HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Src and phosphatidylinositol 3–kinase mediate soluble
E-selectin–induced angiogenesis
Pawan Kumar, Mohammad A. Amin, Lisa A. Harlow, Peter J. Polverini, and Alisa E. Koch
Angiogenesis plays an important role in a
variety of pathophysiologic processes,
including tumor growth and rheumatoid
arthritis. We have previously shown that
soluble E-selectin (sE-selectin) is an important angiogenic mediator. However,
the mechanism by which sE-selectin mediates angiogenesis is still unknown. In
this study, we show that sE-selectin is a
potent mediator of human dermal microvascular endothelial cell (HMVEC) chemotaxis, which is predominantly mediated
through the Src and the phosphatidylinositiol 3–kinase (PI3K) pathways. Further,
sE-selectin induced a 2.2-fold increase in
HMVEC tube formation in the Matrigel in
vitro assay. HMVECs pretreated with the
Src inhibitor (PP2) and the PI3K inhibitor
(LY294002) or transfected with Src antisense oligonucleotides or Akt dominantnegative mutants significantly inhibited
sE-selectin–mediated HMVEC tube formation. In contrast, HMVECs transfected with
an extracellular signal-related kinase 1/2
(ERK1/2) mutant or pretreated with the
mitogen-activated protein kinase (MAPK)
inhibitor PD98059 failed to show sEselectin–mediated HMVEC tube formation. Similarly, in the Matrigel-plug in vivo
assay, sE-selectin induced a 2.2-fold increase in blood vessel formation, which
was significantly inhibited by PP2 and
LY294002 but not by PD98059. sE-selectin induced a marked increase in Src,
ERK1/2, and PI3K phosphorylation. PI3K
and ERK1/2 phosphorylation was significantly inhibited by PP2, thereby suggesting that both of these pathways may be
activated via Src kinase. Even though the
ERK1/2 pathway was activated by sEselectin in HMVECs, it seems not to be
essential for sE-selectin–mediated angiogenesis. Taken together, our data clearly
show that sE-selectin–induced angiogenesis is predominantly mediated through
the Src-PI3K pathway. (Blood. 2003;101:
3960-3968)
© 2003 by The American Society of Hematology
Introduction
Angiogenesis, or new blood vessel formation, is a complex process
that involves endothelial cell proliferation and migration, followed
by capillary tube formation. Each stage in this process can be
modulated by a number of factors present in normal or pathologic
conditions. Vascular endothelial growth factor (VEGF) is one of
the most studied factors that mediates angiogenesis through Src
family kinases and phosphatidylinositiol 3–kinase (PI3K) pathways.1,2 Src kinases are activated by a variety of growth factors and
function downstream of receptor tyrosine kinases.3,4 Eliceiri et al,
showed that both VEGF and fibroblast growth factor (FGF)
stimulate Src activation in avian endothelial cells.1 However, only
VEGF-induced angiogenesis was inhibited by treatment with a
retrovirus that encodes Src-251, a dominant-interfering mutant of
Src. Moreover, overexpression of Src-251 in avian blood vessels
induces apoptotic death, indicating that VEGF-induced activation
of Src is essential for endothelial cell survival and angiogenesis.
Src kinases, once activated, may in turn activate downstream PI3K.
PI3K has been implicated in a number of cellular functions
including cell adhesion, cell survival, and angiogenesis.2,5 The
serine-threonine kinase, Akt, is a downstream target of PI3K.
Cardone et al6 have shown that Akt phosphorylation promotes cell
survival by phosphorylating Bcl-2/Bcl-XL agonist causing cell
death (BAD), forkhead-related protein 1 (FKHR1), and caspase-9.
Akt has also been shown to play an important role in angiogenesis.2
E-selectin is a calcium-dependent lectin that mediates adhesive
interactions of circulating leukocytes with vascular endothelium
during normal and abnormal inflammatory conditions such as
rheumatoid arthritis (RA) and atherosclerosis.7,8 E-selectin is a
single-chain 115-kDa glycoprotein with a lectinlike N terminal
domain, an epidermal growth factor (EGF)–like motif, and a
variable number of repeat units homologous to the consensus
repeats of complement binding proteins.9 The lectin domain of
E-selectin plays a major role in ligand recognition and binds to
sialyl Lex on leukocytes as well as on endothelial cells.10-12
E-selectin is rapidly synthesized and expressed on activated
endothelial cells and is rapidly shed from the cellular surfaces on
cellular activation.7 Elevated levels of soluble E-selectin (sEselectin) have been found in patients with vasculoproliferative
disorders such as RA13 and tumor growth.14,15 We have previously
shown that sE-selectin is a potent angiogenic mediator.16 Recently,
we have shown that sE-selectin mediates monocyte chemotaxis
through the Src- mitogen-activated protein kinase (MAPK) pathway.17 However, the signaling pathways by which sE-selectin
mediates angiogenesis are still unknown.
From the Veterans Administration, Lakeside Division, Chicago Health Care
System, Chicago, IL; the Department of Medicine, Northwestern University
Medical School, Chicago, IL; and the Department of Oral Sciences, University
of Minnesota School of Dentistry, Minneapolis, MN.
included funds from National Institute of Health grants AI-40987 and HL-58695.
Submitted April 29, 2002; accepted January 3, 2003. Prepublished online as
Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-04-1237.
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.
Supported by the Gallagher Professorship for Arthritis Research and funds
from the Veterans’ Administration Research Service. Additional support
3960
Reprints: Alisa E. Koch, Department of Medicine, Division of Rheumatology,
Northwestern Medical School, 303 E Chicago Ave, Ward 3-315, Chicago, IL
60611; e-mail: [email protected].
© 2003 by The American Society of Hematology
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
In this study, we examined the signaling pathways by which
sE-selectin mediates angiogenesis. Our results suggest that sEselectin mediates its signal in HMVECs through the Src-PI3K
pathway. Inhibition of Src, MAPK, and PI3K pathways with PP2,
PD98059, and LY294002, respectively, significantly reduced sEselectin–mediated HMVEC chemotaxis. Similarly, PP2 and
LY294002 significantly inhibited Matrigel in vitro endothelial cell
tube formation and Matrigel in vivo blood vessel formation.
However, PD98059 failed to significantly inhibit in vitro endothelial cell tube formation as well as in vivo blood vessel formation.
Taken together, these results suggest that Src- and PI3K-dependent
signaling pathways are major mediators of sE-selectin–induced
angiogenesis.
Materials and methods
Reagents
Recombinant human sE-selectin was purchased from R&D Systems
(Minneapolis, MN). sE-selectin contained less than 0.1 ng endotoxin per 1
␮g protein content (per the manufacturer). Basic fibroblast growth factor
(bFGF) was purchased from Intergen (Purchase, NY). Hanks balanced salt
solution (HBSS) was obtained from Life Technologies (Bethesda, MD).
Orthovanadate, paranitrophenylphosphate, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), dimethyl sulfoxide (DMSO), bovine serum
albumin (BSA), IGEPAL CA-630, pertusis toxin, and protein A agarose
conjugate were obtained from Sigma Chemical (St Louis, MO). Protease
inhibitor cocktail tablets were obtained from Boehringer Mannheim
(Mannheim, Germany). PP2, LY294002, and PD98059 were purchased
from Calbiochem (San Diego, CA). Rabbit polyclonal antihuman total Src
antibody, phosphospecific Src antibody, rabbit polyclonal antihuman PI3K
antibody, polyclonal rabbit antihuman total Akt antibody, and phosphospecific Akt antibody were purchased from Upstate Biotechnology (Lake
Placid, NY). Antibodies against total extracellular signal-related kinase 1/2
(ERK1/2) and phosphospecific ERK1/2 were purchased from Biosource
International (Camarillo, CA). Antitubulin antibody was purchased from
Oncogene Research Products (Boston, MA). Endothelial cell basal medium
(EBM) and the bullet kit containing media supplements were purchased
from BioWhittaker (Walkersville, MD). Growth factor–reduced Matrigel,
derived from Engelbreth-Holm-Swarm (EHS) mouse tumor, was purchased
from Becton Dickinson (Bedford, MA). Fetal bovine serum (FBS) and
media 199 were obtained from Gibco BRL (Grand Island, NY). Polycarbonate filters with 8-␮M pores were obtained from Poretics (Livemore, CA).
pcDNA3 plasmid, HB101 competent Escherichia coli cells, and Lipofectin
kits were obtained from Invitrogen (Carlsbad, CA). Protein estimation
reagents (bicinchoninic acid [BCA] kit) were purchased from Pierce
Biotechnology (Rockford, IL). [␥-32P] adenosine triphosphate (ATP),
enhanced chemiluminescence (ECL) Western blotting detection reagents,
and mouse horseradish peroxidase–conjugated antibody were obtained
from Amersham Life Sciences (Arlington Heights, IL).
Cell culture, cell lysis, and immunoblotting
HMVECs were purchased from BioWhittaker. HMVECs were cultured in
endothelial cell growth medium (BioWhittaker) containing the bullet kit
and 10% FBS. HMVECs were used between passages 3 and 12. HMVECs
(2 ⫻ 105/well) were plated in 6-well plates and incubated overnight at 37°C
in EBM containing the bullet kit and 10% FBS. HMVECs were further
incubated for 2 hours in EBM containing no bullet and reduced serum (2%
FBS) prior to stimulating with sE-selectin for various time points. At the
end of each time period, supernatants were aspirated and cells lysed in
extraction buffer containing 100 mM tris(hydroxymethyl)aminomethane
(Tris, pH 7.4), 100 mM NaCl, 1 mM ethylenediamine-tetra acetic acid
(EDTA), 1 mM ethyleneglycol-tetra acetic acid (EGTA), 1 mM NaF, 20
mM NaP2O4, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium
dodecyl sulfate (SDS), 0.5% deoxycholate, 1 mM PMSF, and protease
sE-SELECTIN–MEDIATED ANGIOGENESIS
3961
inhibitors (protease inhibitor cocktail tablets, Boehringer Mannheim, 1
tablet/10 mL). For experiments with signaling inhibitors, HMVECs were
preincubated with the respective inhibitor before activation with sEselectin. The protein content of different samples was quantitated using the
bicinchoninic acid (BCA) protein assay kit. Cell lysates were mixed 1:1
with Laemmli sample buffer and boiled for 5 minutes. Of each sample, 20
␮g was subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE).
Separated proteins were electrophoretically transferred from the gel onto
nitrocellulose membranes using a Tris-glycine buffer. To block nonspecific
binding, membranes were incubated with 5% nonfat milk in Tris-buffered
saline containing 0.01% Tween-20 (TBST) for 1 hour at room temperature.
The blots were incubated in respective primary antibody in TBST plus 5%
nonfat milk at 4°C overnight. After washing with TBST, the blots were
incubated with horseradish peroxidase–conjugated sheep anti–mouse immunoglobulin G (IgG; 1:10 000) or with goat anti–rabbit IgG (1:10 000) for 45
minutes at room temperature. An ECL detection system was used to detect
specific protein bands. Blots were scanned and analyzed for the measurement of the band intensities with UN-SCAN-IT version 5.1 software (Silk
Scientific, Orem, UT). The immunoblots were then stripped for 30 minutes
at 55°C in 67 mM Tris (pH 6.7), 2% SDS, 100 mM 2-mercaptoethanol, and
reprobed with the respective pan antibody.
Plasmid constructions and transient transfections
HMVECs were transiently transfected with 4 ␮g pcDNA3 plasmid
containing the dominant-negative mutants of ERK1/2, Akt, p38 MAPK,
or c-Jun (a gift from Dr Cun-Yu Wang, University of Michigan, Ann
Arbor) using Lipofectin kits (Invitrogen) per the manufacturer’s instructions. An empty pcDNA3 plasmid was used as a negative control and a
pcDNA3 plasmid containing CAT was used to optimize transfection
efficiency. At 72 hours after transfections, HMVECs were used in
chemotaxis assays (or Matrigel in vitro HMVEC tube formation assays)
or harvested for Western blotting.
Preparation of oligonucleotides (ODNs) and lipofection
of HMVECs
The ODNs were synthesized and purified by the Northwestern University
Biotechnology Laboratory and modified with phosphorothioate. Sequences
of the c-Src ODNs used in the study are as follows: antisense, GGG CTT
GCT CTT GCT GCT CCC CAT; and sense, ATG GGG AGC AGC AAG
AGC AAG CCC.18 HMVECs were transiently transfected with sense and
antisense ODNs as described earlier.18 At 16 hours after transfection,
HMVECs were used for chemotaxis assays (or Matrigel in vitro HMVEC
tube formation assays) or harvested for Western blotting.
PI3K assay
HMVECs (2 ⫻ 105 cells/well) were plated in 6-well plates in EBM
containing bullet and 10% FBS. Once the cells were 80% to 90% confluent,
they were further incubated in EBM containing reduced serum (2% FBS)
for 2 hours. HMVECs were then stimulated with sE-selectin (50 nM) for 15
minutes at 37°C. At the end of the incubation, cell lysate was prepared. For
inhibitor studies, HMVECs were pretreated with PP2 (10 ␮M) for 2 hours
prior to stimulation with sE-selectin. PP2 was also present during HMVEC
stimulation with sE-selectin. The protein content of each sample was
quantitated using a bicinchoninic acid (BCA) protein assay kit and
normalized according to the protein concentration. Of each sample, 500 ␮g
in 500 ␮L lysis buffer was incubated with 5 ␮L rabbit anti-PI3K antibody
overnight at 4°C with continuous shaking. Protein A agarose conjugate (60
␮L; 50% slurry in phosphate-buffered saline [PBS]) was added to each
sample and further incubated for one hour at 4°C. The immunoprecipitates
were collected by centrifuging at 14 000g for 10 seconds. The immunoprecipitates were then washed 3 times with buffer A (137 mM NaCl; 20 mM
Tris-HCl, pH 7.4; 1 mM CaCl2; 1 mM MgCl2; and 0.1 mM sodium
orthovanadate) containing 1% nonionic detergent IGEPAL CA-630, followed by 3 washes with buffer B (0.1 M Tris-HCl, pH 7.4; 5 mM LiCl; and
0.1 mM sodium orthovanadate) and 3 washes with TNE (10 mM Tris-HCl,
3962
KUMAR et al
pH 7.4; 150 mM NaCl; and 5 mM EDTA) containing 0.1 mM sodium
orthovanadate. The last wash was removed as completely as possible. To each
sample, the following reagents were added sequentially: 50 ␮L TNE, 10 ␮L (20
␮g) phosphatidylinositol (PI, in 10 mM Tris-HCl, pH 7.4, containing 1 mM
EGTA), and 10 ␮L 100 mM MgCl2. The PI reaction was initiated with the
addition of 5 ␮L [␥-32P] ATP. The reaction mixture was incubated at 37°C for 15
minutes with continuous agitation. The reaction was stopped by the addition of
20 ␮L 6 N HCl. Radiolabeled lipid was extracted from the reaction sample by
adding 160 ␮L CH3Cl/MeOH (1:1), vortexing, and then separating the organic
and aqueous phases by centrifugation for 10 minutes at 14 000g. Radiolabeled
lipid (50 ␮L) containing the lower organic phase was spotted onto oxalate-treated
thin layer chromatography (TLC) plates (Fisher Scientific, Pittsburgh, PA) and
developed in CHCl3/MeOH/H2O/NH4OH (60:47:11.3:2). The TLC plates were
dried and autoradiographed.
HMVEC chemotaxis
HMVECs (3.75 ⫻ 104 cells/25 mL EBM and 0.1% FBS) were placed in the
bottom wells of a 48-well Boyden chemotaxis chamber (NeuroProbe, Cabin
John, MD) with gelatin-coated polycarbonate membranes (8-␮M pore size;
Nucleopore, Pleasanton, CA).16 The chambers were inverted and incubated
in a humidified incubator with 5% CO2/95% air at 37°C for 2 hours,
allowing endothelial cell attachment to the membrane. The chambers were
inverted, and the test substances, PBS or positive control bFGF (60 nM) in
PBS, were added, and the chamber was further incubated for 2 hours at
37°C. In the inhibitor studies, HMVECs were pretreated with the respective
inhibitor (10 ␮M PP2, LY294002, or PD98059; or 50 ng/mL PT) for 2
hours, and inhibitors were present in the lower chamber along with
HMVECs during the chemotaxis assay. The membranes were then removed, fixed in methanol for 1 minute, and stained with Diff-Quik (Dade
Behring, Newark, DE). The data are expressed as the number of cells
migrating through the membrane per well (the sum of 3 high-power ⫻ 40
fields per well, averaged for each quadruplicate well).
Matrigel in vitro HMVEC tube formation assay
The Matrigel in vitro assay was used to examine HMVEC tube formation in
response to sE-selectin. Matrigel was plated in 8-well chamber slides after
thawing on ice and allowed to polymerize at 37°C for 30 to 60 minutes.
HMVECs (400 ␮L; 4 ⫻ 104 cells/mL in media 199 containing 2% FBS and
200 mg/mL endothelial cell growth supplement [Becton Dickinson]) was
added to each chamber and treated with bFGF (vehicle control) or
sE-selectin with or without the respective inhibitor. The chamber slides
were then incubated for 16 to 18 hours at 37°C in 5% CO2 humidified
atmosphere. Culture media were aspirated off the Matrigel surface, and the
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
cells were fixed with methanol and stained with Diff-Quick Solution II.
Each chamber was photographed using a Polaroid Microcam camera (Carl
Zeiss, Jena, Germany) at ⫻ 22 magnification. The number of tubes formed
was quantitated as previously described.19 Briefly, a connecting branch
between 2 discreet endothelial cells was counted as one “tube.”
Matrigel-plug angiogenesis in vivo assay
To examine the effect of sE-selectin on angiogenesis in vivo a Matrigelplug assay was used.20,21 C57/BL6 mice were anesthetized by Metofane
inhalation. Each mouse was shaved on its ventral aspect and given a
subcutaneous injection of sterile Matrigel (500 mL/injection) with a
27-gauge needle. Matrigel containing PBS served as the negative control;
Matrigel containing bFGF (1 ng/mL) served as the positive control; and
Matrigel with sE-selectin (100 nM) was the test substance. In the inhibitor
study, 20 ␮M respective inhibitor (PP2, LY294002, or PD98059) was added
along with sE-selectin in the Matrigel before it was injected. After 7 to 10
days, the mice were killed by Metofane inhalation and cervical dislocation.
The Matrigel plugs were then carefully dissected out, with removal of any
surrounding connective tissue, and then analyzed by hemoglobin measurement or by histology.22,23 For histologic analysis, 3 plugs from each group
were embedded in paraffin for tissue sectioning and staining.
Hemoglobin determination in Matrigel plugs. Hemoglobin levels in
the Matrigel plug correlate with blood vessel growth.19 The Matrigel plugs
dissected from the mice were carefully stripped of any remaining peritoneum. The plugs were weighed and homogenized for 5 to 10 minutes on ice.
Supernatant or standard (50 ␮L) was added to a 96-well plate in duplicate,
followed by 50 ␮L tetramethylbenzidine. The plate was allowed to develop
at room temperature for 15 to 20 minutes with gentle shaking, and the
reaction was stopped by adding 150 ␮L 2N H2SO4. Absorbance was read at
450 nm, and hemoglobin levels were normalized by the weight of the plugs.
Masson trichrome staining of Matrigel plugs. Using Masson trichrome
straining, 5-mm sections were deparaffinized and stained.19 In brief,
sections were hydrated in distilled water followed by incubation with Bouin
solution for one hour at 56°C, washing in water, incubation with Weigert
iron solution for 7 minutes, washing with water, and incubation with
Biebrich scarlet-acid fuchsin solution for 2 minutes. Sections were then
rinsed, incubated in phosphomolybdic-phosphotungstic acid solution for 10
minutes, dipped in aniline blue solution for 5 minutes, rinsed, dipped in
glacial acetic acid solution for 3 to 5 minutes, and dehydrated in 2 changes
of 95% alcohol, 100% alcohol, and 100% xylene. The slides were mounted
with Cytoseal 60 (Stephens Scientific, Kalamazoo, MI).
Figure 1. sE-selectin induces HMVEC chemotaxis through Src and PI3K pathways. (A) HMVECs (1 ⫻ 106 cells/mL) in the lower chamber were incubated with various
concentrations of sE-selectin in the upper chamber for 2 hours at 37°C. PBS was used as a negative control and bFGF (60 nM) as a positive control. Results are expressed as
the number of cells migrating through the membrane per well ⫾ SEM from 3 independent experiments. sE-selectin showed a dose-dependent increase in HMVEC chemotaxis
compared with negative control PBS (P ⬍ .05). *Represents a significant difference (P ⬍ .05) between the sE-selectin and negative control PBS. (B) For inhibition studies,
HMVECs were pretreated with the respective inhibitor or inhibitor combination (10 ␮M PP2, LY294002, or PD98059; or 50 ng/mL PT) for 2 hours at 37°C and then assayed in
48-well chemotaxis chambers in response to 50 nM of sE-selectin. Results are expressed as the number of cells migrating through the membrane per well ⫾ SEM from 6
independent experiments. PT in the figure represent pertusis toxin. *Represents a significant difference (P ⬍ .05) between the respective groups. sE-selectin induced HMVEC
chemotaxis predominantly through the Src and PI3K pathways.
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
sE-SELECTIN–MEDIATED ANGIOGENESIS
3963
Statistical analysis
Data were analyzed using Student t test, and P values less than .05 were
considered significant.
Results
sE-selectin mediates HMVEC chemotaxis through the Src
and PI3K pathways
We have previously shown that sE-selectin is a potent angiogenic
mediator.16 To examine the signaling cascade by which sE-selectin
mediates angiogenesis, we performed HMVEC chemotaxis assays,
a facet of the angiogenic response, in 48-well modified Boyden
chambers. sE-selectin induced a dose-dependent increase in HMVEC chemotaxis, reaching significance at 1 nM to 100 nM
(P ⬍ .05, Figure 1A), compared with negative control PBS. bFGF
was used as positive control in all the assays. To further study the
role of different signaling pathways that may be involved in
sE-selectin–mediated HMVEC chemotaxis, HMVECs were pretreated with the respective signaling inhibitor or combination of
Src kinase inhibitor (PP2, 10 ␮M), PI3K inhibitor (LY294002,
10 ␮M), MAPK inhibitor (PD98059, 10 ␮M), or G-protein
inhibitor (PT, 50 ng/mL) for 2 hours prior to performing the
chemotaxis assays. The Src inhibitor (PP2) was most effective in
inhibiting sE-selectin–mediated HMVEC chemotaxis (37%),
whereas the MAPK inhibitor (PD98059), PI3K inhibitor
(LY294002), and G-protein inhibitor (PT) showed 20%, 34%, and
9% inhibition, respectively (Figure 1B). HMVECs pretreated with the
combination of PP2 and PD98059 showed 76% inhibition of chemotaxis, which was further enhanced by the combination of PP2 and
LY294002 (79%) or the combination of PP2, PD98059, and LY294002
(87%). However, the combination of PD98059 and LY294002 showed
47% sE-selectin–mediated HMVEC chemotaxis inhibition.
To further corroborate the results obtained in the HMVEC
chemotaxis studies using signaling inhibitors, we performed a
similar assay using HMVECs transiently transfected with dominantnegative mutants of ERK1/2 and Akt or HMVECs transiently
transfected with c-Src antisense and sense ODNs. A pcDNA3
plasmid with no insert was used as control for the mutant studies.
HMVECs transiently transfected with plasmids with no insert
Figure 2. HMVECs transfected with the Akt dominant-negative mutant or Src
antisense oligonucleotides showed significantly less sE-selectin–mediated
chemotaxis. (A) HMVECs were transiently transfected with pcDNA3 plasmids
containing ERK1/2 or Akt dominant-negative mutants. Plasmids with no insert were
used as a negative control. At 72 hours after transfections, HMVEC chemotaxis was
performed. (B) HMVECs were transiently transfected with c-Src antisense ODN or
control sense ODN. At 16 hours after transfections, HMVEC chemotaxis was
performed. Results are expressed as the mean number of cells migrating through the
membrane per well ⫾ SEM from 4 independent experiments. *Represents a
significant difference (P ⬍ .05) between the respective groups. HMVECs transfected
with an Akt mutant or Src antisense ODN showed significant inhibition of sE-selectin–
mediated chemotaxis, whereas HMVECs transfected with an ERK1/2 mutant failed to
show significant inhibition.
Figure 3. sE-selectin–induced Src phosphorylation is inhibited by the Src
inhibitor PP2. HMVECs were stimulated with 50 nM sE-selectin for various time
points as indicated (m indicates minutes; NS, nonstimulated). In the inhibition study,
HMVECs were pretreated with PP2 (10 ␮M) for 2 hours prior to stimulating with
sE-selectin, and PP2 was also present during the stimulation with sE-selectin. Cell
extracts were prepared, and the protein content in each sample was quantitated.
Samples (20 ␮g each) were resolved by 10% SDS-PAGE and probed with rabbit
antihuman phospho-Src (*p-Src) antibody. To verify equal loading, the blots were
stripped and reprobed with rabbit antihuman total Src antibody. (A) Representative
blot from 3 independent experiments. (B) Ratio of p-Src to total Src band density ⫾
SEM from 3 independent experiments. *Represents a significant difference (P ⬍ .05)
between the respective groups. sE-selectin induced a marked increase in Src
phosphorylation at 10, 20, and 30 minutes after stimulation. Pretreatment of HMVECs
with PP2 significantly inhibited Src phosphorylation.
showed significantly more chemotaxis in response to both sEselectin and positive control bFGF (Figure 2A). HMVECs transfected with the Akt mutants showed significantly less (55%)
sE-selectin–mediated HMVEC chemotaxis, whereas HMVECs
transfected with the ERK1/2 mutants did not show a significant
inhibition (14%). HMVECs transfected with Src antisense ODNs
showed significant inhibition (53%) of sE-selectin–mediated chemotaxis compared with HMVECs transfected with sense ODNs
(Figure 2B). These results from HMVEC chemotaxis assay using
signaling inhibitors, dominant-negative mutants, and sense and
antisense ODNs suggest that sE-selectin may be mediating HMVEC chemotaxis predominantly through the Src and PI3K pathways and partially through the MAPK pathway.
Signaling cascade mediated by sE-selectin in HMVECs
We next examined the signaling pathways involved in sE-selectin–
stimulated HMVECs. sE-selectin induced protein tyrosine phosphorylation of several proteins in the molecular-weight range of 140 to
Figure 4. sE-selectin induces a time-dependent increase in ERK1/2 phosphorylation. HMVECs were stimulated with 50 nM sE-selectin for various time points as
indicated (m indicates minutes; NS, nonstimulated). In the inhibition study, HMVECs
were pretreated with PP2 (10 ␮M) for 2 hours prior to stimulating with sE-selectin, and
PP2 was also present during the stimulation with sE-selectin. Cell extracts were
prepared and the protein content in each sample was quantified. Samples (20 ␮g
each) were resolved by 10% SDS-PAGE and probed with rabbit antihuman
phospho-ERK1/2 (*p-ERK1/2) antibody. To verify equal loading, the blots were
stripped and reprobed with rabbit antihuman ERK1/2 antibody. (A) Representative
blot from 3 independent experiments. (B) The ratio of p-ERK1/2 to ERK1/2 band
density ⫾ SEM from 3 independent experiments. *Represents a significant difference
(P ⬍ .05) between the respective groups. sE-selectin induced a marked increase in
ERK1/2 phosphorylation at 10, 20, and 30 minutes after stimulation. Pretreatment of
HMVECs with PP2 significantly inhibited ERK1/2 phosphorylation.
3964
KUMAR et al
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
Figure 5. sE-selectin induces PI3K and Akt activation in HMVECs. HMVECs were stimulated with sE-selectin (50 nM) for 15 minutes. In the inhibition study HMVECs were
pretreated with PP2 (10 ␮M) for 2 hours prior to stimulation with sE-selectin, and PP2 was also present during the stimulation with sE-selectin. (A) PI3K was
immunoprecipitated from the cell lysate and then the kinase assay performed using PI as the substrate and [␥-32P]–labeled ATP as the donor of phosphate ions. The
[␥-32P]–labeled lipids were resolved by thin layer chromatography (TLC) and autoradiographed. This is a representative blot from 3 independent experiments. sE-selectin
induced a marked increase in PI phosphorylation (*PI3P) compared with nonstimulated cells. PP2-pretreated HMVECs exhibited substantial inhibition of PI3K activation, thus
indicating that PI3K lies downstream of Src kinase. (B) Cell extracts were prepared and the protein content in each sample was quantitated. Samples (20 ␮g) were resolved by
10% SDS-PAGE and probed with mouse monoclonal antihuman phospho-Akt (*p-Akt) antibody. To verify equal loading, the blots were stripped and reprobed with rabbit
anti-Akt antibody. This is a representative blot from 3 independent experiments. (C) The ratio of p-Akt to Akt band density ⫾ SEM from 3 independent experiments. *Represents
a significant difference (P ⬍ .05) between the respective groups. sE-selectin induced a marked increase in Akt phosphorylation. Pretreatment of HMVECs with PP2
significantly inhibited Akt phosphorylation.
150, 130 to 140, 50 to 65, and 30 to 40 kDa (data not shown). As the
predominant band was observed in the molecular-weight range of
Src kinase (⬇ 60 kDa), we were interested to determine whether
Src family kinases were activated in HMVECs upon stimulation
with sE-selectin. We performed a time-course study of Src kinase
activation followed by Src inhibition using the Src inhibitor PP2.
sE-selectin induced a marked increase in Src phosphorylation at 10,
20, and 30 minutes (Figure 3). Pretreatment of HMVECs with PP2
showed substantial inhibition of Src phosphorylation at 20 and 30
minutes. Equal amounts of protein samples were loaded in each
lane, as confirmed by stripping the blots and reprobing with rabbit
antihuman total Src antibody. We next examined whether sEselectin could also activate the ERK1/2 and the p38 MAPK
pathways in HMVECs. sE-selectin induced a time-dependent
increase in phosphorylation of ERK1/2, which peaked at 30
minutes (Figure 4). However, sE-selectin failed to activate p38
MAPK in HMVECs (data not shown). sE-selectin–induced ERK1/2
phosphorylation was significantly inhibited by the pretreatment of
HMVECs with PP2, suggesting that Src lies upstream of ERK1/2
in sE-selectin–mediated endothelial cell signaling (Figure 4).
Because the PI3K/Akt pathway is known to play an important
role in angiogenesis, we also examined whether the PI3K/Akt
pathway is activated in sE-selectin–stimulated HMVECs.2,24 HMVECs stimulated with sE-selectin showed a marked increased in PI
and Akt phosphorylation (Figure 5), thereby indicating that sEselectin, in addition to activating Src and ERK1/2, also activates
the PI3K/Akt pathway in HMVECs. Next, we examined whether
sE-selectin–induced activation of PI3K in HMVECs was mediated
via Src kinases. To study this, we first pretreated HMVECs with the
Src inhibitor PP2 (10 ␮M) for 2 hours prior to preparing the cell
lysate for both the PI3K assay and the Akt phosphorylation studies.
Pretreatment of HMVECs with PP2 substantially inhibited sEselectin–mediated PI3K activation as observed by PI3K assay and
Akt phosphorylation studies (Figure 5). To further examine whether
there was any cross talk between the PI3K/Akt and ERK1/2
pathways in sE-selectin–stimulated endothelial cells, HMVECs
were pretreated with PP2, PD98059, or LY294002 prior to
stimulation with sE-selectin. The Src inhibitor PP2, as observed
earlier, inhibited both Akt and ERK1/2 activation (Figure 6A). The
PI3K inhibitor (LY294002) and the ERK1/2 inhibitor (PD98059)
failed to show ERK1/2 and Akt inhibition, respectively. Similarly,
HMVECs transfected with the ERK1/2 mutants showed inhibition
of sE-selectin–induced ERK1/2 phosphorylation, but not of Akt
phosphorylation. HMVECs transfected with the Akt mutants
showed inhibition of Akt phosphorylation but not ERK1/2 phosphorylation (Figure 6B). These results suggest that sE-selectin mediates
its signal in HMVECs through PI3K/Akt and ERK1/2 pathways via
Src kinases. HMVECs transfected with antisense Src ODNs
showed significant inhibition of sE-selectin–meditated Src phosphorylation compared with sense Src ODNs (Figure 6C). HMVECs
Figure 6. sE-selectin induces phosphorylation of ERK1/2 and Akt independent
of each other. (A) HMVECs were stimulated with 50 nM sE-selectin for 30 minutes.
In the inhibition study, HMVECs were pretreated with either PP2, PD98059, or
LY294002 (10 ␮M) for 2 hours prior to stimulating with sE-selectin. Cell extracts were
prepared and the protein content in each sample was quantitated. Of each sample,
20 ␮g was resolved by 10% SDS-PAGE and probed with rabbit antihuman
phospho-ERK1/2 (*p-ERK1/2) or rabbit antihuman phospho-Akt (*p-Akt) antibodies.
To verify equal loading, the blots were stripped and reprobed with rabbit antihuman
ERK1/2 or Akt antibody. (B) HMVECs transfected with pcDNA3 containing no insert
or ERK1/2 or Akt mutant were stimulated with sE-selectin for 30 minutes and cell
lysates were prepared. Cell extracts were resolved by 10% SDS-PAGE and probed
with rabbit antihuman phospho-ERK1/2 (*p-ERK1/2) or rabbit antihuman phosphoAkt (*p-Akt) antibodies. Equal loading was verified by stripping the blots and
reprobing with rabbit anti-ERK1/2 or Akt antibody. (C) HMVECs transfected with Src
sense or antisense ODNs were stimulated with sE-selectin for 20 minutes and cell
lysates were prepared. Cell extracts were resolved by 10% SDS-PAGE and probed
with rabbit antihuman phospho-Src (*p-Src) antibody. Equal loading was verified by
stripping the blots and reprobing with rabbit anti-Src antibody. (D) Cell lysates from
HMVECs transfected with pcDNA3 containing no insert, Akt, or ERK mutants were
resolved by 10% SDS-PAGE and probed with mouse monoclonal anti-HA antibody.
Equal loading was verified by stripping the blots and reprobing with mouse
monoclonal antitubulin antibody. sE-selectin induced a time-dependent increase in
Akt and ERK1/2 phosphorylation via Src kinase.
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
sE-SELECTIN–MEDIATED ANGIOGENESIS
3965
Figure 7. sE-selectin induces endothelial cell tube
formation on Matrigel in vitro. The Matrigel in vitro
HMVEC tube formation assay was performed in 8-well
chamber slides. (A) Photomicrographs of representative
assays for DMSO; bFGF; sE-selectin; sE-selectin ⫹
PP2; sE-selectin ⫹ LY294002; and sE-selectin ⫹
PD98059. Original magnification, ⫻ 20. (B) Quantitative
data for endothelial cell tube formation are expressed as
number of tubes/well ⫾ SEM from 3 independent experiments. bFGF is used as positive control. *Represents a
significant difference (P ⬍ .05) between the respective
groups. sE-selectin induced significantly more endothelial tube formation than vehicle control, and this tube
formation was inhibited upon treatment with PP2 and
LY294002. However, HMVECs treated with PD98059 did
not show significant inhibition of sE-selectin–induced
endothelial cell tube formation.
transfected with dominant-negative mutants of Akt and ERK1/2
showed similar relative transfection efficiency as assessed by
Western blotting with the tagged protein hemagglutinin (HA)
(Figure 6D).
fected with ERK1/2 mutants or Src sense ODNs did not show
significant inhibition. These results suggest that sE-selectin
mediates HMVEC tube formation in Matrigel in vitro through
the Src and PI3K pathways but not through the MAPK pathway.
The Src inhibitor (PP2) and the PI3K inhibitor (LY294002) inhibit
sE-selectin–mediated tube formation in the Matrigel
assay in vitro
sE-selectin mediates blood vessel formation through Src
and PI3 kinase pathways
We performed 2 assays to examine the signaling pathways
involved in sE-selectin–mediated angiogenesis. In the first
assay, the Matrigel in vitro HMVEC tube formation assay, we
used PP2, LY294002, and PD98059 inhibitors to study the role
of Src kinases, PI3K, and MAPK, respectively. The Src kinase
inhibitor (PP2) and the PI3K inhibitor (LY294002) significantly
inhibited sE-selectin–mediated HMVEC tube formation (47%
and 49%, respectively) in the Matrigel in vitro assay (P ⬍ .05;
Figure 7). However, the MAPK inhibitor (PD98059) failed to
show significant inhibition of HMVEC tube formation (5%)
compared with sE-selectin–treated cells. The same assay was
performed using HMVECs transfected with Akt or ERK1/2
dominant-negative mutants or with HMVECs transfected with
Src sense or antisense ODNs. HMVECs transfected with
pcDNA3 plasmids containing no insert were used as the
negative control (Figure 8). Similar to the inhibitor studies,
HMVECs transfected with the Src antisense ODNs or Akt
dominant-negative mutants showed significant inhibition of
sE-selectin–mediated tube formation, whereas HMVECs trans-
We used a bioassay, the Matrigel-plug in vivo assay, to examine
whether sE-selectin mediates its biologic function through similar
signaling pathways as observed in HMVEC tube formation by the
Matrigel in vitro assay. Each inhibitor (PP2, LY294002, or
PD98059) was incorporated in the Matrigel along with sE-selectin
before Matrigel was injected into the mice. Similar to the results
observed with the in vitro HMVEC tube formation assay, PP2 and
LY294002 significantly (P ⬍ .05) inhibited sE-selectin–mediated
blood vessel formation (65% and 44%, respectively) in the
Matrigel in vivo assay (Figure 9). However, the MAPK inhibitor
PD98059 did not show significant inhibition of sE-selectin–
mediated blood vessel formation (8%). In the same assay system,
the MAPK inhibitor PD98059 significantly inhibited bFGFmediated blood vessel formation (Figure 9C). These results
along with the in vitro tube formation assay as well as HMVEC
chemotaxis assay suggest that that sE-selectin mediates its
biologic function in HMVECs predominantly through Src and
PI3K pathways.
Figure 8. HMVECs transfected with Akt dominant-negative mutants or Src antisense ODNs show significantly less sE-selectin–induced tube formation. HMVECs
were transiently transfected with a pcDNA3 plasmid containing ERK1/2 and Akt dominant-negative mutants or with Src sense or antisense ODN. Plasmid with no insert was
used as a negative control. HMVECs transfected with mutants were used for Matrigel in vitro HMVEC tube formation assays 72 hours after transfection, whereas HMVECs
transfected with Src ODNs were used 16 hours after transfection. (A) Photomicrographs of a representative assay for (i) HMVECs transfected with no insert, (ii) HMVECs
transfected with no insert and stimulated with sE-selectin, (iii) HMVECs transfected with an Akt mutant, (iv) HMVECs transfected with an ERK1/2 mutant, (v) HMVECs
transfected with Src antisense ODNs and stimulated with sE-selectin, and (vi) HMVECs transfected with Src sense ODNs. Original magnification, ⫻ 20. (B) Quantitative data
for HMVEC tube formation are expressed as number of tubes/well ⫾ SEM from 4 independent experiments. *Represents a significant difference (P ⬍ .05) between groups.
HMVECs transfected with Akt dominant-negative mutants and Src antisense ODNs showed significantly less tube formation compared with HMVECs transfected with
no-insert plasmid.
3966
KUMAR et al
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
Figure 9. PP2 and LY294002 inhibit sE-selectin–mediated blood vessel formation in Matrigel plugs in vivo. (A) A representative assay showing Masson trichrome
staining of blood vessels in paraffin sections of Matrigel plugs treated with PBS; bFGF; sE-selectin; sE-selectin ⫹ PP2; sE-selectin ⫹ LY294002; and sE-selectin ⫹ PD98059.
Arrows indicate blood vessels. Original magnification, ⫻ 40. (B) sE-selectin induced significantly more blood vessel formation (*P ⬍ .05, n ⫽ 18) compared with the PBS
group. The values represent the concentration of hemoglobin (g/dL)/Matrigel-plug weight (mg) ⫾ SEM, with n indicating the number of plugs. The Src inhibitor (PP2) and PI3K
inhibitor (LY294002) significantly inhibited sE-selectin–mediated blood vessel formation. However, the MAPK inhibitor (PD98059) failed to significantly inhibit sE-selectin–
mediated blood vessel formation. (C) bFGF-induced blood vessel formation was significantly inhibited by the MAPK inhibitor PD98059.
Discussion
In the present study, we have examined the role of different
signaling pathways by which sE-selectin mediates HMVEC chemotaxis and angiogenesis. We have previously shown that sE-selectin
is a potent angiogenic mediator,16 and sE-selectin levels are
up-regulated in patients with vasculoproliferative disorders such as
RA25 and tumor growth.14,15 However, the signaling pathways by
which sE-selectin mediates HMVEC chemotaxis and angiogenesis
are still unknown. Our results from the present study show that
sE-selectin mediates HMVEC chemotaxis and angiogenesis predominantly through the Src and PI3K pathways.
Previously, Simon et al26 have shown that neutrophil tethering
on intact transmembrane E-selectin transduces signals in endothelial cells through the MAPK signaling pathway, and cross-linking
of endothelial cell–surface E-selectin with antibodies led to upregulation of mRNA for c-fos, an early response gene, through the
Ras-MAPK pathway.27 We have recently shown that sE-selectin is
a potent chemotactic factor for monocytes, and it mediates
monocyte chemotaxis through the Src-MAPK pathway.17 However, to our knowledge this is the first report studying the signaling
cascade involved in soluble E-selectin–mediated endothelial cell
chemotaxis and angiogenesis. HMVECs stimulated with sEselectin showed phosphorylation of a number of proteins corresponding to molecular weights of 140 to 150, 130 to 140, 55 to 65,
and 30 to 40 kDa (data not shown). As the predominant band
obtained using anti–phosphotyrosine antibody was observed in the
molecular-weight range of Src kinase, and the Src family kinases
are known to play an important role in a number of physiologic
functions including monocyte adherence to endothelial cells,28
endothelial cell differentiation,29 endothelial cell survival,30 and
endothelial cell proliferation and angiogenesis,1 we examined the
role of Src kinase in sE-selectin–stimulated HMVECs. sE-selectin
induced a marked increase in HMVEC Src kinase phosphorylation,
which was substantially inhibited by PP2 or by transfecting the
HMVECs with Src antisense ODNs, thus indicating that Src may
be an important mediator in sE-selectin–induced signaling in
HMVECs. We have previously shown in monocytes that sEselectin activates ERK1/2 and p38 MAPK pathways downstream
of Src kinase.17 We next examined whether sE-selectin also
activates ERK1/2 and p38 MAPK in HMVECs. sE-selectin
induced a time-dependent increase in ERK1/2 phosphorylation,
which was significantly inhibited by PP2. However, sE-selectin
failed to activate p38 MAPK in HMVECs. These results suggest
that sE-selectin may be using similar signaling pathways in
HMVECs as observed previously in monocytes,17 except that in
HMVECs sE-selectin does not activate p38 MAPK.
PI3K, a heterodimer of an 85-kDa adaptor subunit and 100-kDa
catalytic subunit,31,32 is activated by a number of growth factors
including VEGF, FGF, platelet-derived growth factor (PDGF), and
EGF.24,33 It has been implicated in the regulation of cell proliferation,24 differentiation,34 cell survival,35 and angiogenesis.2 To test
whether sE-selectin also mediates its signal in HMVECs through
PI3K, we examined the activation of PI3K by studying the
phosphorylation of 2 downstream PI3K substrates: phosphatidylinositol (PI) and the serine-threonine kinase Akt. sE-selectin induced
markedly higher PI and Akt phosphorylation in HMVECs compared with nonstimulated cells. A number of studies have shown
that PI3K and its substrate Akt can be activated by Src family
kinases.1,30,36 In order to examine whether sE-selectin induces PI3K
activation via Src family kinases, HMVECs were pretreated with
PP2 (10 ␮M) for 2 hours prior to stimulation with sE-selectin.
Pretreatment of HMVECs with PP2 significantly inhibited sEselectin–mediated PI phosphorylation as well as Akt phosphorylation (50%-60%). These results indicate that sE-selectin–induced
PI3K/Akt activation is mediated via Src kinase. To further understand whether there was any cross-regulation of PI3K/Akt and
ERK1/2 pathways in sE-selectin–stimulated endothelial cells,
HMVECs were pretreated with the ERK1/2 inhibitor (PD98059) or
the PI3K inhibitor (LY294002) prior to stimulation with sEselectin. Similar studies were done using HMVECs transfected
with ERK1/2 or Akt mutants that were subsequently stimulated
with sE-selectin. Our results indicate that both PI3K/Akt and
ERK1/2 pathways are activated by sE-selectin in HMVECs
independently of each other via Src kinase. A previous study33 has
shown that EGF also uses a similar signaling cascade as observed
with sE-selectin. It may be important to point out that E-selectin
contains an EGF motif in its sequence.9 However, sE-selectin failed
to show EGF receptor phosphorylation in endothelial cells (data
not shown). It is possible that sE-selectin may be using a similar
receptor molecule to mediate its signal. Currently, only the
lectin-binding part of the sE-selectin receptor (sialyl Lex) is known
and is present on leukocytes as well as on endothelial cells.10-12 The
structure of the protein portion of the receptor molecule is unknown.
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
sE-SELECTIN–MEDIATED ANGIOGENESIS
Next, to examine the functional role of sE-selectin, we performed HMVEC chemotaxis assays, Matrigel in vitro HMVEC
tube formation assays, and Matrigel-plug in vivo blood vessel
formation assays. The results from HMVEC chemotaxis using
signaling inhibitors suggest that sE-selectin mediates chemotaxis
predominantly through the Src and PI3K pathways. Inhibition of
the Src pathway by PP2 provided the maximum inhibition (36%),
followed by the PI3K inhibitor LY294002 (33%) and MAPK
inhibitor PD98059 (20%). However, none of the inhibitors completely blocked sE-selectin–mediated HMVEC chemotaxis, thereby
suggesting that sE-selectin may be mediating HMVEC chemotaxis
through multiple pathways. Pretreatment of HMVECs with a
combination of PP2, PD98059, and LY294002 was most effective
and showed 87% inhibition of sE-selectin–mediated HMVEC
chemotaxis, whereas combination of PP2 plus LY294002 or PP2
plus PD98059 showed 79% and 76% inhibition, respectively.
However, combination of PD98059 plus LY294002 was much less
effective and showed only 46% inhibition. These results suggests
that Src may also be activating other pathways that are independent
of PI3K/Akt or ERK1/2 to mediate HMVEC chemotaxis as Src
inhibitor PP2 is needed to mediate the most significant inhibition.
To further corroborate our results using inhibitors, we performed
endothelial cell chemotaxis assays using HMVECs transiently
transfected with dominant-negative mutants of ERK1/2 and Akt or
HMVECs transfected with Src antisense and sense ODNs. HMVECs transfected with the Akt mutant or Src antisense ODNs
showed significant decrease in sE-selectin–mediated HMVEC
chemotaxis (55% and 53%, respectively) compared with HMVECs
transfected with control vector alone or Src sense ODNs. In
contrast, HMVECs transfected with an ERK1/2 mutant did not
show significant inhibition (14%). Taken together, these results
from HMVEC chemotaxis assay using signaling inhibitors as well
as dominant-negative mutants or Src antisense ODNs suggest that
sE-selectin may be mediating HMVEC chemotaxis predominantly
through the Src and PI3K pathways, and partially through the
ERK1/2 MAPK pathway. Abu-Ghazaleh et al37 and Mochizuki et
al38 have also shown that Src and PI3K pathways play a predominant role in the endothelial cell migration in response to VEGF and
angiopoietin 2.
3967
Angiogenesis is critical in the development of tumors2 and
inflammatory disorders such as RA.39,40 We have previously
reported that sE-selectin is a potent angiogenic mediator.16 In the
present study we examined the signaling cascade by which
sE-selectin mediates angiogenesis. We used 2 assays to study
angiogenesis, the Matrigel in vitro HMVEC tube formation assay
and Matrigel-plug in vivo assay. sE-selectin induced a 2.2-fold
increase in endothelial cell tube formation compared with vehicle
control. PP2 and LY294002 significantly (P ⬍ .05) inhibited
sE-selectin–mediated endothelial cell tube formation (47% and
49%, respectively). Similarly, HMVECs transfected with Src
antisense ODNs or Akt dominant-negative mutants showed significant inhibition of sE-selectin–mediated tube formation compared
with HMVECs transfected with control plasmids or Src sense
ODNs. However, HMVECs transfected with an ERK1/2 mutant or
treated with an ERK1/2 inhibitor (PD98059) failed to show
significant inhibition. In the Matrigel in vivo assay, sE-selectin also
induced significant (2.2-fold) increase in blood vessel formation,
which was significantly inhibited (P ⬍ .05) by treatment with PP2
(65%) or LY294002 (44%). Similar to these results obtained in the
Matrigel in vitro tube formation assay, treatment with PD98059
failed to significantly inhibit sE-selectin–mediated blood vessel
formation in Matrigel plugs in vivo (8%). These results suggest that
even though the MAPK pathway is activated by sE-selectin in
HMVECs and might play a partial role in sE-selectin–mediated
HMVEC chemotaxis, sE-selectin–mediated blood vessel formation
both in vitro as well as in vivo is predominantly mediated through
the Src and PI3K pathways. Previously, Hartwell et al41 have
shown that E-selectin–deficient and wild-type mice exhibit similar
blood vessel formation in response to bFGF and tumor necrosis
factor-␣ (TNF-␣). It could be that bFGF and TNF-␣ do not require
E-selectin molecules to mediate angiogenesis. In contrast, Aoki et
al have shown that E-selectin plays an important role in VEGFmediated angiogenesis as antimouse E-selectin antibody suppressed tube formation induced by recombinant VEGF.42 Taken
together, our present data indicate that sE-selectin mediates angiogenesis predominantly through the Src-PI3K-Akt pathway. Our
study also suggests a potentially novel method to inhibit sE-selectin–
mediated angiogenesis via Src and PI3K pathways.
References
1. Eliceiri BP, Paul R, Schwartzberg PL, et al. Selective requirement for Src kinases during VEGFinduced angiogenesis and vascular permeability.
Mol Cell. 1999;4:915-924.
2. Jiang BH, Zheng JZ, Aoki M, et al. Phosphatidylinositol 3-kinase signaling mediates angiogenesis
and expression of vascular endothelial growth
factor in endothelial cells. Proc Natl Acad Sci
U S A. 2000;97:1749-1753.
3. Kypta RM, Goldberg Y, Ulug ET, et al. Association
between the PDGF receptor and members of the
src family of tyrosine kinases. Cell. 1990;62:481492.
4. Mori S, Ronnstrand L, Yokote K, et al. Identification of two juxtamembrane autophosphorylation
sites in the PDGF beta-receptor; involvement in
the interaction with Src family tyrosine kinases.
EMBO J. 1993;12:2257-2264.
5. Toker A, Cantley LC. Signalling through the lipid
products of phosphoinositide-3-OH kinase.
Nature. 1997;387:673-676.
6. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998;282:1318-1321.
7. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood. 1994;84:2068-2101.
8. Bevilacqua MP, Pober JS, Mendrick DL, et al.
Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci
U S A. 1987;84:9238-9242.
15. Benekli M, Gullu IH, Tekuzman G, et al. Circulating intercellular adhesion molecule-1 and Eselectin levels in gastric cancer. Br J Cancer.
1998;78:267-271.
9. Chapman PT, Haskard DO. Leukocyte adhesion
molecules. Br Med Bull. 1995;51:296-311.
16. Koch AE, Halloran MM, Haskell CJ, et al. Angiogenesis mediated by soluble forms of E-selectin
and vascular cell adhesion molecule-1. Nature.
1995;376:517-519.
10. Phillips ML, Nudelman E, Gaeta FCA, et al.
ELAM-1 mediates cell adhesion by recognition of
a carbohydrate ligand, sialyl-Lex. Science. 1990;
250:1130-1132.
11. Lowe JB, Stoolman LM, Nair RP, et al. ELAM-1–
dependent cell adhesion to vascular endothelium
determined by a transfected human fucosyltransferase cDNA. Cell. 1990;63:475-484.
12. Berg EL, Robinson MK, Mansson O, et al. A carbohydrate domain common to both sialyl-Lea and
sialyl-Lex is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. J Biol Chem.
1991;266:14869-14872.
17. Kumar P, Hosaka S, Koch AE. Soluble E-selectin
induces monocyte chemotaxis through Src family
tyrosine kinases. J Biol Chem. 2001;276:2103921045.
18. Morel JC, Park CC, Zhu K, Kumar P, Ruth JH,
Koch AE. Signal transduction pathways involved
in rheumatoid arthritis synovial fibroblast interleukin-18-induced vascular cell adhesion molecule-1
expression. J Biol Chem. 2002;277:34679-34691.
13. Koch AE, Turkiewicz W, Harlow LA, et al. Soluble
E-selectin in arthritis. Clin Immunol Immunopathol. 1993;69:29-35.
19. Volin MV, Harlow LA, Woods JM, et al. Treatment
with sulfasalazine or sulfapyridine, but not
5-aminosalicyclic acid, inhibits basic fibroblast
growth factor-induced endothelial cell chemotaxis. Arthritis Rheum. 1999;42:1927-1935.
14. Hebbar M, Peyrat JP. Significance of soluble endothelial molecule E-selectin in patients with
breast cancer. Int J Biol Markers. 2000;15:15-21.
20. Passaniti A, Taylor RM, Pili R, et al. A simple,
quantitative method for assessing angiogenesis
and antiangiogenic agents using reconstituted
3968
BLOOD, 15 MAY 2003 䡠 VOLUME 101, NUMBER 10
KUMAR et al
basement membrane, heparin, and fibroblast
growth factor. Lab Invest. 1992;67:519-528.
21. Johns A, Freay AD, Fraser W, et al. Disruption of
estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.
Endocrinology. 1996;137:4511-4513.
22. Lijana RC, Williams MC. Tetramethylbenzidine—a substitute for benzidine in hemoglobin
analysis. J Lab Clin Med. 1979;94:266-276.
23. Liem HH, Cardenas F, Tavassoli M, et al. Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3⬘,5,5⬘tetramethylbenzidine dihydrochloride, a safe
substitute for benzidine. Anal Biochem. 1979;98:
388-393.
24. Roche S, Koegl M, Courtneidge SA. The phosphatidylinositol 3-kinase alpha is required for
DNA synthesis induced by some, but not all,
growth factors. Proc Natl Acad Sci U S A. 1994;
91:9185-9189.
25. Jacob CO, McDevitt HO. Tumour necrosis factor-␣ in murine autoimmune ‘lupus’ nephritis.
Nature. 1988;331:356-358.
26. Simon SI, Hu Y, Vestweber D, et al. Neutrophil
tethering on E-selectin activates beta 2 integrin
binding to ICAM-1 through a mitogen-activated
protein kinase signal transduction pathway. J Immunol. 2000;164:4348-4358.
27. Hu Y, Kiely JM, Szente BE, et al. E-selectindependent signaling via the mitogen-activated
protein kinase pathway in vascular endothelial
cells. J Immunol. 2000;165:2142-2148.
28. Chisholm LJ, Dovgan PS, Agrawal DK, et al.
Modulation of monocyte adherence to endothelial
cells by endothelin-1 involvement of Src (p60src)
and JAK1-like kinases. J Vasc Surg. 1996;23:
288-300.
29. Klint P, Kanda S, Kloog Y, et al. Contribution of
Src and Ras pathways in FGF-2 induced endothelial cell differentiation. Oncogene. 1999;18:
3354-3364.
30. Wong BR, Besser D, Kim N, et al. TRANCE, a
TNF family member, activates Akt/PKB through a
signaling complex involving TRAF6 and c-Src.
Mol Cell. 1999;4:1041-1049.
31. Hiles ID, Otsu M, Volinia S, et al. Phosphatidylinositol 3-kinase: structure and expression of the
110 kd catalytic subunit. Cell. 1992;70:419-429.
32. Klippel A, Escobedo JA, Hu Q, et al. A region of
the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo. Mol Cell Biol. 1993;13:5560-5566.
33. Thakker GD, Hajjar DP, Muller WA, et al. The role
of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J Biol Chem. 1999;
274:10002-10007.
34. Qiu Y, Robinson D, Pretlow TG, et al. Etk/Bmx, a
tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3⬘kinase and is involved in interleukin 6-induced
neuroendocrine differentiation of prostate cancer
cells. Proc Natl Acad Sci U S A. 1998;95:36443649.
35. Yao R, Cooper GM. Requirement for phosphati-
dylinositol-3 kinase in the prevention of apoptosis
by nerve growth factor. Science. 1995;267:20032006.
36. Vara JA, Caceres MA, Silva A, et al. Src family
kinases are required for prolactin induction of cell
proliferation. Mol Biol Cell. 2001;12:2171-2183.
37. Abu-Ghazaleh R, Kabir J, Jia H, et al. Src mediates stimulation by vascular endothelial growth
factor of the phosphorylation of focal adhesion
kinase at tyrosine 861, and migration and antiapoptosis in endothelial cells. Biochem J. 2001;
360:255-264.
38. Mochizuki Y, Nakamura T, Kanetake H, et al. Angiopoietin 2 stimulates migration and tube-like
structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn. J Cell
Sci. 2002;115:175-183.
39. Park CC, Morel JC, Amin MA, et al. Evidence of
IL-18 as a novel angiogenic mediator. J Immunol.
2001;167:1644-1653.
40. Koch AE. The role of angiogenesis in rheumatoid
arthritis: recent developments. Ann Rheum Dis.
2000;59:165-171.
41. Hartwell DW, Butterfield CE, Frenette PS, et al.
Angiogenesis in P- and E-selectin-deficient mice.
Microcirculation. 1998;5:173-178.
42. Aoki M, Kanamori M, Yudoh K, et al. Effects of
vascular endothelial growth factor and E-selectin
on angiogenesis in the murine metastatic RCT
sarcoma. Tumour Biol. 2001;22:239-246.