ICAM-2–overexpressing fibroblasts, from apoptosis27; however, no data are available on the role
of ICAM-2 in endothelial apoptosis. Inhibition of EC apoptosis is
an essential step in angiogenesis, and the induction of apoptosis
counteracts angiogenesis.36,37 In this paper we show that ICAM-2–
deficient endothelial cells are more susceptible to apoptosis. The
lack of ICAM-2–dependent survival signals on the ICAM-2–
deficient ECs could therefore contribute to the defect in angiogenesis shown here. The ICAM-2–dependent antiapoptotic effects
described in other cell types were shown to be linked to the
phosphatidylinositol 3-kinase (PI-3K)/Akt pathway,27 which plays
a critical role in regulating EC survival, cell migration, and tube
formation.38 Studies are underway to determine whether this
pathway is involved in ICAM-2–mediated survival signals in ECs.
Angiogenesis involves endothelial cell spreading, migration,
and regulation of cell-cell and cell-matrix adhesion,39 all of which
require significant reorganization of the actin cytoskeleton. The
cytoplasmic tail of ICAM-2 is linked to the actin cytoskeleton by
the ezrin-radixin-moesin (ERM) family, which acts as linkers
between plasma membrane proteins and the cytoskeleton.28 The
absence of ICAM-2 could alter ERM distribution and/or function
and, as a consequence, the actin cytoskeleton itself, hence affecting
cell shape change, migration, and junction formation. We found no
difference in ezrin distribution between ICAM-2–deficient ECs and
control ECs (M.-T.H. and A.M.R., unpublished observation, September 2004). However, the distribution of other members of the
ERM family, or indeed their activation state, was not investigated;
therefore, it is possible that a defect in ERM function in these cells
may exist. ERM controls cell adhesion and migration through the
Rho family of small GTPases,40 which play a critical role in
regulating cytoskeletal dynamics, cell migration, and junction
formation.41 We have previously shown that cross-linking of
ICAM-2 on HUVECs does not activate RhoA.16 The small GTPase
Rac has been shown to be required for tube formation on
Matrigel.30 In this study, we demonstrate that ICAM-2 crosslinking activates Rac in HUVECs, and that endothelial ICAM-2 is
required for Rac activation during tube formation. Although during
spreading Rac activation was similar in ICAM-2–deficient cells
and WT cells, ICAM-2–deficient cells failed to sustain Rac
activation during formation of the tube network. These results
indicate that ICAM-2 is required to maintain Rac activation during
tube formation. The defect in Rac activation could also explain the
delayed endothelial cell migration of ICAM-2⫺/⫺ cells observed in
the in vitro wound-healing assay, compared with WT. It is worth
noting that ICAM-2 expression in the WT-MCEC-SV line was
low; hence, other ICAM-2–independent mechanisms contributing to this phenotype cannot be ruled out. Our findings suggest
that ICAM-2–dependent activation of Rac results in defective
endothelial cell migration. ICAM-2 therefore may regulate
angiogenesis at least in part via its ability to activate Rac at the
migrating/branching stage.
Rac plays multiple roles in endothelial cells; therefore, the
finding that ICAM-2 regulates Rac suggests that other mechanisms
may also contribute to the overall defect in angiogenesis observed
in the ICAM-2–deficient mice. Rac regulates endothelial junction
assembly and function as well as cadherin adhesiveness.42 VEcadherin itself can regulate Rac activation via the guanosine
exchange factor Tiam,43 indicating a bidirectional cross-talk between junctional signaling molecules and small GTPases of the
Rho family. Our results suggest that during the establishment of
endothelial junctions Rac activation requires at least 2 distinct
junctional signaling events, one VE-cadherin–dependent and another ICAM-2–dependent. The VE-cadherin–dependent signals
seem to be constitutively required, since basal levels of active Rac
in VE-cadherin null cells were found to be lower than in WT
cells.44 In the ICAM-2 null cells, on the other hand, Rac activation
was normal at baseline but failed to increase during tube formation,
suggesting that ICAM-2 provides a secondary signal required for
junction stability via Rac activation. Preliminary results suggest
that this may be the case: staining for JAM-A was reduced in
monolayers from ICAM-2⫺/⫺ migrating cells compared with WT
cells (G.M.B., V.A., and A.M.R., unpublished observation). Detailed studies of the morphology of ICAM-2⫺/⫺ cells during
migration and junction formation and on the relationship between
junctional pathways are in progress.
In summary, this study demonstrates that the leukocyte adhesion molecule ICAM-2 is involved in angiogenesis. We have
identified a new pathway in which ICAM-2–mediated signaling
activates the small GTPase Rac and regulates angiogenesis. We
have described several new functions for ICAM-2, namely regulation of survival and cell migration and the ability to support
homophilic interaction. This study also reveals a novel pathway for
Rac activation in endothelial cells. Our results provide new insight
in the regulation of endothelial homeostasis and angiogenesis by
junctional adhesion molecules.
Acknowledgements
We thank Prof Britta Engelhardt (Theodor Kocher Institute,
University of Bern, Switzerland) and Dr Sussan Nourshargh
(Imperial College London, United Kingdom) for access to the
ICAM-2⫺/⫺ mice; Prof Yuti Chernajovsky (Barts and The London,
United Kingdom) and Dr Charlotte Lawson (Imperial College
London, United Kingdom) for the retrovirus-expressing cell line
SVU19.5; Dr Alan Entwistle and Dr Priam Villalonga (Ludwig
Institute, University College London, United Kingdom) and Dr
Elaine Lidington (Imperial College London, United Kingdom) for
helpful scientific and technical advice.
References
1. Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9:685-693.
2. Dejana E, Spagnuolo R, Bazzoni G. Interendothelial
junctions and their role in the control of angiogenesis,
vascular permeability and leukocyte transmigration.
Thromb Haemost. 2001;86:308-315.
3. McLaughlin F, Hayes BP, Horgan CM, et al. Tumor necrosis factor (TNF)-alpha and interleukin
(IL)-1beta down-regulate intercellular adhesion
molecule (ICAM)-2 expression on the endothelium. Cell Adhes Commun. 1998;6:381-400.
4. Staunton DE, Dustin ML, Springer TA. Functional
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 5
cloning of ICAM-2, a cell adhesion ligand for
LFA-1 homologous to ICAM-1. Nature. 1989;339:
61-64.
5. Xie J, Li R, Kotovuori P, et al. Intercellular adhesion molecule-2 (CD102) binds to the leukocyte
integrin CD11b/CD18 through the A domain. J Immunol. 1995;155:3619-3628.
6. Geijtenbeek TB, Krooshoop DJ, Bleijs DA, et al.
DC-SIGN-ICAM-2 interaction mediates dendritic
cell trafficking. Nat Immunol. 2000;1:353-357.
7. Gerwin N, Gonzalo JA, Lloyd C, et al. Prolonged
eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended
hyperresponsiveness. Immunity. 1999;10:9-19.
8. Huang MT, Larbi KY, Gerwin N, Nourshargh S.
ICAM-2 mediates PECAM-1-independent neutrophil transmigration in vivo [abstract]. FASEB J.
2005;854:A1504.
9. Rival Y, Del Maschio A, Rabiet MJ, Dejana E,
Duperray A. Inhibition of platelet endothelial cell
adhesion molecule-1 synthesis and leukocyte
transmigration in endothelial cells by the combined action of TNF-alpha and IFN-gamma. J Immunol. 1996;157:1233-1241.
10. Ozaki H, Ishii K, Horiuchi H, et al. Cutting edge:
combined treatment of TNF-␣ and IFN-␥ causes
redistribution of junctional adhesion molecule in
human endothelial cells. J Immunol. 1999;163:
553-557.
ENDOTHELIAL ICAM-2 REGULATES ANGIOGENESIS
transcription in endothelial cells. J Immunol.
2002;169:1007-1013.
17. Ackermann EJ, Taylor JK, Narayana R, Bennett
CF. The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem. 1999;274:
11245-11252.
18. Wajant H. The Fas signaling pathway: more than
a paradigm. Science. 2002;296:1635-1636.
19. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. A novel assay for apoptosis: flow
cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein
labelled Annexin V. J Immunol Methods. 1995;
184:39-51.
20. Eckes B, Dogic D, Colucci-Guyon E, et al. Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.
J Cell Sci. 1998;111:1897-1907.
21. Rao RM, Clarke JL, Ortlepp S, et al. The S128R
polymorphism of E-selectin mediates neuraminidase-resistant tethering of myeloid cells under
shear flow. Eur J Immunol. 2002;32:251-260.
22. Ren XD, Schwartz MA. Determination of GTP
loading on Rho. Methods Enzymol. 2000;325:
264-272.
23. Vailhe B, Vittet D, Feige JJ. In vitro models of vasculogenesis and angiogenesis. Lab Invest. 2001;
81:439-452.
11. McLaughlin F, Ludbrook VJ, Kola I, Campbell CJ,
Randi AM. Characterisation of the tumour necrosis factor (TNF)-(alpha) response elements in the
human ICAM-2 promoter. J Cell Sci. 1999;112:
4695-4703.
24. Fawcett J, Buckley C, Holness CL, et al. Mapping
the homotypic binding sites in CD31 and the role
of CD31 adhesion in the formation of interendothelial cell contacts. J Cell Biol. 1995;128:12291241.
12. McLaughlin F, Ludbrook VJ, Cox J, et al. Combined genomic and antisense analysis reveals
that the transcription factor Erg is implicated in
endothelial cell differentiation. Blood. 2001;98:
3332-3339.
25. Bazzoni G. The JAM family of junctional adhesion
molecules. Curr Opin Cell Biol. 2003;15:525-530.
13. Gory S, Dalmon J, Prandini MH, et al. Requirement of a GT box (Sp1 site) and two Ets binding
sites for vascular endothelial cadherin gene transcription. J Biol Chem. 1998;273:6750-6755.
26. de Fougerolles AR, Stacker SA, Schwarting R,
Springer TA. Characterization of ICAM-2 and
evidence for a third counter-receptor for LFA-1.
J Exp Med. 1991;174:253-267.
27. Perez OD, Kinoshita S, Hitoshi Y, et al. Activation
of the PKB/AKT pathway by ICAM-2. Immunity.
2002;16:51-65.
14. Lidington EA, Rao RM, Marelli-Berg FM, et al.
Conditional immortalization of growth factorresponsive cardiac endothelial cells from H-2KbtsA58 mice. Am J Physiol Cell Physiol. 2002;282:
C67-C74.
28. Yonemura S, Hirao M, Doi Y, et al. Ezrin/radixin/
moesin (ERM) proteins bind to a positively
charged amino acid cluster in the juxtamembrane cytoplasmic domain of CD44, CD43,
and ICAM-2. J Cell Biol. 1998;140:885-895.
15. Dai L, Claxson A, Marklund SL, et al. Amelioration
of antigen-induced arthritis in rats by transfer of
extracellular superoxide dismutase and catalase
genes. Gene Ther. 2003;10:550-558.
29. Mangeat P, Roy C, Martin M. ERM proteins in cell
adhesion and membrane dynamics. Trends Cell
Biol. 1999;9:187-192.
16. Thompson PW, Randi AM, Ridley AJ. Intercellular
adhesion molecule (ICAM)-1, but not ICAM-2,
activates RhoA and stimulates c-fos and rhoA
30. Connolly JO, Simpson N, Hewlett L, Hall A. Rac
regulates endothelial morphogenesis and capillary assembly. Mol Biol Cell. 2002;13:2474-2485.
31. Newman PJ, Newman DK. Signal transduction
1643
pathways mediated by PECAM-1: new roles for
an old molecule in platelet and vascular cell biology. Arterioscler Thromb Vasc Biol. 2003;23:953964.
32. Martin-Padura I, Lostaglio S, Schneemann M, et
al. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates
monocyte transmigration. J Cell Biol. 1998;142:
117-127.
33. Bazzoni G, Tonetti P, Manzi L, et al. Expression of
junctional adhesion molecule-A prevents spontaneous and random motility. J Cell Sci. 2005;118:
623-632.
34. Melero I, Gabari I, Corbi AL, et al. An anti-ICAM-2
(CD102) monoclonal antibody induces immunemediated regressions of transplanted ICAM-2negative colon carcinomas. Cancer Res. 2002;
62:3167-3174.
35. Dejana E. Endothelial cell-cell junctions: happy
together. Nat Rev Mol Cell Biol. 2004;5:261-270.
36. Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh
DA. Role of Raf in vascular protection from distinct apoptotic stimuli. Science. 2003;301:94-96.
37. Hanahan D, Folkman J. Patterns and emerging
mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353-364.
38. Shiojima I, Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res.
2002;90:1243-1250.
39. Bischoff J. Approaches to studying cell adhesion
molecules in angiogenesis. Trends Cell Biol.
1995;5:69-74.
40. Mackay DJG, Esch F, Furthmayr H, Hall A. Rhoand Rac-dependent assembly of focal adhesion
complexes and actin filaments in permeabilized
fibroblasts: an essential role for ezrin/radixin/
moesin proteins. J Cell Biol. 1997;138:927-938.
41. Ridley AJ, Schwartz MA, Burridge K, et al. Cell
migration: integrating signals from front to back.
Science. 2003;302:1704-1709.
42. Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley AJ. 9Rgr and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci. 2001;114:
1343-1355.
43. Lampugnani MG, Zanetti A, Breviario F, et al. VEcadherin regulates endothelial actin activating
Rac and increasing membrane association of
Tiam. Mol Biol Cell. 2002;13:1175-1189.
44. Carmeliet P, Lampugnani MG, Moons L, et al.
Targeted deficiency or cytosolic truncation of the
VE-cadherin gene in mice impairs VEGF-mediated
endothelial survival and angiogenesis. Cell. 1999;98:
147-157.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2005 106: 1636-1643
doi:10.1182/blood-2004-12-4716 originally published online
May 26, 2005
Endothelial intercellular adhesion molecule (ICAM)−2 regulates
angiogenesis
Miao-Tzu Huang, Justin C. Mason, Graeme M. Birdsey, Valerie Amsellem, Nicole Gerwin, Dorian O.
Haskard, Anne J. Ridley and Anna M. Randi
Updated information and services can be found at:
http://www.bloodjournal.org/content/106/5/1636.full.html
Articles on similar topics can be found in the following Blood collections
Cell Adhesion and Motility (790 articles)
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.