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Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
Center for Vascular Biology and
Departments of Medicine and Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232;
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Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095;
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Theodor Kocher Institute, Universitat Bern, Bern, Switzerland; and
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Department of Veteran’s Affairs, Vanderbilt University Medical Center, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß,
and 
; granulocyte chemotactic protein 2; and epithelial
neutrophil-activating protein-78, can mediate angiogenesis in the
absence of preceding inflammation. To date, the receptor on endothelial
cells responsible for chemotaxis and neovascularization mediated by
these ELR+ CXC chemokines has not been determined. Because
all ELR+ CXC chemokines bind to CXC chemokine receptor 2
(CXCR2), we hypothesized that CXCR2 is the putative receptor for
ELR+ CXC chemokine-mediated angiogenesis. To test this
postulate, we first determined whether cultured human microvascular
endothelial cells expressed CXCR2. CXCR2 was detected in human
microvascular endothelial cells at the protein level by both Western
blot analysis and immunohistochemistry using polyclonal Abs specific
for human CXCR2. To determine whether CXCR2 played a functional role in
angiogenesis, we determined whether this receptor was involved in
endothelial cell chemotaxis. We found that microvascular endothelial
cell chemotaxis in response to ELR+ CXC chemokines was
inhibited by anti-CXCR2 Abs. In addition, endothelial cell
chemotaxis in response to ELR+ CXC chemokines was sensitive
to pertussis toxin, suggesting a role for G protein-linked receptor
mechanisms in this biological response. The importance of CXCR2 in
mediating ELR+ CXC chemokine-induced angiogenesis in vivo
was also demonstrated by the lack of angiogenic activity induced by
ELR+ CXC chemokines in the presence of neutralizing Abs to
CXCR2 in the rat corneal micropocket assay, or in the corneas of
CXCR2-/- mice. We thus conclude that CXCR2 is the
receptor responsible for ELR+ CXC chemokine-mediated
angiogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, and
; granulocyte chemotactic protein-2 (GCP-2); and epithelial
neutrophil-activating protein 78 (ENA-78), are potent inducers of
angiogenesis in vivo (3). In contrast, CXC chemokines that
lack the ELR motif (ELR-), such as platelet
factor 4, IFN-inducible protein-10, and monokine induced by IFN-,
are potent angiostatic factors, and can inhibit neovascularization
mediated by ELR+ CXC chemokines (3, 4). Furthermore, the ELR- CXC chemokines
have also been shown to inhibit neovascularization induced by the
classical angiogenic factors, basic fibroblast growth factor (bFGF) and
vascular endothelial cell growth factor (VEGF) (3). The
receptor responsible for the induction of angiogenesis by
ELR+ CXC chemokines has not yet been
elucidated.
The CXC chemokines have been shown to interact with the CXC chemokine
receptor (CXCR) family of molecules. These receptors are members of the
rhodopsin-like seven-transmembrane G protein-coupled receptor family
(5, 6, 7, 8). The chemokine receptors range in size from 339 to
373 aa, and possess 25–80% homology to one another on the amino acid
level (6, 7, 9, 10). To date, five CXC receptors have been
identified in various human cell lines. CXCR1, which is also known as
IL-8RA, binds IL-8 with high affinity (8), and more
recently has been shown to also bind GCP-2 with a somewhat reduced
affinity (11). CXCR2, also known as IL-8RB, possesses 78%
identity with CXCR1 on the amino acid level with the majority of
divergence between the two proteins being located in the amino
terminus, the carboxyl terminus, and within the second extracellular
loop (6). This divergence in the extracellular regions
(the amino terminus and the second extracellular loop) may partly
explain why CXCR2 shows less selectivity in chemokine binding than does
CXCR1. CXCR2 has been shown to bind all of the
ELR+ CXC chemokines, including IL-8; ENA-78;
GRO-, -ß, and -; neutrophil-activating protein-2 (NAP-2); and
GCP-2 with high affinity (6, 8, 9, 12). CXCR3 has been
shown to bind the ELR- CXC chemokines
IFN-inducible protein-10 and monokine induced by IFN-
(5). CXCR4 has recently been the focus of a number of
studies, as it has been shown to be a cofactor for HIV infection of T
lymphocytes by T cell tropic viruses (13). To date,
stromal cell-derived factor-1 is the only known ligand for CXCR4, and
this ELR- CXC chemokine can inhibit HIV
infection by competing with lymphotropic HIV virus for binding of CXCR4
(14). CXCR5 is found mostly on B cells and is responsible
for B cell chemotaxis mediated by B cell-attracting chemokine 1
(15). There is a sixth receptor that has been shown to
bind CXC chemokines, DARC or the Duffy Ag receptor for chemokines
(16, 17). IL-8, GRO-, and NAP-2 have all been shown to
bind to DARC; however, ligand binding of this receptor is not
restricted to the CXC chemokines, and various CC chemokines, such as
RANTES and monocyte chemotactic protein-1, also bind to DARC with high
affinity (18). Moreover, DARC does not appear to
demonstrate ligand-receptor signal coupling (19). Another
receptor whose ligand binding is restricted to the CXC chemokines is
encoded by an open reading frame from Herpesvirus saimiri
(20). The ECRF3 open reading frame has been shown to
encode a seven-transmembrane receptor, and this receptor has been shown
to bind CXC chemokines, in particular the ELR+
CXC chemokines IL-8, GRO-, and NAP-2 (20). This
receptor is most homologous to CXCR2; however, this homology is only
30% at the amino acid level, even though the CXC chemokine-binding
repertoire is essentially identical (20).
In this study, we investigated the identity of the chemokine receptor
responsible for mediating angiogenesis induced by the
ELR+ CXC chemokines. As mentioned, the
ELR+ CXC chemokine IL-8 can bind both CXCR1 and
CXCR2 with high affinity, while the ELR+ CXC
chemokines GRO-, -ß, -, and ENA-78 have been shown to bind only
CXCR2 with high affinity (8, 21). Because all of these
ELR+ CXC chemokines only bind CXCR2 in common,
and all induce angiogenesis in vivo, we hypothesized that CXCR2 is the
putative receptor present on endothelial cells that mediates
angiogenesis induced by ELR+ CXC chemokines in
vivo. In this study, we demonstrate that CXCR2 is expressed by cultured
human microvascular endothelial cells (HMVEC), and that not only can
neutralizing Abs to CXCR2 inhibit endothelial cell chemotaxis in vitro,
but also inhibit neovascularization induced by the
ELR+ CXC chemokines in the rat CMP assay in vivo.
Furthermore, corneal neovascularization induced by
ELR+ CXC chemokines in
CXCR2-/- mice is impaired as compared with that
induced in wild-type mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human neutrophils were isolated from heparinized venous blood collected from healthy volunteers, by mixing 1:1 with 0.9% saline and separation from mononuclear cells by Ficoll-Hypaque density-gradient centrifugation. Human neutrophils were then isolated by sedimentation in 5% dextran in 0.9% saline (Sigma, St. Louis, MO) and separated from erythrocytes by hypotonic lysis. After washing twice, neutrophils were suspended in HBSS with calcium/magnesium (Life Technologies, Grand Island, NY) at a concentration of 2 x 106 cells/ml. Neutrophils were >99% viable, as determined by trypan blue exclusion. Human dermal microvascular endothelial cells (HMVEC-D) and human lung microvascular endothelial cells (HMVEC-L) were both obtained from Cell Systems (Kirkland, WA), and were propagated in CS-C complete media on attachment factor-coated tissue culture flasks according to the manufacturer’s directions. All endothelial cell lines were stained for factor VIII-related Ag to confirm their identity as being of endothelial origin. Both the HMVEC-L- and HMVEC-D-cultured cells were positive for the expression of factor VIII-related Ag, while human neutrophils were negative following immunohistochemical staining.
To generate 293 cells that expressed human CXCR2, the cDNA for human
CXCR2 was amplified by RT-PCR from total RNA isolated from human
neutrophils using the Access PCR kit (Promega, Madison, WI). The
primers used for amplification were: forward, 5'-GTC AGG ATC CAA GTT
TAC CTC AAA AAT GG-3', and reverse, 5'-CTT AGG TCG ACG GTC TTA GAG AGT
AGT GG-3'. The reverse-transcription reaction was performed at 42°C
for 45 min, followed by denaturation at 94°C for 2 min. PCR was then
performed for 40 cycles as follows: 94°C denaturation for 1 min,
55°C annealing for 1 min, and 68°C elongation for 2 min. The
resulting PCR was subjected to electrophoresis on an agarose gel, and a
band of 
1.1 kb was removed and purified using the Wizard PCR DNA
purification kit (Promega). The PCR product was ligated into the
T-overhang plasmid pTARGET according to the manufacturer’s directions
kit (Promega). This resulted in human CXCR2 expression under control of
the human CMV promoter and allowed for generation of stably transfected
mammalian cells by the presence of a neomycin resistance gene. The
293 cells were transfected with 5µg of CXCR2 plasmid DNA or
control pTARGET DNA by calcium phosphate transfection, as previously
described (22). G418-resistant colonies were expanded, and
expression of CXCR2 was confirmed by FACS analysis using a mAb against
human CXCR2 (R&D Systems, Minneapolis MN).
Abs and Ab generation
A rabbit polyclonal Ab to the carboxyl terminus of human CXCR2
was purchased from Santa Cruz Biotechnologies (Santa Cruz CA).
Anti-rabbit IgG Abs conjugated to HRP were purchased from Bio-Rad
(Hercules CA). For Ab generation, peptides specific to the
amino-terminal region of human or murine CXCR2 were generated by
solid-phase peptide synthesis performed on a Millipore 9050 continuous
flow peptide synthesizer (Millipore, Milford, MA) using Fmoc chemistry.
Briefly, 1 g of polyethylene glycol polystyrene-graft copolymer
peptide synthesis support (PEG-PS resin) and 0.8 mM of each Fmoc-amino
acid active ester were used. Cleavage and deprotection were conducted
in 88% trifluoroacetic acid (TFA), 5% liquefied phenol, 2%
triisopropylsilane, and 5% water for 2–12 h at room temperature. The
free peptide was then precipitated and washed repeatedly with ice-cold
ether and dried under vacuum. The peptide was resuspended in water,
acidified with TFA, and purified by preparative reversed-phase HPLC in
a C4 column (25 x 100 mm, 15 mm, 300A, 
-Pak; Waters,
Millipore, Bedford, MA). The column was eluted at 5 ml/min with a
gradient of 0–60% acetonitrile in 0.1% TFA at an increment of 1.3%
per mm. Fractions were lyophilized and analyzed by analytical
reversed-phase HPLC and mass spectroscopy.
A 17-mer peptide constituting the amino terminus of the mouse CXCR2 (MGEFKVDKFNIEDFFSG) and a 21-mer peptide constituting the amino terminus of human CXCR2 (CMEDFNMESDSFEDFWKGEDL) were synthesized as described above. The peptides contained an additional alanine residue at the carboxyl terminus, and a cysteine residue at the amino terminus for conjugation with keyhole limpet hemocyanin (KLH). Conjugation with Imject maleimide-activated KLH was conducted according to the manufacturer (Pierce, Rockford, IL). Polyclonal antiserum against either murine CXCR2 or human CXCR2 was generated following s.c. or i.m. injections of 100 µg of the KLH-conjugated peptide in CFA, followed by at least three boosters of 100 µg of KLH-conjugated peptide in IFA. Direct ELISA was used to evaluate antisera titers, and sera were drawn when titers had reached greater than 1:1,000,000.
The specificity of the Ab to human CXCR2 was confirmed following receptor neutralization studies on stably transfected cells. This Ab specifically recognized human CXCR2 and prevented binding of IL-8 to 293 cells transfected to overexpress human CXCR2. This Ab did not cross-react with CXCR1, nor did it prevent binding of IL-8 to human CXCR1, when used to detect receptor expression in 293 cells overexpressing human CXCR1. To determine the specificity of the anti-mouse CXCR2 Abs, we tested the ability of the Ab to inhibit neutrophil recruitment in vivo. CBA/J mice or Fischer rats were injected i.p. with 80 ng of recombinant KC (K and C coordinates on the autorad from intial cloning (23)) in combination with either 0.5 ml of the anti-murine CXCR2 antisera or 0.5 ml of normal rabbit serum. Four hours later, animals were sacrificed and peritoneal lavage was performed using 3 ml of PBS with 5 mM EDTA. The concentration of cells within the lavage fluid was counted using a hemacytometer; equal numbers of cells were subjected to cytocentrifugation; and the slides were fixed and stained using the Diff-Quik kit (Baxter Diagnostics, McGaw Park, IL), followed by cell differential determination. It was found that the presence of anti-murine CXCR2 Abs specifically inhibited neutrophil migration in response to KC, while having no effect on monocyte infiltration in both rodent systems.
RT-PCR for CXCR2 gene expression
Cells were isolated as described above. Total RNA was extracted from cells using Trizol reagent (Life Technologies, Grand Island, NY), according to manufacturer’s instructions. RT-PCR was performed using an Access RT-PCR kit (Promega, Madison, WI). ß-actin was used as a housekeeping gene. For ß-actin, the sense primer used was 5'-GTGGGGCGCCCCAGGCACCA; the antisense primer was 5'-GCTCGGCCGTGGTGGTGAAGC (550 bp). For human CXCR2, the sense primer used was 5'-CCGGGCGTGGTGGTGAG; the antisense primer was 5'-TCTGCCTTTTGGGTCTTGTGAATA (385-bp product). PCR products were visualized by agarose gel electrophoresis. To exclude genomic DNA contamination, PCR was performed in the presence or absence of a preceding step that included reverse-transcriptase reaction with the isolated RNA.
Western blot analysis
Total protein extracts were made by scraping endothelial or 293 cell monolayers into TNE lysis buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 2.5 mM EDTA) supplemented with 2 ng/ml aprotinin and 35 ng/ml PMSF or by resuspending isolated neutrophils into supplemented TNE lysis buffer. Cell extracts were incubated on ice for 30 min, followed by centrifugation at 4°C for 30 min. Supernatants were then removed and assayed for total protein content using bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) and comparison with known amounts of BSA. A total of 40 µg of total protein was loaded in each well of a 12% polyacrylamide gel, and extracts were subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene fluoride membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine buffer (20 mM Tris, 150 mM glycine, pH 8, methanol added to a final concentration of 20% (v/v)). Blots were blocked in 5% skim milk in TBST buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature, followed by incubation in rabbit primary Ab sera against human CXCR2 diluted 1/1000 in blocking solution for 2 h at room temperature. Blots were washed for three 10-min washes in TBST and were incubated for 1 h at room temperature in goat anti-rabbit HRP-conjugated secondary Ab (Bio-Rad, Hercules, CA) at a 1/20,000 dilution. Blots were again washed for four 10-min washes in TBST, and proteins were visualized following incubation of the blots in SuperSignal chemiluminescent substrate solution according to the manufacturer’s protocol (Pierce) and exposure to XAR-5 film (Kodak, Rochester, NY).
Immunohistochemistry
Immunolocalization of CXCR2 was performed as previously described (24). Briefly, cytospins of human peripheral blood neutrophils, or Tissue-Teks (Fisher, Pittsburgh, PA) of 90% confluent unstimulated endothelial cell monolayers were fixed in 4% paraformaldehyde in PBS for 10 min, then rinsed twice with PBS. Before staining, the slides were again fixed for 30 min in the presence or absence of 1:1 absolute methanol and 3% H2O2, and rinsed in PBS, and then nonspecific binding sites were blocked with a 1/50 dilution of normal goat serum by incubation at room temperature for 30 min. Following the blocking step, a 1/500 dilution of either control (rabbit) or rabbit anti-human CXCR2 serum was added as a primary Ab, and slides were incubated for 30 min at room temperature. Slides were then rinsed with PBS, overlaid with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit; Vector Laboratories, Burlingame, CA), and incubated for an additional 30 min. Slides were again rinsed two times with PBS, and were then treated with streptavidin-conjugated peroxidase (Vector ABC Elite Kit; Vector Laboratories) for 30 min at room temperature. Following three washes with PBS, the slides were subjected to colorimetric detection using the substrate chromogen 3,3'-diaminobenzidine (Vector Laboratories). Slides were incubated for 5–10 min in 3,3'-diaminobenzidine solution at room temperature to allow color development, and rinsed with distilled water to quench the reaction. Mayer’s hematoxylin was used as a counterstain.
Endothelial cell chemotaxis
Endothelial cell chemotaxis assays were performed essentially as
previously described (3). HMVEC-L cells were harvested by
trypsinization, resuspended in CS-C medium without growth factors with
2% FBS added (Cell Systems), and preincubated with either
anti-human CXCR2 antiserum or normal rabbit serum at a final
dilution of 1/250 at 37°C for 1 h. Alternatively, endothelial
cells were preincubated in 2.5 mM pertussis toxin (PTx; Sigma) at
37°C for 1 h. Following preincubation, an aliquot of 160 µl
containing 5 x 105 cells/ml was added to
each of the lower wells of a 12-well chemotaxis chamber (Neuro Probe,
Cabin John, MD). The chambers were assembled (by placing 0.1%
gelatin-coated 5-µm pore-size filters over the lower wells, followed
by a gasket and the upper chamber) and incubated at 37°C in a
CO2 incubator for 2 h in an inverted
position. The chambers were then turned upright, and 100-µl aliquots
of various chemotactic agents in solution were added to the upper wells
of the chamber in the following concentrations: 80 ng/ml IL-8, ENA-78,
or GRO-; 40 ng/ml bFGF or VEGF. The chambers were placed in a 37°C
CO2 incubator for 2 h, at which time the
filters were removed, the bottom of the filters were scraped to remove
cells that did not undergo chemotaxis, and then the filters were
subjected to Diff-Quik (Baxter) staining. The number of endothelial
cells that had migrated through the filters into the upper chambers was
calculated by counting the total number of cells in 15 separate fields
of view under x40 power. Results were expressed as the number of
endothelial cells that migrated per high power field (HPF) after
subtracting the background (unstimulated control) to demonstrate
specific migration. Experiments were repeated independently at least
three times.
Rat corneal micropocket assay of angiogenesis
To address the ability of neutralizing CXCR2 Abs to inhibit angiogenesis in vivo, the corneal micropocket assay was performed in the rat eye, as previously described (3). Human IL-8, human ENA-78, murine KC, or murine macrophage-inflammatory protein (MIP)-2 was diluted in PBS plus 0.25% human serum albumin to a final concentration of 80 ng per pellet. VEGF or bFGF was diluted as above to a final concentration of 50 ng per pellet. Rabbit anti-murine CXCR2 antiserum or normal rabbit serum was added at a 1/1000 dilution to each of the above chemokines and cytokines, and mixed with an equal volume of Hydron casting solution (Hydro Med Sciences, New Brunswick, NJ). Five-microliter aliquots were pipetted onto the flat surface of a sterile polypropylene specimen container and were allowed to polymerize overnight under UV light in a laminar flow hood. Before implantation, the pellets were rehydrated with normal saline. Animals were given 150 mg/kg ketamine and 250 µg/kg atropine as an anesthetic, and the rat corneas were anesthetized with 0.5% proparacaine hydrochloride ophthalmic solution, followed by implantation of the Hydron pellet into an intracorneal pocket (1–2 mm from the limbus). Six days after implantation, animals received heparin (1000 U) and ketamine (150 mg/kg) i.p. 30 min before sacrifice, followed by perfusion with 10 ml of colloidal carbon via the left ventricle. Corneas were then harvested and photographed. No inflammatory response was observed in any of the corneas treated with the above cytokines. Sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were considered positive neovascularization responses. Negative responses were characterized by either no vessel growth or by the presence of only an occasional hairpin loop or sprout that displayed no evidence of sustained growth.
Mouse corneal angiogenesis assay
Hydron pellets incorporating sucralfate with either vehicle alone, bFGF (3 pmol/pellet, gift from Scios, Sunnyvale, CA), murine rMIP-2 (20 pmol/pellet, gift from Elias Lolis at Yale University School of Medicine, New Haven, CT), or human rIL-8 (20 pmol/pellet, purchased from R&D Systems, Minneapolis, MN) were made as described (25). Pellets were surgically implanted into corneal stromal micropockets created 1 mm medial to the lateral corneal limbus of C57BL/6 CXCR2-/- and CXCR2+/+ mice (9–10 wk old, derived from the strain developed at Genentech (South San Francisco, CA) by Cacalano et al. (26), a gift from Dr. Robert Terkeltaub, and maintained by Ann Richmond). Five days postimplantation, corneas were photographed at an incipient angle of 35–50o from the polar axis in the meridian containing the pellet, using a Zeiss split lamp. Images were digitalized and processed by subtractive color filters (Adobe Photoshop 4.0; Adobe Systems, Mountain View, CA): the fraction of the total corneal image that was vascularized (vascular area), and the ratio of pixels marking neovascular capillaries, both within the vascularized region (regional vascular density) and within the total corneal image (total vascular density), were calculated using the Bioquant software (Nashville, TN).
Statistical analysis
Data were analyzed on a PC computer using the Statview 5.0 statistical package (Abacus Concepts, Berkeley, CA). Comparisons were made using the unpaired t test or the Mann-Whitney test for nonparametric data. Data were considered statistically significant if p values were 0.05 or less.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We sought to determine the mechanism by which the
ELR+ CXC chemokines mediate the angiogenic
activity of endothelial cells, by examining the presence and the role
of the chemokine receptor CXCR2, in angiogenesis. Because all
ELR+ CXC chemokines induce angiogenic activity,
and CXCR2 on neutrophils appears to bind all of these chemokines, we
first evaluated whether CXCR2 mRNA was expressed in HMVEC, as compared
with neutrophils. Total RNA was extracted from HMVEC-L and HMVEC-D, as
well as neutrophils. RT-PCR analysis demonstrated the presence of an
appropriate band for CXCR2 mRNA by gel electrophoresis in both HMVEC
lines, as well as in neutrophils (Fig. 1
, IA). ß-actin served as an internal control (Fig. 1, IB). No bands were visualized using the same primers for
CXCR2 under conditions in which reverse transcriptase was excluded from
the reaction before PCR (Fig. 1, IC). This finding supported
the notion that the RT-PCR product/bands seen in Fig. 1, IA,
was not due to genomic DNA contamination of the specimens before PCR.
To further confirm that this mRNA was transcribed into protein, we used
an Ab specific for human CXCR2 in Western blot analysis to determine
whether this receptor was expressed in microvascular endothelial cells
at the protein level. The molecular mass of CXCR2 has been reported to
be a 44- to 46-kDa band in Western blot analysis by other investigators
(27, 28). Total protein extracts of human neutrophils,
HMVEC-D, HMVEC-L, control-transfected 293 cells, and CXCR2-transfected
293 cells were made in TNE lysis buffer. An aliquot containing 50 µg
of total protein was subjected to SDS-PAGE and transferred to
polyvinylidene fluoride membrane for Western blot analysis using a
rabbit polyclonal Ab specific to human CXCR2. A protein band of
molecular mass of 50 kDa was specifically recognized by the human
CXCR2 Ab in lysates from human neutrophils and CXCR2-transfected 293
cells (positive controls), while this band was absent in
control-transfected 293 cells (Fig. 1, II). A protein band
of similar molecular mass was also recognized by the Ab in both HMVEC-D
and HMVEC-L protein extracts (Fig. 1, II). The presence of
this 50-kDa protein species in endothelial cell extracts was also
confirmed by Western blot analysis using a second commercially
available Ab to human CXCR2 (data not shown).
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, IIB), which was absent when
normal rabbit serum was used as a primary Ab control (Fig. 2, IIA). In a similar manner, HMVEC-L demonstrated specific
staining for expression of CXCR2 in the presence of anti-human
CXCR2-specific Ab (Fig. 2, IID), as compared with the
absence of staining in the presence of control Ab (Fig. 2, IIC). CXCR2 protein was also detected in HMVEC-D monolayers
following immunohistochemical staining (data not shown). These findings
support the presence of an immunoreactive protein consistent with CXCR2
from HMVEC that appears to be similar to that which has been detected
on human neutrophils.
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A function of an angiogenic factor is to induce the chemotaxis of
endothelial cells. We have previously shown that the
ELR+ CXC chemokines are potent agonists of
endothelial cell chemotaxis, while the ELR- CXC
chemokines inhibit chemotaxis mediated by both
ELR+ CXC chemokines and bFGF (3). To
determine whether this chemotaxis was mediated through ligand binding
and subsequent signal transduction via CXCR2, we performed chemotaxis
assays in the presence or absence of neutralizing Ab to human CXCR2, as
compared with normal rabbit serum as a control. As shown in Fig. 3, addition of 10 nM of the
ELR+ CXC chemokines IL-8 or ENA-78 induced
specific HMVEC-L chemotaxis (118.4 ± 15.6 and 108.1 ± 9.6
cells/HPF, respectively). bFGF (5 nM) was also shown to induce similar
levels of endothelial cell chemotaxis in our assay system to those
observed with the ELR+ CXC chemokines (119.4
± 16.1 cells/HPF). To determine whether CXCR2 was mediating the
endothelial cell chemotactic effect of ELR+ CXC
chemokines, we addressed whether specific Abs to human CXCR2 inhibited
HMVEC-L chemotaxis. HMVEC-L were preincubated in the presence of
anti-human CXCR2 or normal control Abs, and then used in the
chemotaxis assay. As compared with cells incubated with angiogenic
stimuli alone, HMVEC-L chemotaxis in response to IL-8 or ENA-78 was
attenuated by 97% or 99%, respectively (p <
0.001), by the presence of neutralizing Ab to human CXCR2. In contrast,
preincubation of HMVEC-L with control Abs yielded chemotaxis results
similar to those obtained in the presence of the
ELR+ CXC chemokines alone (123.8 ± 17.7 and
101.7 ± 14.4 cells/HPF for IL-8 and ENA-78, respectively). A
similar inhibition of HMVEC-L chemotaxis in response to the
ELR+ CXC chemokine GRO- was also observed in
the presence of anti-CXCR2 Abs (data not shown). Moreover,
preincubation of endothelial cells with Abs to human CXCR2 had no
significant effect on HMVEC-L chemotaxis in response to bFGF
(Fig. 3). A similar inhibition of chemotaxis by anti-human CXCR2 in
response to ELR+ CXC chemokines was also
found when HMVEC-D were used (data not shown).
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Endothelial cell chemotaxis in response to ELR+ CXC chemokines is inhibited by PTx
To confirm that signal transduction through CXCR2, a G
protein-linked receptor, was responsible for the endothelial cell
chemotactic response to the ELR+ CXC chemokines,
we tested the ability of PTx to inhibit this specific migration. The
chemotactic response of HMVEC-L to either IL-8, ENA-78, bFGF, or VEGF
in the presence or absence of PTx is shown in Fig. 4. Endothelial cell chemotaxis in
response to IL-8 or ENA-78 was attenuated by 97% and 91%,
respectively (p < 0.001), in the presence of
2.5 mM PTx. In contrast, HMVEC-L chemotaxis in response to bFGF or VEGF
was unaltered in the presence of PTx (p = 0.25
and 0.28, respectively). These data indicate that HMVEC-L chemotaxis in
response to ELR+ CXC chemokines, but not to bFGF
or VEGF, is mediated by a signal transduction pathway associated with
PTx-sensitive G proteins. These data further support CXCR2 as the
candidate receptor mediating endothelial cell chemotaxis to
ELR+ CXC chemokines.
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To ascertain that CXCR2 was responsible for angiogenesis mediated
by the ELR+ CXC chemokines in vivo, we performed
the corneal micropocket assay of neovascularization in the rat. Various
angiogenic agents, including human IL-8, KC (the murine homologue of
human GRO-), MIP-2 (the murine homologue of human GRO-ß, ),
human ENA-78, and the nonchemokine angiogenic factors bFGF and VEGF,
were embedded in Hydron pellets in the presence of anti-murine
CXCR2 or control Abs. All the ELR+ CXC chemokines
tested, IL-8, ENA-78, KC, and MIP-2, induced neovascularization in the
presence of control Abs (Fig. 5C and Table I). However, in the presence of
neutralizing anti-murine CXCR2 Abs, angiogenesis induced by
ELR+ CXC chemokines was markedly inhibited (Fig. 5D and Table I). In contrast, anti-murine CXCR2 Abs
failed to inhibit angiogenesis induced by either bFGF or VEGF (Fig. 5, E and F, and Table I). These findings support the
contention that ELR+ CXC chemokine-induced
angiogenesis in vivo is dependent upon CXCR2.
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To further demonstrate the importance of CXCR2 in
ELR+ CXC chemokine-induced angiogenesis, we
performed corneal micropocket assays in
CXCR2-/- mice and their wild-type counterparts.
Vigorous neovascular responses toward Hydron pellets containing bFGF,
murine MIP-2, or human IL-8 were observed in wild-type mice (Fig. 6A, top panels). In
contrast, neovascular responses to the ELR+ CXC
chemokines were drastically reduced in CXCR2-/-
mice, while angiogenesis in CXCR2-/- mouse
corneas that had been implanted with pellets containing bFGF remained
vigorous (Fig. 6A, bottom panel). The degree of
neovascularization of each cornea was estimated by analysis of
digitized images, as described in Materials and Methods. A
reduction in the vascularized area (Fig. 6B), the regional
vascular density (Fig. 6C), and the total vascular density
(Fig. 6C) was observed in response to IL-8 and MIP-2 in
CXCR2-/- corneas as compared with their
wild-type counterparts. In contrast, there was no significant
difference in these parameters between CXCR2-/-
mice and wild-type controls when bFGF was used as the angiogenic
stimulus. These data further support the importance of CXCR2 in
mediating angiogenesis induced by the ELR+ CXC
chemokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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overexpression is associated with tumor progression and
metastasis in a variety of tumor models, such as nonsmall cell lung
carcinoma (31, 32), gastric carcinoma (33),
and melanoma (34). Moreover, survival of nude mice bearing
ovarian tumors was inversely correlated with the expression of IL-8 by
these tumors (35). The overexpression of these
ELR+ CXC chemokines has also been shown to be
associated with angiogenesis of psoriasis (36), idiopathic
pulmonary fibrosis (24), and granulation tissue of burn
wounds (37). To act as an angiogenic agent, the
ELR+ CXC chemokines should be able to induce the
chemotaxis of endothelial cells following specific ligand-receptor
interactions. We hypothesized that the chemokine receptor CXCR2 was
present on microvascular endothelial cells and is responsible for the
angiogenic effect mediated by ELR+ CXC
chemokines. This contention is supported by the fact that CXCR2 is
known to bind all the ELR+ CXC chemokines that
induce angiogenesis, including IL-8; ENA-78; GRO-, -ß, and -;
NAP-2; and GCP-2 (8). The expression of microvascular
endothelial cell CXCR2 has also been found in various disorders that
are associated with a predominance of neovascularization. CXCR2
expression has been localized to malignant melanoma cells and
microvascular endothelium within melanomas (34), as well
as to microvessels in head and neck squamous cell carcinoma
(38), and to both large and small vessel endothelial cells
in human breast carcinoma (39). CXCR2 expression has also
been localized to microvascular endothelial cells of granulation tissue
associated with burn wounds (37).
Our results indicate that the seven-transmembrane G protein-linked
chemokine receptor CXCR2 can be found in microvascular endothelial
cells of both dermal and lung origin. This result is in contrast to
that reported by other researchers, who have indicated that CXCR1, but
not CXCR2 expression could be detected on large vessel endothelial
cells (40). It is unlikely that CXCR1 is responsible for
the angiogenic activity attributable to the ELR+
CXC chemokines, because only IL-8 and GCP-2 have the potential to bind
to and signal via CXCR1 (11). Our data demonstrate that
microvascular endothelial cell chemotaxis in response to the
ELR+ CXC chemokines IL-8 and ENA-78 was
unaffected by the presence of Abs to human CXCR1, indicating that
although this receptor may be expressed on HMVEC-L, it is not
responsible for endothelial cell chemotaxis mediated by
ELR+ CXC chemokines. Additionally, there is no
known rodent homologue of CXCR1; therefore, neovascularization induced
by the ELR+ CXC chemokines in the rodent corneal
micropocket assay for angiogenesis may not be mediated through CXCR1
binding. This notion was confirmed in the studies using
CXCR2-/- mice, in which we found that murine
MIP-2 failed to induce an angiogenic response, as compared with a
positive angiogenic response in the wild-type mice. However, we also
found that the angiogenic response to human IL-8 was not completely
inhibited in the CXCR2-/- mice. We cannot
exclude that this response was due to a nonspecific angiogenic effect
related to inflammation/immunity in response to the use of a human Ag
in the mouse cornea. In addition, we cannot exclude that this small
angiogenic response may be due to another receptor in the mouse that
binds IL-8, but not MIP-2. The Abs that recognize human CXCR2 used in
our experiments were from two separate sources, and have been shown to
be specific to CXCR2 and do not cross-react with CXCR1. Thus, the
results of our Western blot and immunohistochemical staining cannot be
due to cross-reaction with CXCR1 present on endothelial cells. Our
results indicating the presence of CXCR2 on microvascular endothelial
cells are further supported by the fact that this Ab can functionally
inhibit endothelial cell chemotaxis in response to not only IL-8, but
also ENA-78 and GRO-, which do not bind CXCR1.
Our results also indicate that the chemotactic response to the
ELR+ CXC chemokines is inhibited by PTx, which is
consistent with a mechanism whereby the ELR+ CXC
chemokines induce endothelial cell chemotaxis through a PTx-sensitive G
protein signal transduction pathway. PTx, isolated from the bacteria
Bordetella pertussis, is known to inhibit signal
transduction mediated by certain G proteins, such as the
Gi/Go proteins, following
ADP ribosylation of the G subunit of the heterotrimeric G protein
species (41, 42). Signal transduction mediated by ligand
binding of CXCR2 has been shown to be dependent on the interaction of
the chemokine receptor with the PTx-sensitive Gi2
G protein in neutrophils and 293 cells stably transfected to express
CXCR2 (43, 44). The failure of PTx to inhibit endothelial
cell chemotaxis mediated by bFGF in our assay system is consistent with
the specificity of PTx to inhibit G protein-mediated signal
transduction pathways, and further supports the contention that
ELR+ CXC chemokines induce angiogenesis through
interaction with and activation of CXCR2.
There have been many discrepancies found in the literature regarding CXCR2 expression by endothelial cells. It is possible that endothelial cells may express different chemokine receptor repertoires depending on the cell culture conditions, the degree of confluence (45), or matrix the cells are grown on at the time of analysis of receptor expression. In addition, it has been demonstrated that binding of IL-8 to the endothelium in vivo shows heterogeneity in different segments of the vascular tree and within similar types of vessels located in different organs (46). Therefore, the disparate results concerning the expression of CXCR1 or CXCR2 on endothelium could be a result of different sources of endothelial cells, or different isolation and culture techniques. Rot et al. (46) have also reported that dermal microvascular endothelial cells that have previously demonstrated the capacity to bind IL-8 in vivo lose their ability to bind IL-8 during isolation and subsequent cell culture, suggesting that some phenotypic changes may occur in endothelial cells as a result of these culture techniques. While these studies concluded that IL-8 binding to endothelial cells was a result of association of the ligand with glycosaminoglycan residues on the surface of the cells (46), our data would support the contention that CXCR2 is also present on microvascular endothelial cells and that this receptor is critically involved in mediating endothelial cell chemotaxis and angiogenesis in response to ELR+ CXC chemokines.
DARC has also been shown to be expressed on postcapillary endothelial cells (47, 48), and therefore could potentially be the putative receptor mediating angiogenesis induced by the ELR+ CXC chemokines. This is unlikely, because although this receptor does bind all the ELR+ CXC chemokines known to induce angiogenesis, it also binds other chemokines of the CC family, which to date have no known angiogenic properties. In addition, there have been no indications that DARC mediates signal transduction events following ligand binding of chemokines (19), and because of this observation, it has been proposed to act as a sponge that binds excess free chemokines during inflammatory responses. Moreover, our finding for the fundamental role of CXCR2 in mediating endothelial cell migration in vitro and angiogenesis in vivo would suggest that DARC is unlikely to play a direct role in endothelial cell chemotaxis to ELR+ CXC chemokines. Similarly, although CXCR4 is readily expressed on endothelial cells (45, 49, 50), CXCR4 is not known to bind the ELR+ CXC chemokines and can thus be excluded as the receptor responsible for ELR+ CXC chemokine-induced angiogenesis.
The identification of CXCR2 as the receptor responsible for mediating angiogenesis induced by the ELR+ CXC chemokines has important implications. As mentioned, CXCR2 expression has been shown to be associated with neovascularization in the context of certain diseases such as psoriasis and tumorigenesis. Antagonists specific for CXCR2 could be used therapeutically to inhibit ELR+ CXC chemokine-dependent neovascularization, and improve the prognosis of patients with diseases that are associated with marked angiogenesis. Inhibition of neovascularization has been shown to drastically inhibit the progression and metastasis of various cancers; thus, inhibition of ELR+ CXC chemokine-mediated angiogenesis could also be advantageous as adjuvant cancer therapy. We have shown that neutralization of IL-8 or ENA-78 using specific neutralizing Abs to these molecules can inhibit the growth of nonsmall cell lung carcinoma in SCID mice (31, 32). These treatments, however, only reduced tumor growth by 40–60%. This is most likely the result of the fact that these tumors produce multiple ELR+ CXC chemokine ligands contributing to their tumorigenicity. Inhibition of ligand binding to CXCR2, or inhibition of the signal transduction events following activation of the CXCR2 receptor would in essence simultaneously inhibit the biological effect of multiple ELR+ CXC chemokines. Future work from our laboratory will focus on the inhibition of tumor growth and metastasis by antagonizing the function of the CXCR2 receptor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert M. Strieter, Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles School of Medicine, 900 Veteran Avenue, 14-154 Warren Hall, Box 711922, Los Angeles, CA 90095.
3 Abbreviations used in this paper: GRO, growth-related oncogene; bFGF, basic fibroblast growth factor; CXCR, CXC chemokine receptor; DARC, Duffy Ag receptor for chemokines; ENA, epithelial neutrophil-activating protein; GCP, granulocyte chemotactic protein; HMVEC, human microvascular endothelial cell; HMVEC-D, human dermal microvascular endothelial cell; HMVEC-L, human lung microvascular endothelial cell; HPF, high power field; KLH, keyhole limpet hemocyanin; MIP, macrophage-inflammatory protein; NAP, neutrophil-activating protein; PTx, pertussis toxin; TFA, trifluoroacetic acid; VEGF, vascular endothelial cell growth factor.
Received for publication December 28, 1999. Accepted for publication July 31, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, GROß, GRO, neutrophil-activating peptide-2, and epithelial cell-derived neutrophil-activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor. J. Biol. Chem. 271:20545., inhibits non-small cell lung carcinoma tumor growth and metastasis. Hum. Gene Ther. 11:247.[Medline]
differentially stimulate calcium influx through IL-8 receptors A and B. J. Biol. Chem. 271:20540. with interleukin-8 receptors. J. Biol. Chem. 271:12783.This article has been cited by other articles:
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 |
|
  J. Gavard, X. Hou, Y. Qu, A. Masedunskas, D. Martin, R. Weigert, X. Li, and J. S. Gutkind A Role for a CXCR2/Phosphatidylinositol 3-Kinase {gamma} Signaling Axis in Acute and Chronic Vascular Permeability Mol. Cell. Biol., May 1, 2009; 29(9): 2469 - 2480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. de Kruijf, J. van Heteren, H. D. Lim, P. G.M. Conti, M. M. C. van der Lee, L. Bosch, K.-K. Ho, D. Auld, M. Ohlmeyer, M. J. Smit, et al. Nonpeptidergic Allosteric Antagonists Differentially Bind to the CXCR2 Chemokine Receptor J. Pharmacol. Exp. Ther., May 1, 2009; 329(2): 783 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Singh, A. Sadanandam, K. C. Nannuru, M. L. Varney, R. Mayer-Ezell, R. Bond, and R. K. Singh Small-Molecule Antagonists for CXCR2 and CXCR1 Inhibit Human Melanoma Growth by Decreasing Tumor Cell Proliferation, Survival, and Angiogenesis Clin. Cancer Res., April 1, 2009; 15(7): 2380 - 2386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Kim, R. Romero, C. J. Kim, A. L. Tarca, S. Chhauy, C. LaJeunesse, D.-C. Lee, S. Draghici, F. Gotsch, J. P. Kusanovic, et al. Villitis of Unknown Etiology Is Associated with a Distinct Pattern of Chemokine Up-Regulation in the Feto-Maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-Fetal Graft-versus-Host Disease J. Immunol., March 15, 2009; 182(6): 3919 - 3927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Andreasen and N. H. Carbonetti Role of Neutrophils in Response to Bordetella pertussis Infection in Mice Infect. Immun., March 1, 2009; 77(3): 1182 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Singh, M. Varney, and R. K. Singh Host CXCR2-Dependent Regulation of Melanoma Growth, Angiogenesis, and Experimental Lung Metastasis Cancer Res., January 15, 2009; 69(2): 411 - 415. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Brant and J. P. Fabisiak Nickel and the Microbial Toxin, MALP-2, Stimulate Proangiogenic Mediators from Human Lung Fibroblasts via a HIF-1{alpha} and COX-2-Mediated Pathway Toxicol. Sci., January 1, 2009; 107(1): 227 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. C. Keeley, B. Mehrad, and R. M. Strieter Chemokines as Mediators of Neovascularization Arterioscler. Thromb. Vasc. Biol., November 1, 2008; 28(11): 1928 - 1936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nedachi, H. Fujita, and M. Kanzaki Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle Am J Physiol Endocrinol Metab, November 1, 2008; 295(5): E1191 - E1204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gelati, A. C. Aplin, E. Fogel, K. D. Smith, and R. F. Nicosia The Angiogenic Response of the Aorta to Injury and Inflammatory Cytokines Requires Macrophages J. Immunol., October 15, 2008; 181(8): 5711 - 5719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. K. Pedersen and M. A. Febbraio Muscle as an Endocrine Organ: Focus on Muscle-Derived Interleukin-6 Physiol Rev, October 1, 2008; 88(4): 1379 - 1406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Strieter Out of the Shadows: CXC Chemokines in Promoting Aberrant Lung Cancer Angiogenesis Cancer Prevention Research, October 1, 2008; 1(5): 305 - 307. [Full Text] [PDF]
|
|
|
|
|
|
H. Sun, W.-C. Chung, S.-H. Ryu, Z. Ju, H. T. Tran, E. Kim, J. M. Kurie, and J. S. Koo Cyclic AMP-Responsive Element Binding Protein- and Nuclear Factor-{kappa}B-Regulated CXC Chemokine Gene Expression in Lung Carcinogenesis Cancer Prevention Research, October 1, 2008; 1(5): 316 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Coleman, B. E. Rendon, M. Zhao, M.-W. Qian, R. Bucala, D. Xin, and R. A. Mitchell Cooperative Regulation of Non-Small Cell Lung Carcinoma Angiogenic Potential by Macrophage Migration Inhibitory Factor and Its Homolog, D-Dopachrome Tautomerase J. Immunol., August 15, 2008; 181(4): 2330 - 2337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yu, Y. Su, S. R. Opalenik, T. Sobolik-Delmaire, N. F. Neel, S. Zaja-Milatovic, S. T. Short, J. Sai, and A. Richmond Short tail with skin lesion phenotype occurs in transgenic mice with keratin-14 promoter-directed expression of mutant CXCR2 J. Leukoc. Biol., August 1, 2008; 84(2): 406 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sukkar, J. Jenkins, J. Sanchez, and E. M. Wagner Inhibition of CXCR2 attenuates bronchial angiogenesis in the ischemic rat lung J Appl Physiol, May 1, 2008; 104(5): 1470 - 1475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawanishi, Y. Matsui, M. Ito, J. Watanabe, T. Takahashi, K. Nishizawa, H. Nishiyama, T. Kamoto, Y. Mikami, Y. Tanaka, et al. Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer Clin. Cancer Res., May 1, 2008; 14(9): 2579 - 2587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Raghuwanshi, M. W. Nasser, X. Chen, R. M. Strieter, and R. M. Richardson Depletion of {beta}-Arrestin-2 Promotes Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer J. Immunol., April 15, 2008; 180(8): 5699 - 5706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Amura, K. S. Brodsky, B. Gitomer, K. McFann, G. Lazennec, M. T. Nichols, A. Jani, R. W. Schrier, and R. Brian Doctor CXCR2 agonists in ADPKD liver cyst fluids promote cell proliferation Am J Physiol Cell Physiol, March 1, 2008; 294(3): C786 - C796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Matsuda, S. Fukuda, K. Matsumoto, and H. Saito Th1/Th2 Cytokines Reciprocally Regulate In Vitro Pulmonary Angiogenesis via CXC Chemokine Synthesis Am. J. Respir. Cell Mol. Biol., February 1, 2008; 38(2): 168 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Bieche, C. Chavey, C. Andrieu, M. Busson, S. Vacher, L. Le Corre, J.-M. Guinebretiere, S. Burlinchon, R. Lidereau, and G. Lazennec CXC chemokines located in the 4q21 region are up-regulated in breast cancer Endocr. Relat. Cancer, December 1, 2007; 14(4): 1039 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Petreaca, M. Yao, Y. Liu, K. DeFea, and M. Martins-Green Transactivation of Vascular Endothelial Growth Factor Receptor-2 by Interleukin-8 (IL-8/CXCL8) Is Required for IL-8/CXCL8-induced Endothelial Permeability Mol. Biol. Cell, December 1, 2007; 18(12): 5014 - 5023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ribaux, J. A. Ehses, N. Lin-Marq, F. Carrozzino, M. Boni-Schnetzler, E. Hammar, J.-C. Irminger, M. Y. Donath, and P. A. Halban Induction of CXCL1 by Extracellular Matrix and Autocrine Enhancement by Interleukin-1 in Rat Pancreatic {beta}-Cells Endocrinology, November 1, 2007; 148(11): 5582 - 5590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. W. Horton, Y. Yu, S. Zaja-Milatovic, R. M. Strieter, and A. Richmond Opposing Roles of Murine Duffy Antigen Receptor for Chemokine and Murine CXC Chemokine Receptor-2 Receptors in Murine Melanoma Tumor Growth Cancer Res., October 15, 2007; 67(20): 9791 - 9799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. K. Pedersen, T. C. A. Akerstrom, A. R. Nielsen, and C. P. Fischer Role of myokines in exercise and metabolism J Appl Physiol, September 1, 2007; 103(3): 1093 - 1098. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Nakao, Y. Hata, M. Miura, K. Noda, Y. N. Kimura, S. Kawahara, T. Kita, T. Hisatomi, T. Nakazawa, Y. Jin, et al. Dexamethasone Inhibits Interleukin-1{beta}-Induced Corneal Neovascularization: Role of Nuclear Factor-{kappa}B-Activated Stromal Cells in Inflammatory Angiogenesis Am. J. Pathol., September 1, 2007; 171(3): 1058 - 1065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Sanchez, A. Moldobaeva, J. McClintock, J. Jenkins, and E. Wagner The role of CXCR2 in systemic neovascularization of the mouse lung J Appl Physiol, August 1, 2007; 103(2): 594 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Proost, A. Mortier, T. Loos, J. Vandercappellen, M. Gouwy, I. Ronsse, E. Schutyser, W. Put, M. Parmentier, S. Struyf, et al. Proteolytic processing of CXCL11 by CD13/aminopeptidase N impairs CXCR3 and CXCR7 binding and signaling and reduces lymphocyte and endothelial cell migration Blood, July 1, 2007; 110(1): 37 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Struyf, M. D. Burdick, E. Peeters, K. Van den Broeck, C. Dillen, P. Proost, J. Van Damme, and R. M. Strieter Platelet Factor-4 Variant Chemokine CXCL4L1 Inhibits Melanoma and Lung Carcinoma Growth and Metastasis by Preventing Angiogenesis Cancer Res., June 15, 2007; 67(12): 5940 - 5948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mohsenin, M. D. Burdick, J. G. Molina, M. P. Keane, and M. R. Blackburn Enhanced CXCL1 production and angiogenesis in adenosine-mediated lung disease FASEB J, April 1, 2007; 21(4): 1026 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.P. Hess, A.E. Hamilton, S. Talbi, C. Dosiou, M. Nyegaard, N. Nayak, O. Genbecev-Krtolica, P. Mavrogianis, K. Ferrer, J. Kruessel, et al. Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators Biol Reprod, January 1, 2007; 76(1): 102 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Bonnett, E. J. Cornish, A. G. Harmsen, and J. B. Burritt Early Neutrophil Recruitment and Aggregation in the Murine Lung Inhibit Germination of Aspergillus fumigatus Conidia Infect. Immun., December 1, 2006; 74(12): 6528 - 6539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wislez, N. Fujimoto, J. G. Izzo, A. E. Hanna, D. D. Cody, R. R. Langley, H. Tang, M. D. Burdick, M. Sato, J. D. Minna, et al. High expression of ligands for chemokine receptor CXCR2 in alveolar epithelial neoplasia induced by oncogenic kras. Cancer Res., April 15, 2006; 66(8): 4198 - 4207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. N. Gomperts and R. M. Strieter CXC Chemokines in Angiogenesis and Metastases Am. Assoc. Cancer Res. Educ. Book, April 1, 2006; 2006(1): 11 - 18. [Full Text] [PDF]
|
|
|
|
|
|
J. A. Belperio, M. P. Keane, M. D. Burdick, B. N. Gomperts, Y. Y. Xue, K. Hong, J. Mestas, D. Zisman, A. Ardehali, R. Saggar, et al. CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury J. Immunol., November 15, 2005; 175(10): 6931 - 6939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Numasaki, M. Watanabe, T. Suzuki, H. Takahashi, A. Nakamura, F. McAllister, T. Hishinuma, J. Goto, M. T. Lotze, J. K. Kolls, et al. IL-17 Enhances the Net Angiogenic Activity and In Vivo Growth of Human Non-Small Cell Lung Cancer in SCID Mice through Promoting CXCR-2-Dependent Angiogenesis J. Immunol., November 1, 2005; 175(9): 6177 - 6189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Mestas, M. D. Burdick, K. Reckamp, A. Pantuck, R. A. Figlin, and R. M. Strieter The Role of CXCR2/CXCR2 Ligand Biological Axis in Renal Cell Carcinoma J. Immunol., October 15, 2005; 175(8): 5351 - 5357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Ruppel, D. Willison, H. Kataoka, A. Wang, Y.-W. Zheng, I. Cornelissen, L. Yin, S. M. Xu, and S. R. Coughlin Essential role for G{alpha}13 in endothelial cells during embryonic development PNAS, June 7, 2005; 102(23): 8281 - 8286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Moldobaeva and E. M. Wagner Difference in proangiogenic potential of systemic and pulmonary endothelium: role of CXCR2 Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1117 - L1123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pastore, F. Mascia, F. Mariotti, C. Dattilo, V. Mariani, and G. Girolomoni ERK1/2 Regulates Epidermal Chemokine Expression and Skin Inflammation J. Immunol., April 15, 2005; 174(8): 5047 - 5056. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Brat, A. C. Bellail, and E. G. Van Meir The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis Neuro-oncol, April 1, 2005; 7(2): 122 - 133. [Abstract] [PDF]
|
|
|
|
 |
|
 T. Akerstrom, A. Steensberg, P. Keller, C. Keller, M. Penkowa, and B. K. Pedersen Exercise induces interleukin-8 expression in human skeletal muscle J. Physiol., March 1, 2005; 563(2): 507 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Schruefer, N. Lutze, J. Schymeinsky, and B. Walzog Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8 Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1186 - H1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Ehlert, C. A. Addison, M. D. Burdick, S. L. Kunkel, and R. M. Strieter Identification and Partial Characterization of a Variant of Human CXCR3 Generated by Posttranscriptional Exon Skipping J. Immunol., November 15, 2004; 173(10): 6234 - 6240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Weber, A. Schober, and A. Zernecke Chemokines: Key Regulators of Mononuclear Cell Recruitment in Atherosclerotic Vascular Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): 1997 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Struyf, M. D. Burdick, P. Proost, J. Van Damme, and R. M. Strieter Platelets Release CXCL4L1, a Nonallelic Variant of the Chemokine Platelet Factor-4/CXCL4 and Potent Inhibitor of Angiogenesis Circ. Res., October 29, 2004; 95(9): 855 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Liehn, A. Schober, and C. Weber Blockade of Keratinocyte-Derived Chemokine Inhibits Endothelial Recovery and Enhances Plaque Formation After Arterial Injury in ApoE-Deficient Mice Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1891 - 1896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J J Haringman, J Ludikhuize, and P P Tak Chemokines in joint disease: the key to inflammation? Ann Rheum Dis, October 1, 2004; 63(10): 1186 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Henke, A. Varga, S. De, C. B. Deatrick, J. Eliason, D. A. Arenberg, P. Sukheepod, P. Thanaporn, S. L. Kunkel, G. R. Upchurch Jr, et al. Deep Vein Thrombosis Resolution Is Modulated by Monocyte CXCR2-Mediated Activity in a Mouse Model Arterioscler. Thromb. Vasc. Biol., June 1, 2004; 24(6): 1130 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Scapini, M. Morini, C. Tecchio, S. Minghelli, E. Di Carlo, E. Tanghetti, A. Albini, C. Lowell, G. Berton, D. M. Noonan, et al. CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A J. Immunol., April 15, 2004; 172(8): 5034 - 5040. [Abstract] [Full Text] [PDF]
|
|
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M. Pold, L. X. Zhu, S. Sharma, M. D. Burdick, Y. Lin, P. P. N. Lee, A. Pold, J. Luo, K. Krysan, M. Dohadwala, et al. Cyclooxygenase-2-Dependent Expression of Angiogenic CXC Chemokines ENA-78/CXC Ligand (CXCL) 5 and Interleukin-8/CXCL8 in Human Non-Small Cell Lung Cancer Cancer Res., March 1, 2004; 64(5): 1853 - 1860. [Abstract] [Full Text] [PDF]
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M. P. Keane, J. A. Belperio, Y. Y. Xue, M. D. Burdick, and R. M. Strieter Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer J. Immunol., March 1, 2004; 172(5): 2853 - 2860. [Abstract] [Full Text] [PDF]
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M. Strasly, G. Doronzo, P. Capello, D. Valdembri, M. Arese, S. Mitola, P. Moore, G. Alessandri, M. Giovarelli, and F. Bussolino CCL16 activates an angiogenic program in vascular endothelial cells Blood, January 1, 2004; 103(1): 40 - 49. [Abstract] [Full Text] [PDF]
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I. U. Schraufstatter, K. Trieu, M. Zhao, D. M. Rose, R. A. Terkeltaub, and M. Burger IL-8-Mediated Cell Migration in Endothelial Cells Depends on Cathepsin B Activity and Transactivation of the Epidermal Growth Factor Receptor J. Immunol., December 15, 2003; 171(12): 6714 - 6722. [Abstract] [Full Text] [PDF]
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 S. T. Tarzami, W. Miao, K. Mani, L. Lopez, S. M. Factor, J. W. Berman, and R. N. Kitsis Opposing Effects Mediated by the Chemokine Receptor CXCR2 on Myocardial Ischemia-Reperfusion Injury: Recruitment of Potentially Damaging Neutrophils and Direct Myocardial Protection Circulation, November 11, 2003; 108(19): 2387 - 2392. [Abstract] [Full Text] [PDF]
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N. Kaminski, J. A. Belperio, P. B. Bitterman, L. Chen, S. W. Chensue, A. M.K. Choi, S. Dacic, J. H. Dauber, R. M. du Bois, J. J. Enghild, et al. Idiopathic Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): S1 - 105. [Full Text] [PDF]
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S. Srisuma, S. S. Biswal, W. A. Mitzner, S. J. Gallagher, K. H. Mai, and E. M. Wagner Identification of Genes Promoting Angiogenesis in Mouse Lung by Transcriptional Profiling Am. J. Respir. Cell Mol. Biol., August 1, 2003; 29(2): 172 - 179. [Abstract] [Full Text] [PDF]
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 R. J. Phillips, M. D. Burdick, M. Lutz, J. A. Belperio, M. P. Keane, and R. M. Strieter The Stromal Derived Factor-1/CXCL12-CXC Chemokine Receptor 4 Biological Axis in Non-Small Cell Lung Cancer Metastases Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1676 - 1686. [Abstract] [Full Text] [PDF]
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P. Lu, Y. Nakamoto, Y. Nemoto-Sasaki, C. Fujii, H. Wang, M. Hashii, Y. Ohmoto, S. Kaneko, K. Kobayashi, and N. Mukaida Potential Interaction between CCR1 and Its Ligand, CCL3, Induced by Endogenously Produced Interleukin-1 in Human Hepatomas Am. J. Pathol., April 1, 2003; 162(4): 1249 - 1258. [Abstract] [Full Text] [PDF]
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M. Numasaki, J.-i. Fukushi, M. Ono, S. K. Narula, P. J. Zavodny, T. Kudo, P. D. Robbins, H. Tahara, and M. T. Lotze Interleukin-17 promotes angiogenesis and tumor growth Blood, April 1, 2003; 101(7): 2620 - 2627. [Abstract] [Full Text] [PDF]
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A. L. Miller, R. M. Strieter, A. D. Gruber, S. B. Ho, and N. W. Lukacs CXCR2 Regulates Respiratory Syncytial Virus-Induced Airway Hyperreactivity and Mucus Overproduction J. Immunol., March 15, 2003; 170(6): 3348 - 3356. [Abstract] [Full Text] [PDF]
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A. Li, S. Dubey, M. L. Varney, B. J. Dave, and R. K. Singh IL-8 Directly Enhanced Endothelial Cell Survival, Proliferation, and Matrix Metalloproteinases Production and Regulated Angiogenesis J. Immunol., March 15, 2003; 170(6): 3369 - 3376. [Abstract] [Full Text] [PDF]
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 J. Heidemann, H. Ogawa, M. B. Dwinell, P. Rafiee, C. Maaser, H. R. Gockel, M. F. Otterson, D. M. Ota, N. Lugering, W. Domschke, et al. Angiogenic Effects of Interleukin 8 (CXCL8) in Human Intestinal Microvascular Endothelial Cells Are Mediated by CXCR2 J. Biol. Chem., February 28, 2003; 278(10): 8508 - 8515. [Abstract] [Full Text] [PDF]
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E. S. White, K. R. Flaherty, S. Carskadon, A. Brant, M. D. Iannettoni, J. Yee, M. B. Orringer, and D. A. Arenberg Macrophage Migration Inhibitory Factor and CXC Chemokine Expression in Non-Small Cell Lung Cancer: Role in Angiogenesis and Prognosis Clin. Cancer Res., February 1, 2003; 9(2): 853 - 860. [Abstract] [Full Text] [PDF]
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M. P. Keane, S. C. Donnelly, J. A. Belperio, R. B. Goodman, M. Dy, M. D. Burdick, M. C. Fishbein, and R. M. Strieter Imbalance in the Expression of CXC Chemokines Correlates with Bronchoalveolar Lavage Fluid Angiogenic Activity and Procollagen Levels in Acute Respiratory Distress Syndrome J. Immunol., December 1, 2002; 169(11): 6515 - 6521. [Abstract] [Full Text] [PDF]
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 R. M. Strieter, J. A. Belperio, and M. P. Keane CXC Chemokines in Angiogenesis Related to Pulmonary Fibrosis Chest, December 1, 2002; 122(6_suppl): 298S - 301S. [Abstract] [Full Text] [PDF]
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J. Middleton, A. M. Patterson, L. Gardner, C. Schmutz, and B. A. Ashton Leukocyte extravasation: chemokine transport and presentation by the endothelium Blood, December 1, 2002; 100(12): 3853 - 3860. [Abstract] [Full Text] [PDF]
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I. U. Schraufstatter, K. Trieu, L. Sikora, P. Sriramarao, and R. DiScipio Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells J. Immunol., August 15, 2002; 169(4): 2102 - 2110. [Abstract] [Full Text] [PDF]
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A. B. LENTSCH The Duffy antigen/receptor for chemokines (DARC) and prostate cancer. A role as clear as black and white? FASEB J, July 1, 2002; 16(9): 1093 - 1095. [Abstract] [Full Text] [PDF]
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 M. Gouwy, S. Struyf, F. Mahieu, W. Put, P. Proost, and J. Van Damme The Unique Property of the CC Chemokine Regakine-1 to Synergize with Other Plasma-Derived Inflammatory Mediators in Neutrophil Chemotaxis Does Not Reside in Its NH2-Terminal Structure Mol. Pharmacol., July 1, 2002; 62(1): 173 - 180. [Abstract] [Full Text] [PDF]
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K. Wakasugi, B. M. Slike, J. Hood, K. L. Ewalt, D. A. Cheresh, and P. Schimmel Induction of Angiogenesis by a Fragment of Human Tyrosyl-tRNA Synthetase J. Biol. Chem., May 31, 2002; 277(23): 20124 - 20126. [Abstract] [Full Text] [PDF]
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B. Endlich, D. Armstrong, J. Brodsky, M. Novotny, and T. A. Hamilton Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury J. Immunol., April 1, 2002; 168(7): 3586 - 3594. [Abstract] [Full Text] [PDF]
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S. S. Cheng, N. W. Lukacs, and S. L. Kunkel Eotaxin/CCL11 Suppresses IL-8/CXCL8 Secretion from Human Dermal Microvascular Endothelial Cells J. Immunol., March 15, 2002; 168(6): 2887 - 2894. [Abstract] [Full Text] [PDF]
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C. Choi, O. Kutsch, J. Park, T. Zhou, D.-W. Seol, and E. N. Benveniste Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells Mol. Cell. Biol., February 1, 2002; 22(3): 724 - 736. [Abstract] [Full Text] [PDF]
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 C. Sachidanandan, R. Sambasivan, and J. Dhawan Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury J. Cell Sci., January 7, 2002; 115(13): 2701 - 2712. [Abstract] [Full Text] [PDF]
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P. Proost, E. Schutyser, P. Menten, S. Struyf, A. Wuyts, G. Opdenakker, M. Detheux, M. Parmentier, C. Durinx, A.-M. Lambeir, et al. Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties Blood, December 15, 2001; 98(13): 3554 - 3561. [Abstract] [Full Text] [PDF]
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L. W. Shepard, M. Yang, P. Xie, D. D. Browning, T. Voyno-Yasenetskaya, T. Kozasa, and R. D. Ye Constitutive Activation of NF-kappa B and Secretion of Interleukin-8 Induced by the G Protein-coupled Receptor of Kaposi's Sarcoma-associated Herpesvirus Involve Galpha 13 and RhoA J. Biol. Chem., November 30, 2001; 276(49): 45979 - 45987. [Abstract] [Full Text] [PDF]
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