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INTRODUCTION |
Angiogenesis, the formation of new vessels from existing capillary
beds, is a crucial process involved in various physiological and
pathophysiological conditions, including embryonal development, wound
healing, chronic inflammation, and tumor growth. It comprises a
multistep sequence of basement membrane proteolysis, endothelial cell
(EC)1 proliferation and
chemotaxis, and organization and maturation of tubular structures.
Angiogenesis results from an increase in angiogenic mediators (such as
vascular endothelial growth factor (VEGF)) or a reduction in
angiostatic factors, including interferon (IFN)-
, as well as the
release of proteases, which degrade the extracellular matrix (1, 2).
Interleukin-8 (IL-8, CXCL-8), an ELR (Glu-Leu-Arg) motif positive
(ELR+) CXC chemokine (3, 4), has been shown to exert direct
angiogenic effects on EC in vitro and in vivo
(5-7). IL-8 exerted a more pronounced chemotactic effect on dermal
microvascular EC compared with macrovascular EC (2). In corneal
micropocket assays in rats (4, 5, 7), angiogenesis was strongly induced
by local administration of IL-8 and variably accompanied by an
inflammatory infiltrate, raising the possibility of an indirect
angiogenic mechanism by IL-8-mediated release of angiogenic mediators
(7).
IL-8 was initially identified as a major proinflammatory cytokine. In
Crohn's disease and ulcerative colitis, total mucosal IL-8 mRNA
and protein are significantly up-regulated in the colonic mucosa
(8-11), in direct proportion with the degree of inflammation (12).
IL-8 acts as a potent chemoattractant for neutrophils, the major
cellular component of acute inflammatory infiltrates (8). In addition
to this proinflammatory function, there is growing evidence that IL-8
exerts effects on nonimmune cells, including the vascular endothelium.
Studies from human gastrointestinal carcinomas have suggested a pivotal
role for IL-8 in tumor angiogenesis and tumor growth. IL-8 expression
correlates with vascularity in gastric carcinomas (13), and microvessel
counts in histologically normal tissue adjacent to colonic
adenocarcinoma indicated distant angiogenic effects mediated by soluble
factors (14). Notably, IL-8 is highly expressed in hyperplastic mucosa
adjacent to colon cancer, supporting an indirect angiogenic effect of
colon cancer cells (15). Moreover, IL-8 is apparently involved in
development of distant metastasis from colorectal cancer (16).
Interestingly, both colonic epithelial cells (17) and microvascular EC
of human gut secrete IL-8 in a regulated fashion in vitro
(18), suggestive of a cross-talk between those cells in the human
intestinal mucosa (19). Substantial evidence exists that IL-8 is a
critical factor in angiogenesis in a multitude of human tumors.
Strategies blocking the angiogenic activity of IL-8 have proven
effective to inhibit angiogenesis, metastasis, and tumor progression in
human tumors in murine models (20-28).
The biological activity of IL-8 is mediated by binding to two highly
related receptors, CXCR1 and CXCR2, on target cells. The chemokine
receptor CXCR2 promiscuously binds all known angiogenic ELR+ CXC chemokines, including IL-8, the growth-regulated
oncogene family members (GRO-
, -
, and -
), NAP-2, GCP-2, and
ENA-78 with high affinity (29, 30). In contrast, CXCR1 specifically
binds only IL-8 and GCP-2 (30, 31). Both CXCR1 and CXCR2 are members of
the seven-transmembrane domain rhodopsin-like G protein-coupled receptor superfamily and share 78% amino acid sequence homology (32).
The detection of the IL-8 receptors, CXCR1 and CXCR2 in cultured EC, by
means of RNA message and functional protein expression, has
demonstrated conflicting results (33), as the degree of monolayer
confluence and the extracellular matrix likely regulate CXC receptor
expression in EC (34). In addition, several cytokines, including tumor
necrosis factor (TNF)-
are potent inducers of CXCR2 on human
microvascular EC (35). EC subsets from different vascular beds
demonstrate heterogeneous responsiveness to cytokines and
differentially express adhesion molecules and secretory products (36-39). Therefore, we sought to define the expression of IL-8 receptors in human intestinal microvessels and characterize the functional angiogenic response of human intestinal microvascular EC
(HIMEC) to IL-8.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
Monoclonal anti-CXCR1 (Clone 5A12)
and anti-CXCR2 (Clone 6C6) antibodies were purchased from BD Pharmingen
(San Diego, CA). Mouse IgG (R&D Systems, Minneapolis, MN) served as an
isotype control. Recombinant human IL-8 and cytokines were
purchased from R&D Systems. Endothelial cell growth supplement was from
Upstate Biotech Inc. (Lake Placid, NY). MCDB-131 medium, porcine
heparin, bacterial lipopolysaccharide (LPS) (Escherichia
coli O111:B4), concanavalin A, trypsin inhibitor (type II-S), and
PSF (penicillin/streptomycin/fungizone) were from Sigma. Fetal bovine
serum (FBS) and RPMI 1640 medium were obtained from BioWhittaker
(Walkersville, MD). Bovine serum albumin (BSA, Fraction V) was obtained
from Fisher Scientific (Fair Lawn, NJ). Human plasma fibronectin was
purchased from Chemicon International (Temecula, CA). The specific
inhibitors of p42/44 mitogen-activated protein kinase (MAPK) (PD98059)
and phosphoinositide 3-kinase (wortmannin and LY294002) were purchased
from Calbiochem (La Jolla, CA), and MatrigelTM was obtained
from BD Discovery Labware (Bedford, MA).
Cell Culture--
Macroscopically normal intestinal specimens
for HIMEC isolation were obtained from patients undergoing scheduled
bowel resection. The use of human tissues was approved by the
Institutional Review Board of the Medical College of Wisconsin. HIMEC
were isolated as previously described (40). In brief, mucosal strips
from resected normal colon were washed, minced, and digested in
collagenase type II solution (Worthington, Lakewood, NJ; 2 mg/ml). EC
were extruded by mechanical compression and plated onto
fibronectin-coated tissue culture dishes in growth medium (MCDB-131
medium supplemented with 20% (v/v) FBS and endothelial cell growth
supplement, porcine heparin (130 mg/ml), and 2.5% (v/v) PSF solution).
After 7-10 days of culture, microvascular EC clusters were physically
isolated, and a pure culture was obtained. HIMEC cultures were
recognized by microscopic phenotype, modified lipoprotein uptake
(Dil-ac-LDL, Biomedical Technology, Inc., Stoughton, MA), and
expression of Factor VIII-associated antigen. All experiments were
carried out using HIMEC cultures between passages 8 and 12.
Peripheral blood mononuclear cells (PBMC) from healthy donors were
purified by Ficoll-Hypaque (Amersham Biosciences) density centrifugation. Whole blood samples were aseptically drawn into heparinized vials, mixed (1:1) with phosphate-buffered saline (PBS, pH
7.4), and centrifuged over a Ficoll density gradient. Cell viability
was
95% as assessed by trypan blue exclusion. PBMC were washed,
plated, and cultured overnight in RPMI 1640 containing 2 mM
glutamine, 25 mM HEPES, 2 µg/ml LPS, 4 µg/ml
concanavalin A, and 5% (v/v) FBS. Cells were washed and subjected to
RNA isolation.
PCR Primers--
Unlabeled oligonucleotides with the following
sequences were purchased from Operon (Alameda, CA): CXCR1 sense
5'-GGGGCCACACCAACCTTC-3' and antisense 5'-AGTGCCTGCCTCAATGTCTCC-3'
(product size 363 bp; GenBankTM accession number:
BC028221); CXCR2 sense 5'-GGGCAACAATACAGCAAACT-3' and antisense
5'-GCACTTAGGCAGGAGGTCTT-3' (499 bp; accession number: NM_001557);
-actin sense 5'-CCAGAGCAAGAGAGGCATCC-3' and antisense 5'-CTGTGGTGGTGAAGCTGTAG-3' (436 bp; accession number: BC016045).
RNA Isolation and cDNA Synthesis--
Total RNA was isolated
from subconfluent HIMEC cultures by a single step guanidinium
thiocyanate/phenol-chloroform extraction using TRIzol reagent
(Invitrogen, Carlsbad, CA). Genomic DNA was subsequently removed by
DNase I (Amplification Grade, Invitrogen). For reverse transcription,
cDNA was generated using 1 µg of total RNA according to the
manufacturer's protocol (Superscript II reverse transcription kit,
Invitrogen) in a total volume of 20 µl.
PCR Amplification--
PCR was performed using RoboCycler
(Stratagene, La Jolla, CA). For each reaction, 1.25 µl of cDNA
was amplified using specific primers (25 pmol) in a final volume of 50 µl. For CXCR1, PCR was performed for 35 cycles of 94 °C (45 s),
62 °C (30 s), and 72 °C (60 s) followed by a final 72 °C step
(6 min). For CXCR2 and
-actin, the samples were subjected to 35 cycles of 94 (60 s) and 62 °C (150 s), followed by a final 72 °C
step (6 min). PCR products were separated on a 1% agarose gel and
visualized by ethidium bromide staining. DNase-treated total RNA
samples (no reverse transcriptase) were used as a negative
control, whereas cDNA of stimulated PBMC served as a positive
control. Equal loading of cDNA was confirmed by amplification of
-actin.
Flow Cytometry--
HIMEC monolayers were washed and detached
with ice-cold 20 mM EDTA (pH 7.6), PBS and
resuspended in fixation buffer (PBS containing 0.05% (w/v)
NaN3, 0.5% (w/v) BSA, and 1% (w/v) paraformaldehyde). After washing in wash buffer (fixation buffer without
paraformaldehyde), cells were incubated with primary antibodies (10 µg/ml), washed, incubated with fluorescein isothiocyanate-conjugated
goat anti-mouse antibody (BD Biosciences) in the dark and
analyzed (FACScan; BD Biosciences). Mouse isotype IgG served as a
negative control (R&D Systems).
Immunoblot Analysis--
Neutrophils from healthy donors were
purified as described previously (41). Cell viability and purity was
95% as assessed by trypan blue exclusion and DiffQuik staining
(Baxter Scientific, McGraw, IL). Neutrophils and HIMEC from two
different isolations were lysed in lysis buffer (50 mM
Tris-Cl, 2 mM EDTA, 2 mM EGTA, 75 mM NaCl, 25 mM NaF, 25 mM
-glycerophosphate, 1 mM Na3VO4, 1 mM Na2MoO4, 1 mM
phenylmethanesulfonyl fluoride, 1% Nonidet P-40, and 5 µg/ml of each
leupeptin, aprotinin, and pepstatin (all from Sigma) on ice. Lysates
were cleared by centrifugation, and total protein concentration was
determined by Bradford assay (Bio-Rad). 10 µg of neutrophil protein
and 30 µg of HIMEC extracts were separated by SDS-PAGE, blotted onto
nitrocellulose, and blocked for 1 h at room temperature in 3%
(w/v) BSA, 3% (w/v) nonfat dry milk in Tris-buffered saline (50 mM Tris-HCl, pH 7.4) containing 0.1% (v/v) Tween 20 (TBS-T). After washing in TBS-T, blots were incubated with polyclonal
rabbit anti-human anti-CXCR2 antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA) overnight at 4 °C. Immunodetection was performed
using horseradish peroxidase-conjugated goat anti-rabbit antibodies
(Zymed Laboratories Inc., South San Francisco, CA) and
enhanced chemiluminescence (ECL, Amersham Biosciences). For detection
of extracellular signal-regulated kinase (ERK) 1/2, subconfluent HIMEC
monolayers were stimulated with recombinant human IL-8 (100 ng/ml) at
different time points. HIMEC were washed with ice-cold PBS and lysed as
described above. 15 µg of total protein per lane was separated and
blotted, and blocked blots were incubated with antibodies specific for
phosphorylated and total ERK 1/2 (Cell Signaling Tech., Beverly, MA) at
4 °C overnight. Immunodetection was performed as described above.
Immunofluorescence--
F-Actin polymerization was assessed in
subconfluent HIMEC seeded on fibronectin-coated glass chamber slides
(LabTek; Nalge Nunc, Naperville, IL). Cells were cultured in MCDB-131
containing 10% FBS, stimulated with 100 ng/ml recombinant human IL-8
(15 min), and fixed with 4% formaldehyde in PBS for 20 min. Cells were
washed and permeabilized with Triton X-100 (0.2% (v/v) in PBS) for 10 min, blocked with 2.5% (w/v) BSA/PBS, and stained with fluorescein
phalloidin (Molecular Probes, Eugene, OR). After washing, slides were
air dried and mounted with Fluromount-G (Southern Biotechnology,
Birmingham, AL) and examined with a fluorescence microscope (Olympus
BX-40) using a fixed shutter speed to allow for comparison of
fluorescence intensity. In some experiments, stress fiber assembly was
blocked by preincubation of cells with 10 µg/ml neutralizing CXCR2
antibody (30 min at 37 °C).
Endothelial Cell Chemotaxis Assay--
Polycarbonate filters (8 µm pore size, BD Biosciences) were coated with human fibronectin (10 µg/ml) at 4 °C overnight. HIMEC were trypsinized, washed with
chemotaxis buffer (MCDB-131 + 1% (w/v) BSA) containing soybean trypsin
inhibitor (10 mg/ml), and resuspended in chemotaxis buffer. 5 × 105 cells were added to the upper chamber, and chemotaxis
buffer (1000 µl) containing VEGF (50 ng/ml) or recombinant human IL-8 were filled into the lower compartment of the 12-well plates. After
3 h of incubation at 37 °C (5% CO2), cell culture
inserts were removed and the upper side of the membrane was gently
wiped. Filters were stained with DiffQuik (Baxter Scientific, McGraw, IL), air dried, and mounted onto glass slides. Migrated HIMEC adherent
to the lower side of the membrane were counted (10 random high-power
fields (×200) per condition). In inhibition studies, resuspended cells
were incubated with neutralizing anti-CXCR2 antibodies (4 µg/ml) for
30 min at 37 °C, isotype control antibodies, or inhibitors of MAPK
kinase and PI3K as indicated in the figure legends. Control cells
remained free of inhibitors. Cell viability was >95% as assessed by
trypan blue exclusion. Each condition was assessed in triplicate.
Proliferation Assay--
Cellular DNA synthesis was
assessed by [3H]thymidine uptake. HIMEC were kept in
medium without endothelial cell growth supplement (5% (v/v) FBS) for
48 h, trypsinized, and seeded onto fibronectin-coated 24-well
plates (4 × 104 cells per well). After adherence (3 h, 37 °C), cells were stimulated for 18 h with IL-8 (1-100
ng/ml) in medium. Following 12 h of stimulation, cells were pulsed
with [3H]thymidine (1 µCi/ml; Amersham Biosciences).
After washing twice, cells were fixed for 10 min on ice with 5% (v/v)
trichloroacetic acid. DNA was then released from precipitated material
by alkaline lysis in 0.5 N NaOH, and supernatants were
quantified in a
-counter.
In Vitro Tube Formation Assay--
Endothelial tube formation
was assessed using MatrigelTM, a solubilized extracellular
basement membrane matrix extracted from the Engelbreth-Holm-Swarm mouse
sarcoma, as described previously (42). Multiwell dishes (24-well) were
coated with 250 µl of complete medium containing 5 mg/ml Matrigel and
HIMEC resuspended in complete growth medium were seeded at a density of
5 × 104. The growth medium was supplemented with the
MAPK kinase inhibitors PD98059 (10 and 20 µM) LY294002
(10 and 20 µM), or wortmannin (50 or 100 nM).
Control cells remained free of inhibitors. Cells were cultured on
MatrigelTM for 16 h and endothelial tube formation was
assessed by inverted phase-contrast microscopy. Five high-power fields
per condition were examined and experiments were repeated in two
independent HIMEC cultures. Control cells receiving Me2SO
only served as a vehicle control (not shown).
Immunohistochemistry--
Full thickness normal colonic and
ileal mucosa was fixed in 4% (w/v) paraformaldehyde/PBS overnight,
saturated in 20% (w/v) sucrose/PBS, embedded in OCT compound (Sakura,
Japan), and snap frozen in liquid nitrogen. 6-µm frozen sections were
stained with monoclonal mouse anti-human antibodies (anti-CXCR2 (Clone
6C6)) at 4 °C overnight using the Cell and Tissue Staining kit (R&D Systems). After immunodetection of horseradish peroxidase conjugates with diaminobenzidine, sections were briefly counterstained with Mayer's hematoxylin and mounted. Positive staining is visible as a
dark-brown precipitate. Isotype IgG was used as a negative control.
Statistical Analysis--
Statistical analysis was performed by
analysis of variance using StatView for Macintosh (version 4.51; Abacus
Concepts, Inc., Berkeley, CA). p < 0.05 was considered
significant, and data shown are mean ± S.E.
 |
RESULTS |
HIMEC Constitutively Express CXCR2, but Not CXCR1 mRNA--
To
assess CXCR1 and CXCR2 expression in cultured HIMEC, we performed
reverse transcriptase-PCR using receptor-specific primers (Fig.
1). The mRNA for CXCR2 was detected
in unstimulated HIMEC. Moreover, CXCR2 mRNA was markedly increased
upon challenge with TNF-
/LPS (100 IU/ml and 1 µg/ml,
respectively). TNF-
is known to up-regulate IL-8 receptors in human
microvascular EC (35), providing a possible mechanistic explanation for
its in vivo proangiogenic effects (43). Incubation of HIMEC
with IFN-
, an angiostatic cytokine in vitro (44, 45), led
to a slight decrease in detectable mRNA for CXCR2, a receptor shown
to mediate angiogenesis in vivo (32, 46). The message for
CXCR1 could not be amplified in HIMEC cultures from 6 patients using up
to 45 PCR cycles or re-amplification of PCR products (data not shown),
whereas stimulated PBMC (positive control) from healthy donors yielded
a robust signal. 12-Hour stimulation with proinflammatory stimuli
(TNF-
, 100 IU/ml; LPS, 1 µg/ml; IFN-
, 100 IU/ml; combination of
TNF-
/LPS and IL-1
, 100 IU/ml) failed to induce detectable CXCR1
mRNA.

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Fig. 1.
HIMEC express mRNA for CXCR2, but not
CXCR1. Detection of CXCR1 and CXCR2 mRNA in HIMEC by
semiquantitative reverse transcriptase-PCR using specific primers.
HIMEC constitutively express mRNA for CXCR2, but not CXCR1.
Stimulation (12 h) of HIMEC with a combination of TNF- (100 IU/ml)
and LPS (1 µg/ml) led to marked up-regulation of CXCR2, whereas
IFN- (100 IU/ml), IL-1 (100 IU/ml), and TNF- alone did not.
These stimuli failed to up-regulate mRNA for CXCR1. PBMC, positive
control; -actin, loading control; no reverse transcriptase, negative
control. Representative figure for HIMEC isolates from six different
patients.
|
|
HIMEC Express CXCR2 Protein--
We confirmed CXCR2 expression in
HIMEC by flow cytometry. Unstimulated HIMEC expressed low levels of
CXCR2 when compared with isotype control antibody (Fig.
2A). Staining with anti-CXCR1
antibody did not produce any signal above background (data not shown). Furthermore, we confirmed CXCR2 expression by Western blot analysis of
total HIMEC lysates with a polyclonal anti-CXCR2 antibody, demonstrating a specific band in lysates of two different HIMEC populations. Neutrophil lysates from healthy donors served as a
positive control (Fig. 2B).

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Fig. 2.
HIMEC express CXCR2 receptors. CXCR2
expression on cultured HIMEC was detected by flow cytometry
(A) and Western blot analysis (B). CXCR2 is
localized to the HIMEC cell surface (A, solid
black), as compared with an isotype control antibody
(A, empty) using flow cytometry. Staining with
anti-CXCR1 antibodies did not produce any signal above background (data
not shown). Total lysates of two different HIMEC isolates (HIMEC 1, HIMEC 2; 30 µg of total protein each) were used to detect CXCR2 in
Western blot analysis (B). NP, neutrophil lysate
(positive control; 15 µg of total protein).
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Human Mucosal Microvessels Express CXCR2 in Vivo--
To further
assess expression of CXCR2, the putative receptor for ELR+
chemokine-mediated CXCR2 angiogenesis in vivo, we
performed immunostaining. CXCR2 was detected by immunohistochemistry of
both colonic (Fig. 3A) and
small intestinal (Fig. 3B) normal full thickness specimens. Microvessels in close proximity to colorectal adenocarcinoma were similarly stained as was normal colon (not shown). Isotype controls did
not produce detectable specific signal (Fig. 3, C and
D).

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Fig. 3.
CXCR2 is expressed in human intestinal
microvessels in vivo. CXCR2 detection by
immunostaining of frozen sections of colonic (A) and small
intestinal (B) microvessels from normal full-thickness
specimens. A positive signal is visible in mucosal and submucosal
microvessels as a dark brown precipitate (diaminobenzidine;
arrows; bright field microscopy; original
magnification, ×400). Isotype controls did not produce any specific
signal (C and D). Representative figures shown
are from three independent experiments.
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IL-8 Induces Stress Fibers in HIMEC--
Chemokines typically
elicit activation and migration of target cells. Having shown that
HIMEC express an IL-8 receptor, we next assessed induction of stress
fibers as a biological function for chemokine receptor signaling by
incubation with fluorescein phalloidin. In unstimulated HIMEC, F-actin
staining was limited to the cell periphery and intercellular junctions
(arrow, Fig. 4A).
IL-8 (100 ng/ml) lead to a rapid (starting at ~1 min) and sustained
(30 min) F-actin reorganization in HIMEC, showing prominent stress
fiber bundles (arrows, Fig. 4B) and a marked
increase in fluorescence intensity. In addition, cellular retraction
could be observed at the periphery (arrowheads) of some
cells, leading to gaps in the monolayer (Fig. 4B). Stress
fiber induction in response to IL-8 utilizes CXCR2, as preincubation of
HIMEC with neutralizing anti-CXCR2 antibodies markedly attenuated
IL-8-induced F-actin polymerization (Fig. 4C).

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Fig. 4.
Stress fiber assembly in HIMEC by IL-8 is
attenuated by anti-CXCR2. Effect of IL-8 on stress fiber
polymerization in HIMEC as assessed by fluorescence staining with
fluorescein phalloidin, a substance specifically detecting F-actin.
Confluent monolayers of HIMEC were grown on fibronectin-coated glass
chamber slides, stimulated with IL-8 (100 ng/ml, 15 min) prior to
staining. A, minimal stress fibers in unstimulated HIMEC
(negative control). B, IL-8, a CXCR2 ligand, strongly
increases stress fiber assembly (arrows denote stress fiber
bundles). At some intercellular junctions, a marked retraction
(arrowheads) of endothelial cells is notable, leading to
scattered interruptions of the continuity of the endothelial cell
monolayer. Preincubation of HIMEC with neutralizing anti-CXCR2
antibodies (10 µg/ml, 15 min, 37 °C) markedly attenuated stress
fiber assembly by IL-8 (note less fluorescence intensity and less
pronounced stress fiber induction, C). Fluorescence
microscopic images; original magnification, ×400; fixed shutter speed.
Figure shows representative images from one of three independent
experiments.
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IL-8 Is a Potent Chemoattractant for HIMEC Migration--
Having
demonstrated IL-8 activation of stress fibers in HIMEC, we assessed its
ability to stimulate cell migration/chemotaxis using fibronectin-coated
polycarbonate filters (Transwell). IL-8 concentrations as low as 1 ng/ml induced maximal chemotactic responses of HIMEC, comparable with
those induced by the potent angiogenic factor VEGF (50 ng/ml). In
accordance with observations on human dermal microvascular EC (2),
there is an optimal concentration for IL-8-induced chemotaxis of human
microvascular EC, as the number of cells migrating across the filter
decreases with higher concentrations of IL-8 (Fig.
5A). Consistent with our
findings in F-actin polymerization, preincubation of HIMEC with
neutralizing antibodies to CXCR2 (4 µg/ml) significantly decreased
chemotactic activity (Fig. 5B), as compared with incubation
with isotype control antibodies (4 µg/ml).

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Fig. 5.
IL-8 is a potent chemoattractant for HIMEC
migration in vitro. Effect of IL-8 on HIMEC migration
assessed by a Transwell chemotaxis assay. HIMEC migrating across
fibronectin-coated polycarbonate filters (pore size, 8 µm) were
quantified using modified Wright's stain and bright field microscopy.
IL-8 elicits strong chemotactic properties in HIMEC, comparable with
VEGF (50 ng/ml; positive control). Note the biphasic dose response, as
higher concentrations of IL-8 (100 ng/ml and higher) do not exhibit
marked chemotaxis in HIMEC (A). The inhibitory effects of
anti-CXCR2 antibodies on HIMEC were studied at 10 ng/ml IL-8, a
concentration shown to effectively evoke a chemotactic response in
HIMEC. HIMEC were preincubated with neutralizing anti-CXCR2 antibodies
(4 µg/ml, 30 min, 37 °C) or isotype control antibodies (4 µg/ml). Preincubation with anti-CXCR2 significantly inhibited
IL-8-induced chemotactic response in HIMEC (B). *,
p < 0.01 versus positive control. No
IL-8, negative control; IL-8 (10 ng/ml), positive
control. All conditions were assessed in triplicate, and data are
expressed as mean number of migrated cells per high-power field
(×200) ± S.E.
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IL-8 Enhances Proliferation Rate of HIMEC--
Angiogenesis
requires the directed migration and increased cellular proliferation of
EC. Proliferation rates of HIMEC, assessed by
[3H]thymidine uptake, were significantly higher following
12 h of IL-8 stimulation (Fig. 6).
Comparable with our chemotaxis data, IL-8-induced proliferation was
maximal at concentrations of 10 ng/ml. Thus, IL-8 was able to produce a
marked increase in proliferation rates, suggesting a pivotal role for
IL-8-induced EC proliferation in vivo.

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Fig. 6.
IL-8 enhances the proliferation rate of HIMEC
in vitro. IL-8-induced HIMEC proliferation was assessed by
[3H]thymidine uptake. 4 × 104
HIMEC/well were stimulated for 12 h with increasing concentrations
of IL-8 (0, 1, 10, and 100 ng/ml) and pulsed for 6 h with 1 µCi/ml [3H]thymidine. [3H]Thymidine
uptake in total cell lysates was measured in a -counter. IL-8
enhanced HIMEC proliferation in a biphasic fashion, with an apparent
maximum at 10 ng/ml IL-8. A concentration of 100 ng/ml IL-8 did not
result in a significant increase in proliferation. All conditions were
assessed in triplicate, and data are expressed as percent of control
[3H]thymidine uptake (0 ng/ml IL-8) ± S.E. *,
p < 0.05 versus control.
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IL-8 Enhances Phosphorylation of ERK 1/2
(p42/44 MAPK)--
Chemokine activation of MAPK
phosphorylation in target cells has been demonstrated previously (47).
We next assessed the ability of IL-8 to induce MAPK phosphorylation by
immunoblot analysis. IL-8 (100 ng/ml) leads to a marked phosphorylation
of extracellular signal-regulated kinase (ERK 1/2, p42/44 MAPK),
reaching its maximum after 30 min of stimulation (Fig.
7). This phosphorylation is not
transient, as ERK 1/2 remains strongly phosphorylated after 60 min of
stimulation (Fig. 4). Equal loading of protein was assured using the
Bradford assay and Coomassie staining of the polyacrylamide gel after
separation of proteins.

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Fig. 7.
IL-8 enhances phosphorylation of ERK 1/2
(p42/44 MAPK) in HIMEC. Activation of ERK 1/2, a member of the
MAPK superfamily, in HIMEC by IL-8 was assessed using Western blot
analysis. Total cell lysates from cultured HIMEC stimulated with IL-8
(100 ng/ml) were subjected to SDS-PAGE and immunoblotted with
phospho-specific anti-ERK 1/2 antibodies. IL-8 enhances phosphorylation
of ERK 1/2 in a time-dependent fashion. Note the strong and
sustained increase of phosphorylated ERK 1/2 at 15 min of stimulation
with IL-8, as compared with total ERK. Coomassie staining of the
polyacrylamide gel confirms equal amounts of total protein loaded. The
representative figure shown is from one of three independent
experiments.
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|
In Vitro Tube Formation of HIMEC Requires Activation of ERK
1/2 and PI3K--
The role of ERK 1/2 and PI3K in in
vitro angiogenesis of HIMEC was assessed by tube formation assays.
HIMEC in complete growth medium were seeded onto a three-dimensional
extracellular matrix preparation (MatrigelTM) and incubated
for 16 h at 37 °C. Where indicated, HIMEC were incubated with
inhibitors of MAPK kinase (PD98059) and PI 3-kinase (wortmannin and
LY294002), respectively. Naive HIMEC seeded onto Matrigel in complete
growth medium display formation of robust tube-like structures within
8 h (not shown), with further maturation after 16 h (Fig.
8, panel A). Inhibitors of
both MAPK kinase and PI3K at concentrations specific for their
signaling targets exhibited a marked inhibitory effect on the formation
of tube-like structures by HIMEC, visible by the disruption of
tube-like structures and cells remaining coherent in spherical clusters
(Fig. 8, panels B-H). These results indicate
that activation of both ERK 1/2 and PI3K are required for endogenous
in vitro tube formation in HIMEC, defining the crucial role
of both signaling pathways in functional angiogenesis of HIMEC.

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Fig. 8.
In vitro tube formation of HIMEC
requires activation of ERK 1/2 and PI3K. In vitro
endothelial tube formation assays employing MatrigelTM as a
three-dimensional extracellular matrix. HIMEC in complete growth medium
(5 × 104) were seeded onto 24-well plates containing
MatrigelTM (5 mg/ml). Where indicated, the medium was
supplemented with specific inhibitors of MAPK kinase (PD98059, 10 and
20 µM) or PI3K (wortmannin, 50 and 100 nM; or
LY294002, 10 and 20 µM), respectively. Control cells
receiving Me2SO only served as a vehicle control (not
shown). Naive HIMEC seeded in complete growth medium display robust
tube formation (A). All three inhibitors markedly abrogated
spontaneous tube formation of HIMEC seeded in complete growth medium.
Note the cells remaining adherent in spherical clusters, lacking
mature tube-like structures (B-H).
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|
Inhibitors of MAPK Kinase and PI3K Elicit Inhibitory Effects on
IL-8-induced Chemotaxis in HIMEC--
To specifically assess the role
of ERK 1/2 and PI3K in the propagation of the intracellular signal
evoked by IL-8, HIMEC were incubated with inhibitors of MAPK kinase
(PD98059; 10 and 20 µM) and of PI3K (wortmannin; 50 and
100 nM) and subjected to a chemotaxis assay using 10 ng/ml
IL-8, a concentration shown to evoke a robust chemotactic response in
HIMEC. Both inhibitors used at target-specific concentrations resulted
in a dose-dependent decrease in the chemotactic activation
of HIMEC by IL-8, as compared with the vehicle control (Me2SO) (Fig. 9). Supported
by the notion that HIMEC require activation of both ERK 1/2 and PI3K
for in vitro angiogenesis, these data indicate that IL-8, a
major angiogenic CXC chemokine, utilizes the ERK 1/2 and PI3K signaling
pathways to exhibit its biological effect in HIMEC.

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Fig. 9.
IL-8-induced chemotaxis in HIMEC is
diminished by specific inhibitors of ERK 1/2 and PI3K. Chemotaxis
assay using 10 ng/ml IL-8 and specific inhibitors of ERK 1/2 and PI3K.
As shown above, IL-8 acts as a potent chemoattractant on HIMEC. This
chemotactic response was potently diminished by PD98059 (10 and 20 µM) and wortmannin (50 and 100 nM) in a
dose-dependent fashion. *, p < 0.05 versus positive control; n.s., not significant.
No IL-8, negative control; IL-8 (10 ng/ml),
positive control. All conditions were assessed in triplicate, and data
are expressed as mean number of migrated cells per high-power field
(×200) ± S.E.
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DISCUSSION |
Recently, angiogenesis has evoked intense clinical interest. Novel
anti-angiogenic drugs that potentially could help in the prevention
("angioprevention") (48) or treatment of advanced tumor stages by
inducing tumor regression or inhibiting tumor progression have
undergone clinical investigation. In colorectal adenocarcinoma,
angiogenesis is linked to prognostic parameters, including recurrence
rate, lymph node metastasis, and disease-free survival (49-51).
However, most multicenter trials (phase II and III) investigating
angiogenesis inhibitors, many of them focusing on VEGF and its
receptors (52), have failed to improve the clinical course. SU5416
(semaxanib), a VEGF receptor inhibitor, recently failed in phase III
studies in the treatment of advanced colorectal cancer. Other phase III
failures were observed for matrix metalloproteinase inhibitors (BB2516,
AG3340, and Bay-12-9566) in the treatment of various solid tumors.
Although VEGF and its receptors may be important in the development of
new blood vessels, these trials suggest that malignancy-associated neovascularization in humans is dependent on multiple angiogenic and
angiostatic factors. Additional mechanisms to be considered in the
inhibition of tumor neovascularization will involve angiostatin (53),
endostatin (54), and cyclooxygenase (55). Accordingly, as a plausible
mechanism for the failure of VEGF-based anti-angiogenesis trials, a
local decrease in angiostatic factors, as well as an increase in
established angiogenic factors, including ELR+ CXC
chemokines such as IL-8, may contribute to the development of
malignancy-associated neovascularization. Our study demonstrates that
human intestinal microvascular EC do not express the high specificity/affinity IL-8 receptor (CXCR1), and IL-8-induced
angiogenesis in the human intestine is likely dependent on CXCR2, a
receptor targeted by multiple angiogenic ELR+ CXC
chemokines. This is in accordance with the observations of Addison
et al. (32) that CXCR2, not CXCR1 is the receptor
most likely responsible for ELR+ CXC chemokine-induced
angiogenesis in microvascular EC.
VEGF, a major angiogenic factor, was recently shown to utilize several
intracellular signaling cascades, including ERK 1/2, p70 S6 kinase, and
PI3K (phosphoinositide 3-OH kinase) through its receptor Flk-1/KDR in
human EC (56, 57). Recent investigation has focused on the role of PI3K
in the migration of various cell types (58), including EC (59, 60).
Specifically, PI3K is crucial for mitogen-induced cell motility
responses of EC, including actin reorganization and chemotaxis (61).
Wortmannin, a multicyclic fungal metabolite and a selective inhibitor
of PI3K, is a potent inhibitor of angiogenesis in vivo (62)
and EC chemotaxis (61). In turn, the intracellular lipid kinase PI3K is
now appreciated as a critical mediator of angiogenesis through its
downstream targets PKB (63) and Akt, a serine/threonine protein kinase (64), as overexpression of the catalytic subunit of PI3K as well as of
constitutively active Akt has revealed increased in vivo
angiogenesis in the chicken chorioallantoic membrane assay (65).
Activation of ERK 1/2 by chemokines seems to be a signaling event
downstream of PI3K, because phosphorylation of ERK 1/2 in response to
several chemokines can be inhibited by PI3K inhibitors (58, 66).
In addition, PI3K is believed to exert oncogenic properties involved in
cellular transformation, aberrant cell cycle progression, and
tumorigenesis (68).
Specific inhibitors of both ERK 1/2 and PI3K activation displayed a
marked inhibitory effect on biological endothelial functions in HIMEC.
In an in vitro angiogenesis assay employing Matrigel as an
extracellular matrix, the PI3K inhibitors wortmannin and LY294002 as
well as the MAPK kinase inhibitor, PD98059, markedly abrogated the
spontaneous tube formation of HIMEC. Similarly, inhibitors of both
mentioned pathways suppressed the chemotactic activity of IL-8 (10 ng/ml) in a dose-dependent fashion. Given the notion
that major proangiogenic factors share intracellular signaling pathways
required for angiogenesis, the development of tailored
endothelium-specific drugs inhibiting MAPK kinase and PI3K may emerge
as novel pharmacologic strategies to prevent tumor-associated angiogenesis.
The importance of IL-8 in tumor progression, metastasis, and
angiogenesis has been demonstrated by several groups using neutralizing anti-IL-8 antibodies in the treatment of human adenocarcinomas transplanted into athymic nude mice (22, 23, 26). However, it is
important to consider variation in the chemokine biology of rodents
compared with humans, where mice express IL-8 receptors, but a murine
analog of IL-8 has not yet been identified. Models of tumorigenesis in
mice cannot as of yet directly assess the contribution of IL-8 through
gene targeted deletion. Therefore, utilization of human cell systems
will remain an essential component of defining receptor-mediated growth
pathways involving IL-8.
The biological effects of IL-8 on EC are concentration dependent in a
biphasic fashion, as higher concentrations of IL-8 reduce its
chemotactic properties. This observation was demonstrated in human
dermal microvascular cells and human macrovascular EC, although human
macrovascular EC displayed lower overall levels of chemotactic response
toward different concentrations of IL-8 (2). Similarly, EC
proliferation rates induced by IL-8 revealed a biphasic pattern, a
phenomenon previously described for human macrovascular EC (69). The
biphasic functional response to IL-8 was not unexpected, as it has been
shown for other chemokines, including chemotactic response to the
ELR
CXC chemokine stromal cell-derived factor-1 (70).
Another possible IL-8 receptor, the Duffy antigen, was originally
identified as a blood group antigen required for invasion by the
malarial parasites, Plasmodium vivax and Plasmodium
knowlesi (71-73). Further studies revealed that a
chemokine-binding protein expressed on red blood cells is identical to
the Duffy antigen, which was subsequently renamed Duffy antigen
receptor for chemokines (DARC) (74). DARC nonselectively binds the
ELR+ CXC chemokines IL-8, GRO-
(MGSA), and NAP-2 as well
as the nonangiogenic CC chemokines RANTES (regulated on activation
normal T cell expressed and secreted) and MCP-1 with comparably high
affinity (75). DARC does not couple to G-proteins, and binding of
chemokines to DARC does not evoke any intracellular signal (76).
Although DARC is expressed on microvascular EC of Duffy-negative
individuals (77), its molecular role in neovascularization is unknown
(32, 46, 78). Consistent with this, it has been suggested that DARC
might act as a chemokine sink to bind excess chemokines during inflammatory responses (32), thus sustaining chemokine action in a
buffering fashion. Alternatively, DARC might serve as a competing decoy
receptor for chemokines, limiting excessive response to high amounts of
chemokines in inflammation (46).
CXCR2 apparently plays a major role in IL-8-induced signaling, as
different biological effects of IL-8 on intestinal EC can be markedly
attenuated by preincubation with neutralizing anti-CXCR2 antibodies.
These data suggest that in addition to VEGF signaling, which frequently
has been evaluated as a single target in recent anti-angiogenesis
trials, there are additional pathways of tumor-induced angiogenesis in
human cancers. Targeting multiple receptors with redundant function may
be essential for the inhibition of angiogenesis clinically. Blocking of
CXCR2 signaling by antibodies, or delivery of selective CXCR2
antagonistic compounds (79) or peptides (67) may emerge as an
additional therapeutic approach in anti-angiogenesis treatment of
colonic adenocarcinoma.