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* Laboratory of Molecular Immunoregulation,
Division of Basic Sciences, ABL-BRP, Data Management Services, Inc., Frederick, Maryland 21702, USA; and
NCI, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA
1Correspondence: NCI-Frederick Cancer Research and Development Center, Bldg. 560, Rm. 21–89A, Frederick, MD 21702-1201, USA. E-mail: oppenhei{at}ncifcrf.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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but not IP-10 induced calcium mobilization in adherent
ECs, suggesting that signaling events associated with calcium
mobilization are separable from those required for chemotaxis. Taken
together, our data indicated that functional differences among EC types
is dependent on the level of the expression of CXC chemokine receptors.
Whether this heterogeneity in receptor expression by ECs reflects
distinct differentiation pathways remains to be established.—Salcedo,
R., Resau, J. H., Halverson, D., Hudson, E. A., Dambach, M.,
Powell, D., Wasserman, K., Oppenheim, J. J. Differential
expression and responsiveness of chemokine receptors (CXCR1–3) by
human microvascular endothelial cells and umbilical vein endothelial
cells.
Key Words: chemokines • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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are
angiogenic (1)
. They all interact with CXCR2 and IL-8
interacts with CXCR1 on neutrophils as well. Although it is well
established that IL-8 can act as an angiogenic factor
(1
2
3
4)
, the expression of IL-8 receptors on endothelial
cells (ECs) remains controversial. Several lines of evidence indicate
that IL-8 plays a direct role in angiogenesis. IL-8 has been shown to
bind specifically to ECs (4)
and to induce chemotaxis of
bovine ECs (5)
. Injection of IL-8 into rat corneas induced
vascularization in the absence of inflammatory responses (1
, 6)
, suggesting that corneal ECs might express IL-8 receptors. A
recent report described the expression of CXCR1–4 mRNA in ECs grown
under subconfluent conditions (7)
, suggesting that culture
conditions may affect the expression of CXC chemokine receptors. Other
lines of evidence support an indirect role for IL-8 in angiogenesis.
According to Hu et al. (4)
, IL-8-induced corneal
vascularization was consistently accompanied by an inflammatory
reaction that might involve the release of other angiogenic factors.
IL-8 failed to induce the chemotaxis of human umbilical vein
endothelial cells (HUVECs) (4)
. In addition, the lack of
IL-8 receptor expression at the RNA level on HUVECs and HMECs was
consistent with the inability of these cells to bind IL-8
(9)
. Moreover, by RNase protection assay, we detected only
CXCR4 expression on HUVECs, which led to the identification of SDF-1
and ß as non-ELR+ angiogenic factors
(10)
. This latter observation is consistent with findings
of impaired mesenteric vascularization in CXCR4 knock out mice
(11)
The ELR- CXC chemokines, interferon-inducible
protein 10 (IP-10) and macrophage-derived interferon gamma-inducible
chemokine (MIG), are reported to be angiostatic (1
, 12
, 13)
. These chemokines induce chemotaxis of activated T cells via
interactions with CXCR3 (14)
. Although CXCR3 mRNA was
reported to be expressed in ECs (7)
, its expression at the
protein level has not been determined.
ECs from different sources are known to be heterogeneous; they
differ in their expression of adhesion proteins, secretory products,
and responsiveness to cytokines (15
16
17
18)
. In addition, ECs
exhibited diversity in signaling responses to PKC activation
(15)
. In view of the apparent limited receptor expression
and chemokine responsiveness of HUVECs, we decided to evaluate CXC
chemokine receptor expression at the protein level on HMECs, based on
the hypothesis that they might be more representative of ECs involved
in somatic angiogenesis. The functional capacity of these receptors as
evidenced by chemotaxis, calcium flux, and receptor internalization was
also studied.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. HUVECs were isolated as described previously
(10)
. All endothelial cell types were cultured on collagen
type I-coated plastic (Biocoat) with EGM medium (Clonetics,
Walkersville, Md.) containing 5% fetal calf serum (FCS), VEGF (10
ng/ml), bFGF (10 ng/ml), glutamine (2 mM), and gentamicin (100 U/ml).
All experiments were performed on subcultures between the second to
fifth ‘in vitro’ passage.
Chemokines and antibodies
Recombinant human interleukin-8 (IL-8), stromal-derived
factor-1 (SDF-1), interferon-induced T cell activation (ITAC),
MIG, IP-10, B cell activation chemokine (BCA-1), vascular endothelial
growth factor (VEGF), and basic fibroblast growth factor (bFGF) were
purchased from Pepro Tech, Inc. (Rocky Hill, N.J.). Monoclonal
anti-human CXCR-1 and CXCR-2 were kindly provided by Dr. Jean Kim of
Genentech (San Francisco, Calif.), anti-human CXCR3 (49801–111) and
anti-human CXCR4 (12G5) were purchased from R&D Systems (Minneapolis,
Minn.). Mouse IgG (Coulter, Miami, Fla.) was used as the negative
control.
Immunofluorescence flow cytometry
Indirect immunofluorescence was performed on either
saponin-permeabilized or nonpermeabilized HMECs and HUVECs by exposing
cells to saturating amounts of mouse antibodies to human CXCR1, -2, -3,
or -4. Fluorescein-conjugated F(ab)2 fragments of
goat anti-mouse (Sigma, St. Louis, Mo.) diluted 1:50 were used as the
secondary antibody. After staining, cells were analyzed using a FACScan
flow cytometer (Becton Dickinson, Mountain View, Calif.).
Assessment of receptor distribution by confocal laser microscopy
Monolayers of subconfluent HMECs were fixed with 2%
paraformaldehyde for 15 min. Thereafter, cells were permeabilized with
0.15% saponin in buffer containing 0.5% gelatin for 30 min, washed
twice with phosphate-buffered saline (PBS), and subjected to staining
with corresponding primary antibody in gelatin buffer containing
rhodamine-conjugated concanavalin A (Con-A; Molecular Probes, Eugene,
Oreg.) for 30 min. After washing, cells were incubated with
fluorescein-conjugated rabbit anti-mouse for 30 min. Finally, cells
were washed and stained with DAPI (Sigma) for 10 min and slides were
then examined using a Zeiss 310 Confocal Laser Scanning Microscope.
Nomarski, rhodamine (543 nM, red), DAPI (UV 364 nM, blue), and
fluorescein (488 nM) images were prepared for each specimen and
subsequently superimposed using the Nomarski image as a base.
Endothelial cell migration assay
HMEC and HUVEC chemotaxis was performed using micro Boyden’s
chambers. Briefly, polycarbonate filters of 5 µm pore size
(Nucleopore, NeuroProbe, Cabin John, Md.) were coated with fibronectin
(10 µg/ml) (Sigma) overnight at 4°C. Binding buffer containing
1.0% bovine serum albumin (BSA) in RPMI 1640 with or without
chemokines was placed in the lower compartment of the chamber and
0.5 x 106 cells/ml resuspended in binding
medium were then added to the upper compartment. The chambers were
incubated for 3 h at 37°C. After the filters were removed, the
upper surface was scraped, fixed with methanol, and stained with
Leukostat (Fisher Scientific, Pittsburgh, Pa.). Membranes were analyzed
using the BIOQUANT program R&M Biometrics, Inc., Nashville, Tenn.) and
the results were expressed as the mean number of migrated cells/ten
fields at 10x magnification. For inhibitory assays, cells were
preincubated with different concentrations of monoclonal antibodies
(mAbs) during 10 min; thereafter, cells were added to the upper
compartment of the Boyden chamber.
Assessment of receptor internalization by flow cytometry
HMECs plated on gelatin were incubated with either IL-8 (100
ng/ml), SDF-1 (100 ng/ml) or IP-10 (1 µg/ml) for 30 min, washed
once with ice-cold PBS, and trypsinized for 3 min on ice. Thereafter,
cells were washed in cold PBS containing 0.2% FCS and stained for 30
min with antibodies to either CXCR1, -2, -3, or -4. After washing,
cells were incubated with fluorescein-conjugated goat anti-mouse
antibodies. Samples were analyzed using a FACScan flow cytometer
(Becton Dickinson).
Calcium mobilization
Calcium mobilization was performed using a Zeiss 410 confocal
laser scanning microscope on adherent ECs maintained at 37°C in a
micro-incubator (20/20 Technology Inc.). HMECs were first grown on 35
mm petri dishes, covered with No. 1 coverslips (MatTek, Inc.),
coated with gelatin, and subsequently loaded with Fluo-3 (1 µM)
(Molecular Probes) for 30 min at 37°C in 5%
CO2. The cells were washed four times with PBS
without calcium containing 0.5% BSA. Saline buffer
(CaCl2 and MgCl2) was added
to the cells and the petri dishes were transferred to the
micro-incubator. Nomarski and fluorescent images were recorded
throughout each experiment. The chemokines were added and digitized
images were stored, recorded every minute for 4 min after stimulation,
and the number of pixels and relative intensity was determined using an
Optimas 5.0 image analysis program (Bothell, Wash.) as described by
Klineberg et al. with modifications (20)
. The measurements
were exported to Microsoft Excel for statistical analysis. The figures
were obtained using a Codonics NP1600 printer. Ionomycin was used for
the positive control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50–70% confluence, the cell surface expression
of chemokine receptors was analyzed three times per donor by
immunofluorescence as described in Materials and Methods. As shown in
Fig. 1A
, CXCR1, CXCR2, CXCR3, and CXCR4 were all expressed on the
cell surface of HMECs with specific mean fluorescence intensities of
39.6, 9.1, 42.5, and 74.2, respectively. However, the low levels of
signal for some of the donors and our previous observation that
chemokine receptors can be internalized (10)
led us to
perform an analysis of permeabilized cells. Cell permeabilization
revealed an increased fluorescence intensity with specific mean
fluorescence intensity of 209.5 for CXCR1, 37.8 for CXCR2, 87.3 for
CXCR3, and 395.0 for CXCR4 (Fig. 1B
), indicative of the
presence of intracellular pools as well as cell surface expression of
CXCR1, CXCR2, CXCR3, and CXCR4 in HMECs. Whether these intracellular
pools represent internalized receptor or the novo synthesized or
degraded remains to be determined.
|
To confirm this observation, we performed confocal microscopy of HMECs.
As shown in Fig. 1C
, D
, E
, F
, G
, HMECs were stained with Con-A
rhodamine (red), which binds to lectin receptors on the cell surface
and outlines the cells. Dapi staining (blue) was used to locate and
identify DNA and nuclei. In addition, the cells were stained with
control Ab and specific Abs to CXC chemokine receptors, which were
detected using a fluorescein FITC-conjugated rabbit anti-mouseIgG
(green). Overlaying of the corresponding images (e.g., RGB image)
produced red cells in the absence of receptor expression, orange cells
for low receptor expression and colocalization with lectins, yellow
cells for high receptor expression and colocalization with lectins, and
green cells for receptor expression without lectin colocalization. In
Fig. 1C
, Con-A rhodamine staining is shown for the mouse IgG
control. In Fig. 1D
, intense expression of CXCR1 on HMECs is
shown to be distributed both intracellularly and on the cell surface.
Figure 1E
shows very weak staining of CXCR2 on HMECs. Figure 1F
shows the level of fluorescence staining achieved using
the CXCR3 antibody, which apparently was greater than that of CXCR2
(Fig. 1E
) but lower than that of CXCR1 (Fig. 1D
).
Finally, Fig. 1G
shows high expression of CXCR4 on HMECs at
a level that in this experiment is comparable to that of CXCR1 (Fig. 1D
). Thus, analysis of receptor expression on HMECs from a
representative donor showed low levels of CXCR2, intermediate levels of
CXCR3, and high levels of CXCR1 and CXCR4.
IL-8 elicited HMEC migration, receptor internalization, and
calcium fluxes
The functional capacity of the IL-8 receptors expressed by HMECs
was evaluated first by chemotaxis assays. Consistent with the
expression of IL-8 receptors on HMECs, we observed a dose-dependent
chemotactic response of these cells to IL-8. Significantly elevated,
maximal chemotactic responses were observed at concentrations of 1
ng/ml, 5 ng/ml, and 10 ng/ml (Fig. 2A
). The interaction of chemokines with their receptors
typically results in receptor redistribution and/or internalization;
therefore, we studied the effects of IL-8 on binding to their receptors
on HMECs by immunofluorescence. After 30 min of cell stimulation with
IL-8 at 37°C, immunostaining showed that IL-8 induced significant
internalization of CXCR1 (Fig. 2B
) and CXCR2 (Fig. 2C
), as evidenced by a decrease in the specific mean
fluorescence intensity (from 62.7 to 9.9 for CXCR1 and from 14.4 to 5.2
for CXCR2).
|
Calcium mobilization is another functional capability displayed by many
chemokine receptors; therefore, we studied the capacity of IL-8 to
induce calcium fluxes in HMECs. Calcium fluxes were detectable in HMECs
provided these cells were adherent to collagen. We were unable to
detect calcium fluxes in ECs when they were in suspension (data not
shown). IL-8 (10 ng/ml) induced calcium fluxes in HMECs at 1 min after
stimulation (Fig. 2F
), which was detected as an
increase in cell-associated fluorescence intensity over the basal
fluorescence obtained prior to stimulation shown in Fig. 2E
.
The fluorescence intensity was clearly decreased by 3 min after
stimulation, as shown in Fig. 2G
and H
. Thus,
under the appropriate conditions IL-8 can induce rapid and transient
calcium mobilization in HMECs.
The migration of HMECs toward IL-8 is both CXCR1 and -2 dependent
To evaluate the contribution of CXCR1 and CXCR2 to EC chemotaxis,
we used neutralizing antibodies (Abs) to CXCR1 and CXCR2, alone or in
combination. As shown in Fig. 3
, the Ab to CXCR2 partially blocked the IL-8-induced chemotactic
response of HMECs by 30% (P<0.039) whereas CXCR1
antibody was more effective (P<0.001) and inhibited the
chemotactic response by 70%. When both antibodies were used
together, we observed more than 90% inhibition (P<0.001).
The antibodies were used at 10 µg/ml, a concentration that showed a
maximal inhibitory response (data not shown). Although CXCR2 was less
abundantly expressed than CXCR1 on HMEC, our data indicated that this
receptor contributed considerably to the chemotactic response of HMECs
to IL-8. The data were analyzed by means of a two way ANOVA with the
Games-Howell procedure used for pairwise comparisons.
|
IP-10, ITAC, and MIG elicited chemotactic responses from HMECs
The detection of CXCR3 on HMECs prompted us to investigate the
potential chemotactic effects of known CXCR3 ligands. As shown in
Fig. 4A
, at relatively high concentrations the ligands for CXCR3,
including IP-10, ITAC, and MIG, all induced significantly elevated
levels of HMEC chemotaxis. In contrast to IL-8, which exhibited maximal
chemotactic activity at 1 to 10 ng/ml (Fig. 2A
), and
SDF-1, which exhibited maximal chemotactic activity at 10 ng/ml
(4A), the chemotactic response of HMECs toward IP-10 peaked at 500
ng/ml (P= 0.002). ITAC exhibited maximal chemotactic
responses at 100 ng/ml (P=0.039) and 500 ng/ml
(P=0.004); and MIG at 100 ng/ml (P=0.007) as
assessed by a two-way ANOVA with the Games-Howell procedure used for
pairwise comparisons (Fig. 4A
). Thus, high concentrations of
IP-10, ITAC, and MIG are chemotactic for HMECs. Due to the lack of
availability of neutralizing CXCR3 antibodies, we could not evaluate
the contribution of CXCR3 on the chemotactic responses mediated by
CXCR3 ligands on ECs or IL-2-activated T cells, which were used as a
control (data not shown).
|
We evaluated the binding of IP-10 and SDF-1 on expression of their
respective receptors on HMECs by immunofluorescence. After 30 min at
37°C of HMEC stimulation with either IP-10 or SDF-1,
immunostaining for CXCR3 or -4 was performed. As shown in Fig. 4B
and C
, IP-10 and SDF-1 induced significant
internalization of CXCR3 (Fig. 4B
) and CXCR4 (Fig. 4C
), respectively, as shown by the specific mean
fluorescence intensity, which decreased from 45.3 to 12.0 for CXCR3 and
from 111.9 to 13.4 for CXCR4.
We also tested the capacity of IP-10 and SDF-1 to induce
calcium fluxes in HMECs. As shown in Fig. 4D
, E
, F
, G
, H
, SDF-1
(100 ng/ml) induced calcium fluxes in adherent HMECs after 1 min of
stimulation (Fig. 4F
), which was detected as an
increase in cell-associated fluorescence intensity over the basal
(Fluo-3 alone) level of intensity obtained prior to stimulation with
SDF-1 (Fig. 4E
). The fluorescence intensity started to
decrease at 3 min after stimulation (Fig. 4G
, H
).
In contrast to SDF-1 and IL-8, IP-10 did not induce calcium fluxes
in HMECs over a dose range from 1 ng/ml to 3000 ng/ml (data not shown).
IP-10, ITAC, or MIG inhibited the chemotactic responses of
HMECs to IL-8
IP-10 and MIG are reported inhibitors of the
chemotactic response of bovine ECs toward IL-8 in vitro
(5
). Our experiments indicated that IP-10, ITAC, and
MIG per se can induce chemotactic responses from HMECs;
therefore, we evaluated the effect of these chemokines on IL-8-induced
chemotaxis of HMECs. IL-8 at an optimal chemotactic dose (10 ng/ml) was
tested with the addition of increasing concentrations of either IP-10
(Fig. 5A
), ITAC (Fig. 5B
) or MIG (Fig. 5C
).
The combination of CXCR3 ligands with IL-8 did not show an increased
chemotaxtic response by HMECs. In contrast, however, the chemotactic
response of HMECs to IL-8 was inhibited in a dose-dependent fashion by
IP-10, or ITAC or MIG above 50 ng/ml (P<0.020). A two-way
ANOVA indicated that IP-10, ITAC, and MIG effects were significant
(P<0.001). Post hoc tests were done using the
Games-Howell procedure and indicated IP-10 effects were significantly
higher than ITAC (P=0.005) or MIG-induced inhibition
(P<0.001), whereas no statistically significant difference
was found between the inhibitory potency of ITAC and MIG
(P=0.442).
|
HMECs and HUVECs exhibited disparate chemotactic responses to IL-8
Our findings regarding CXCR1 and -2 protein
expression and responsiveness by HMECs is in contrast with our previous
attempts to detect these receptors on HUVECs (10)
. To
verify that HUVECs are indeed hyporesponsive to IL-8 in
vitro, we isolated HUVECs from 10 donors and compared their
chemotactic responses to IL-8 vs. HMECs from seven different donors
(Fig. 6A
, B
). In contrast to HMECs, which all migrated to IL-8, only
30% of the HUVEC preparations exhibited significant chemotactic
responses to IL-8. Overall, HMECs exhibited significantly higher
chemotaxis indexes that ranged from 2.7 to 4.0 vs. 1.2 to 2.8 of HUVECs
(P<0.01; Fisher-Behrens conservative t test)
(6C). Thus, HMECs exhibited higher chemotactic responses to IL-8 than
HUVECs.
|
HMECs express higher levels of IL-8 receptors than HUVECs
The above data suggested a disparity at the level of IL-8
receptor expression by HMECs and HUVECs. We therefore compared CXCR1
and -2 protein expression by flow cytometry in several preparations of
HUVECs and HMECs. Consistent with the variability of the chemotactic
responses of HUVECs to IL-8, there was high degree of heterogeneity in
IL-8 receptor expression levels (Fig. 7
). The mean channel fluorescence for CXCR1 and -2 for 10 HUVEC
preparations was 66 (±20) and 49 (±15), respectively, whereas for
seven HMEC preparations the mean channel fluorescence intensity was 261
(±22) and 156 (±30) for CXCR1 and –2, respectively. These results
showed significantly higher mean fluorescence intensities for HMECs
than HUVECs (P<0.001 for CXCR-1, and P=0.009 for
CXCR-2 as assessed by the Fisher-Behrens conservative t
test). Thus, both the proportion and the number of receptors per HUVEC
were lower than HMECs.
|
Similar levels of expression of CXCR4 but not of CXCR3 on
HMECs and HUVECs
We compared the expression of CXCR3 and -4 on HUVECs and
HMECs by flow cytometry in permeabilized ECs as shown in Fig. 8
. CXCR3 was expressed on all HMECs, with a mean fluorescence intensity
of 173.9 (±30.2), whereas only 60% of the HUVEC preparations
expressed CXCR3, with a mean fluorescence intensity of 81.4 (±19.9).
Thus, the mean fluorescence intensity for HMECs was higher than for
HUVECs (P=0.032); for CXCR-3, CXCR4 was the most abundantly
expressed CXC chemokine receptor on ECs of either origin, with mean
fluorescence intensity of 306 (±19) for HMECs and 289.4 (±20) for
HUVECs. Thus, there was no significant difference regarding the levels
of expression of CXCR4 on these two cell types (P=0.206).
Comparisons were done by Fisher-Behrens conservative t test.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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22
23
24
25)
, or can be stimulated
by an increase in angiogenic mediators to express cell surface
activation markers and release proteases that degrade the basement
membrane (13
, 26
27
28
29
30
31)
. This enables ECs to migrate
directionally into surrounding tissues in response to a gradient of
angiogenic factors. Subsequent events (not fully characterized) lead to
proliferation of ECs, formation of tubules, generation of new basement
membranes, and anastomosis of newly formed vessels to preexisting
vessels. In the case of chemokines, angiogenic
ELR+ ligands such as IL-8, ENA 78, and GRO-
are also counterbalanced at inflamed and injured sites by the presence
of angiostatic IP-10, an ELR- chemokine
(32)
.
Although IL-8 and IP-10 have been extensively studied in in
vivo and in vitro angiogenesis models, the expression
of receptors for these chemokines on ECs remains controversial
(1
, 4
, 5
, 7
8
9
10)
. We have compared the expression of
receptors for the CXC chemokine family on human microvascular ECs with
HUVECs to test the hypothesis that HMECs may be better indicators of
the role of chemokines in somatic angiogenesis and to evaluate the
prediction that differences in receptor expression are responsible for
different functional abilities of various EC types. By FACS, confocal
microscopy, detection of calcium flux, receptor internalization, and
chemotaxis we found that all HMEC and a minority of HUVEC preparations
display functional CXC type receptors 1 through 3. As predicted, HMECs
also expressed more of these receptors per cell than HUVECs.
CXCR4 was expressed abundantly on all HUVECs and HMECs examined, which
is consistent with the observation that CXCR4 plays a more fundamental
developmental role in the establishment and subsequent maintenance of
vasculature. Indeed, CXCR4 knockout mice have defective vasculogenesis
within their gastrointestinal tracts (11)
. Although CXCR2
expression was low when compared with the expression of CXCR1 on HMECs,
the inhibitory effects of neutralizing antibodies indicated that CXCR2
participates in the chemotaxis of ECs toward IL-8. In contrast, ECs of
either type tested did not migrate or proliferate in response to BCA-1,
the ligand for CXCR5, suggesting that this receptor is not expressed by
ECs (data not shown).
The detection of CXCR3 on HMECs, prompted us to investigate the
potential chemotactic effects of known CXCR3 ligands. Despite published
reports that CXCR3 ligands such as IP-10 inhibit angiogenesis and
actually block the chemotactic effects of angiogenic factors such as
IL-8 and VEGF on ECs, we observed that at higher concentrations, IP-10
and ITAC (at 500 ng/ml) and MIG (at 100 ng/ml) also induced in
vitro EC chemotaxis of HMECs. This apparently conflicts with
previous reports indicating that IP-10 was not chemotactic for bovine
endothelial cells (1)
. The underlying basis of this
contradiction is unclear at this time. The capacity of a chemokine to
induce chemotaxis of ECs has been correlated with the ability of that
chemokine to induce ‘in vivo’ angiogenesis. The
chemotactic effects of CXCR3 ligands represent an exception. Our data
showing that angiostatic chemokines such as IP-10 also induced EC
migration resembles the biphasic effects also reported for
thrombospondin, a well-known angiostatic factor that at high doses also
induced migration of murine capillary and bovine aortic endothelial
cells, and at lower doses inhibited angiogenesis induced by bFGF
(33
34)
. These observations raise the possibility that
the presence of various chemokine combinations may have different and
complicated pathophysiological consequences, which could be determined
by a dynamic balance of angiogenic vs. angiostatic chemokines. This is
supported by our data showing that combinations of angiostatic ligands
inhibited the chemotaxis of ECs elicited by IL-8, in agreement with a
previous report (1)
. Consequently, although IP-10, ITAC,
and MIG bind a different receptor than angiogenic chemokines, they may
antagonize the effect of angiogenic chemokines perhaps by interfering
with downstream signal transduction pathways of angiogenic chemokine
receptors. Alternatively, it is possible that the interference might be
based on protein–protein interaction between angiostatic with
angiogenic ligands having a dominant-negative effect. These
possibilities are being further investigated.
Taken together, our data indicate that in the presence of appropriate stimuli, HMECs (unlike HUVECs) are highly responsive and consistently express CXCR1, -2, and -3. HMECs may therefore provide a better model for in vitro studies of angiogenesis than HUVECs. The basis for the apparent heterogeneity among these EC types remains unclear. It is possible that HMECs and HUVECs are the products of divergent differentiation pathways. Whether this heterogeneity in receptor expression by ECs reflects distinct differentiation pathways remains to be established. Although we have not been able to change the phenotypes of HUVECs to resemble those of HMECs, we cannot rule out the possibility that differences in their functional responsiveness represent a consequence of different endogenous conditions typically encountered by microvascular and large blood vessel ECs. We are continuing to explore various means of inducing chemokine receptor expression on HUVECs to learn more about the regulation of chemokine receptor expression and are investigating the basis for the inhibitory effects of angiostatic chemokines.
|
ACKNOWLEDGMENTS |
|---|
Received for publication December 6, 1999.
Revision received March 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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