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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1969-1979
Inflammatory Cytokines and Vascular Endothelial Growth
Factor Stimulate the Release of Soluble Tie Receptor From
Human Endothelial Cells Via Metalloprotease Activation
By
Rachel Yabkowitz,
Susanne Meyer,
Tabitha Black,
Gary Elliott,
Lee
Anne Merewether, and
Harvey K. Yamane
From the Departments of Mammalian Cell Molecular Biology,
Experimental Hematology, Protein Structure, and Protein Chemistry,
Amgen Inc, Thousand Oaks, CA.
 |
ABSTRACT |
Activation of endothelial cells, important in processes such as
angiogenesis, is regulated by cell surface receptors, including those
in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be
modulated by phosphorylation, turnover, or proteolytic release of a
soluble extracellular domain. Previously, we demonstrated that release
of soluble tie-1 receptor from endothelial cells by phorbol myristate
acetate (PMA) is mediated through protein kinase
C and a Ca2+-dependent protease. In this
study, the release of soluble tie-1 was shown to be stimulated by
inflammatory cytokines and vascular endothelial growth factor
(VEGF), but not by growth factors such as basic fibroblast
growth factor (bFGF) or transforming growth factor 
(TGF). Release of soluble tie by tumor necrosis factor (TNF) or VEGF occurred within 10 minutes of
stimulation and reached maximal levels within 60 minutes. Specificity
was shown by fluorescence-activated cell sorting (FACS) analysis;
endothelial cells exhibited a significant decrease in cell surface
tie-1 expression in response to TNF, whereas expression of epidermal
growth factor receptor (EGF-R) and CD31 was stable. In
contrast, tie-1 expression on megakaryoblastic UT-7 cells was
unaffected by PMA or TNF. Sequence analysis of the cleaved receptor
indicated that tie-1 was proteolyzed at the
E749/S750 peptide bond in the proximal
transmembrane domain. Moreover, the hydroxamic acid derivative BB-24
demonstrated dose-dependent inhibition of cytokine-, PMA-, and
VEGF-stimulated shedding, suggesting that the tie-1 protease was a
metalloprotease. Protease activity in a tie-1 peptide cleavage assay
was (1) associated with endothelial cell membranes, (2) specifically
activated in TNF-treated cells, and (3) inhibited by BB-24.
Additionally, proliferation of endothelial cells in response to VEGF,
but not bFGF, was inhibited by BB-24, suggesting that the release of
soluble tie-1 receptor plays a role in VEGF-mediated proliferation.
This study demonstrated that the release of soluble tie-1 from
endothelial cells is stimulated by inflammatory cytokines and VEGF
through the activation of an endothelial membrane-associated metalloprotease.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SERVING BOTH BARRIER and conduit
functions, the vasculature plays a critical role in maintaining normal
organ function and exacerbating pathological processes. Endothelial
cell activation, proliferation, migration, and matrix remodeling all
are essential components of inflammation and angiogenesis, processes
accompanying diseases such as cancer, diabetic retinopathy, and
rheumatoid arthritis.1,2 The response of endothelial cells
to the cytokines and growth factors that mediate these pathways is
dependent on the expression and activity of their corresponding cell
surface receptors. Consequently, the regulation of endothelial cell
receptor function has become an area of expanding research focus and effort.
Receptor tyrosine kinases (RTK) play pivotal roles in the
proliferation, activation, and differentiation of a spectrum of cell
types.3 Endothelial cells express several members of the RTK family, including receptors for vascular endothelial growth factor
(VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor
(EGF).4 The VEGF receptor
(VEGF-R), Flk-1, stimulates endothelial cell
proliferation, migration, and tubule formation in vitro.5
In vivo, Flk-1 activation stimulates increased vascular permeability
and induction of angiogenesis. FGF-R activation by basic FGF
(bFGF) has also been implicated in angiogenesis by
inducing endothelial cell migration, proliferation, and matrix
reorganization.6 These studies suggest that the activation
of RTKs initiates a cascade of signaling pathways leading to the
functional and phenotypic changes associated with vascular remodeling.
The importance of this process is underscored by evidence that the
vasculature is largely quiescent in healthy adults.1 Endothelial cell RTK activation is therefore more strongly associated with disease and its coordinate pathology. For example, in the absence
of angiogenesis, solid tumor growth is severely limited, but tumors
circumvent this restriction by expressing endothelial cell mitogens,
including bFGF and VEGF.7 However, tumor growth in vivo is
inhibited if RTK signaling is prevented, such as by dominant-negative
mutations of the VEGF-R, Flk-1.8 Thus, endothelial cell RTK
activation is an important component of disease progression and the
regulation of RTKs increasingly a focal point for new approaches in
drug development.
The tie-1 RTK is expressed on endothelial cells and a subset of
hematopoietic progenitor cells.4 Tie-1 and tie-2 (tek) form
a subfamily of RTKs possessing EGF-like repeats that distinguish it
from other RTK subfamilies. Although tie-1 expression on
CD34+ hematopoietic stem cells decreases during myeloid
differentiation, in megakaryocyte differentiation a significant
percentage of CD34 cells express tie-1.9
Treatment of megakaryoblastic leukemia cells with phorbol myristate
acetate (PMA) also upregulates tie-1 expression.10 During development, tie-1 expression is
prominent in tissues that give rise to the vasculature. Tie-1 knockout
mice fail to assemble an intact vasculature and die as a result of edema and hemorrhage between E13.5 and 14.5.11 In adults,
tie expression increases during wound healing, in proliferating ovarian capillaries, and in the vasculature surrounding malignant glioblastoma and melanoma.12-14 Although the ligand for tie-1 has yet to
be described, angiopoietin-1, the tie-2 ligand, was recently identified by Davis et al.15 Angiopoietin-1 appears to be involved in
the regulation of cell-cell interactions and maintenance of the
vascular architecture. In contrast to bFGF or VEGF, angiopoietin-1 does not directly stimulate endothelial cell growth. Angiopoietin-1 knock-out mice recapitulate the loss of vascular organization and
embryonic lethality observed with tie-2 knock-outs,16,17 and a mutated tie-2 has been linked with some forms of inherited venous
malformations.18 Recently, adenoviral-mediated delivery of
soluble tie-2 was shown to inhibit tumor growth and metastasis in
vivo,19 suggesting that tie-2 is important in the
development of tumor vasculature. These data suggest that the tie
family of RTKs is critical in the organization and integrity of the
vascular wall.
In this study, we show that the release of soluble tie-1 from
endothelial cells is regulated by inflammatory cytokines and VEGF via
the activation of a hydroxamic acid-sensitive metalloprotease. These
data suggest that tie-1 plays a role in the modulation of cell-cell
interactions and vascular permeability that accompany inflammation.
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MATERIALS AND METHODS |
Reagents.
All cytokines and growth factors were purchased from R&D Systems
(Minneapolis, MN) unless stated otherwise. PMA and lipopolysaccharide (LPS) were obtained from Sigma (St Louis, MO).
Phosphoramidon, Pefabloc, leupeptin, and aprotinin were purchased from
Boehringer Mannheim (Indianapolis, IN). VEGF was purchased from
PeproTech (Rocky Hill, NJ). Purified recombinant bFGF was provided by
Dr Tsutomu Arakawa (Amgen, Thousand Oaks, CA). Purified recombinant soluble CD14 was provided by Dr Mike Kelley (Amgen). The
hydroxamate-based metalloprotease inhibitor BB-24 was a kind gift from
British Biotech Pharmaceuticals (Oxford, UK). Antibodies (Abs) to tie-1
and recombinant soluble tie have been described
previously.20 Abs against CD31 and CD62E were purchased
from Becton Dickenson (Franklin Lakes, NJ). Anti-EGF-R was from
Oncogene Sciences (Uniondale, NY), and anti-FGF-R1 was from Upstate
Biotechnology (Lake Placid, NY). The tie-1a peptide,
DNP-NH-AEGPVQESRAAEEG-COOH, was synthesized and verified as previously
described,21 lyophilized, and reconstituted to 10 mmol/L in
dimethyl sulfoxide (DMSO) before use. Additionally, a
truncated peptide (tie-1b) representing the correct cleavage product,
DNP-NH-AEGPVQE-COOH, was also synthesized and run as a positive control
in all peptide cleavage assays. Both peptides were synthesized with a
dinitrophenyl (DNP) group at the N-terminus for detection at 350 nm in
high-performance liquid chromatography (HPLC) assays.
Cells.
Human umbilical vein endothelial cells (HUVECs) and dermal
microvascular endothelial cells (MDECs) were purchased from Cell Systems (Kirkland, WA). Lung microvascular endothelial cells were purchased from Clonetics Corp (San Diego, CA). Endothelial cells were
grown in the media provided by the vendor and cultures were routinely
used between passages 2 and 6. CHO-tie cells, CHO stable transfectants
expressing full length tie-1 receptor, have been described in detail
previously.20 CHO-tie cells were grown in Dulbecco's
modified Eagle's medium (DMEM) containing 10% dialyzed fetal bovine serum (FBS). UT-7 cells, a megakaryoblastic
leukemic cell line,22 were grown in Iscove's medium
containing 10% FBS and 2 ng/mL granulocyte-macrophage
colony-stimulating factor (GM-CSF).
Enzyme-linked immunosorbent assays (ELISAs).
Sandwich ELISAs for the detection of soluble tie-1 were
performed as previously described.20 Aliquots of
supernatants from endothelial cells treated with cytokines, growth
factors, or control media were assayed in duplicate. Where indicated,
cells were incubated for 10 minutes with 200 nmol/L GF109203X
(Calbiochem, San Diego, CA) or 20 µmol/L PD098059 (New England
BioLabs, Beverly, MA) before adding 10 ng/mL PMA, tumor necrosis factor
 (TNF), or 20 ng/mL VEGF for 2 hours. For
inhibition experiments, cultures were preincubated with increasing
concentrations of the metalloprotease inhibitor BB-24 for 15 minutes
before the addition of the indicated cytokines for 1 hour. Plates were
developed with the TMB substrate kit (KPL, Gaithersburg, MD), and
absorbance was read at 450 nm. Results are expressed as the mean ± SD.
Fluorescence-activated cell sorting (FACS) analysis.
HUVECs treated with 10 ng/mL TNF or PMA for 1 hour were washed with
ice-cold phosphate-buffered saline (PBS) and harvested using
versine-EDTA. In some experiments, cells were preincubated with 5 µmol/L BB-24 or DMSO (1:438 dilution) as a control for 10 minutes
before treatment with TNF. Cells were stained for 45 minutes on ice
with the following Abs: monoclonal antibody (MoAb) 42G10 for tie-1,
antihuman EGF-R, antihuman CD31, MoAb #9 for axl, antihuman CD62E
(E-selectin), and antichicken FGF-R1. Appropriate isotype-matched Ab
controls were used in each experiment. UT-7 cells were treated for 1 hour with 10 ng/mL PMA, TNF, interleukin-1 (IL-1), or VEGF
before staining with tie-1 MoAb 42G10. Samples were processed and
analyzed on a FACScan flow cytometer (Becton Dickinson, Milpitas, CA)
as previously described.20
Tie-1 cleavage site analysis.
HUVECs or CHO-tie cells were grown in 100-mm dishes to confluency.
Cultures were treated with 10 ng/mL TNF or PMA for 1 hour, washed,
and lysed as previously described.20 An affinity matrix for
isolation of the C-terminal fragment of tie-1 was prepared by
covalently cross-linking a C-terminal peptide antibody20 to
protein A Sepharose (Pharmacia Biotech, Piscataway, NJ) with dimethylpimelimidate.23 Lysate was loaded onto the affinity matrix and bound material eluted with 0.1 mol/L glycine, 10 mmol/L CHAPS, pH 2.7. Fractions were immediately neutralized, and those containing the cleaved tie receptor were pooled, concentrated, and
separated on a 14% sodium dodecyl sulfate (SDS)-polyacrylamide gel
followed by transfer to polyvinylidene difluoride membrane (PVDF) for N-terminal microsequencing.
HUVEC membrane preparation.
HUVECs in 100-mm dishes were incubated in the presence or absence of 20 ng/mL TNF for 1 hour before harvesting with 5 mmol/L EGTA/EDTA.
Washed, pelleted cells were resuspended at 5 × 106/mL
in lysis buffer (10 mmol/L NaPO4, pH 7.5, 1 mmol/L
MgCl2, 30 mmol/L NaCl, 1 mmol/L Pefabloc, 200 µmol/L
phosphoramidon) and homogenized on ice in a 5-mL Dounce until less than
20% of the cells remained intact (~50 strokes). The homogenate was
cleared of cell debris by centrifugation at 1,500 rpm for 5 minutes.
The supernatant was then centrifuged at 200,000g for 1 hour at
4°C. The cytosolic 200,000g supernatant fraction was
carefully transferred into a new tube and the membrane pellet was
resuspended at approximately 1 mg/mL in lysis buffer (PBS containing
10% glycerol, 1 mmol/L Pefabloc, 200 µmol/L phosphoramidon, 1.3 µmol/L aprotinin, and 5 µmol/L leupeptin). Both fractions were
stored at  80°C until analysis in the peptide cleavage assay.
Tie-1 peptide cleavage assays.
Fifty microliters (45 µg) of HUVEC cytosol or membrane extract was
mixed with 500 µg/mL tie-1a peptide, DNP-NH-AEGPVQESRAAEEG-COOH, in
PBS, pH 7.5, containing Ca2+ and Mg2+ and
incubated for 1 hour at 37°C. In some experiments, 5 µmol/L BB-24
was added to the reaction mix. Reactions were quenched by the addition
of 10 µL of 0.5 mol/L EDTA/EGTA. Samples were separated on a 33 × 4.6 mm Micra NPS C18 HPLC column and eluted with a linear gradient of 1% to 16% acetonitrile in 0.1% trifluoroacetic acid (TFA) over 30 minutes. Cleavage reaction products were followed by
monitoring absorbance at 350 nm. The tie-1a peptide alone or a mixture
of the tie-1a and tie-1b peptides were incubated in buffer and analyzed
in parallel to facilitate identification of the cleavage products.
Proliferation assays.
HUVECs in growth media were plated into 96-well plates at 1 × 105/mL and incubated overnight before serum starvation in
DMEM containing 0.5% FBS and 1% bovine serum albumin (BSA) for 24 hours. Where indicated, cells were pretreated with increasing
concentrations of BB-24 for 1 hour before the addition of bFGF (1 ng/mL) or VEGF (20 ng/mL) and incubated overnight. The next day, BrdU
was added to each well and incorporation was measured after 24 hours
according to the manufacturer's protocol (BrdU Cell Proliferation
ELISA; Boehringer Mannheim, Indianapolis, IN). Plates were read at 450 nm. All data points were assayed in triplicate and results were expressed as the mean ± SD.
| |
RESULTS |
Previously, our lab demonstrated that treatment of endothelial cells
with PMA stimulated the rapid release of soluble tie-1 receptor into
the media.20 Because PMA is a biological response modifier
with pleiotrophic effects on cell activation and function, we focused
this study on soluble tie-1 release in response to specific cytokines
and growth factors. Figure 1A shows that
the inflammatory cytokines TNF and IL-1, as well as the
growth/permeability factor VEGF, induced the significant release of
soluble tie-1 from HUVECs. In contrast, bFGF, transforming growth
factor (TGF), TGF, IL-8, and IL-6 did not
stimulate soluble tie-1 release above background levels. Interestingly,
soluble tie-1 release mediated by TNF, IL-1, or VEGF was
approximately 50% of that seen in the presence of PMA. Soluble tie-1
was also released from HUVECs in response to LPS + soluble CD14, but
not in response to LPS or soluble CD14 alone (Fig 1B). The release of
soluble tie-1 was also evaluated in microvascular endothelial cells
(Fig 1C). In dermal microvascular endothelial cells, tie-1 release was
less pronounced overall than in HUVECs, especially in response to VEGF. However, in lung microvascular endothelial cells, the extent of tie-1
release in response to inflammatory cytokines and VEGF was quite
similar to that observed in HUVECs. These data suggested that the
release of soluble tie-1 from endothelial cells is specifically triggered by exposure to inflammatory cytokines and VEGF, but not by
mitogenic growth factors such as bFGF or TGF.
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| Fig 1.
Soluble tie-1 is released from endothelial cells by
inflammatory cytokines or VEGF. Cells were incubated with PMA or the
indicated factors (all at 10 ng/mL except for VEGF at 100 ng/mL, LPS
at 20 ng/mL, and soluble CD14 at 100 ng/mL) for 1 hour. After
treatment, supernatants were collected and assayed for soluble tie-1 by
sandwich ELISA as described in Materials and Methods. (A and B) HUVECs
release soluble tie in response to PMA, TNF, IL-1, VEGF, or the
combination of LPS and soluble CD14. (C) Soluble tie release by HUVECs
was compared with release by microvascular dermal endothelial cells
(MDEC) and microvascular lung endothelial cells (MLEC). ( ) The basal
level of soluble tie release, cells incubated with media alone. Results
are expressed as the percentage control (±SD), where 100% control
represents soluble tie released by HUVECs in response to PMA (A and C)
or TNF (B).
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The kinetics of soluble tie-1 release in response to PMA are fairly
rapid, with maximum release occurring within 30 minutes.20 In comparison, the kinetics of tie-1 release in response to TNF was
somewhat slower; only minimal release was observed after 10 minutes and
maximal release was not observed until 1 hour of exposure (Fig 2A). In contrast, VEGF stimulated a
more rapid release of tie-1 from HUVECs. Significant release was
detected after only 5 minutes of VEGF exposure. Release of soluble
tie-1 slowed after 30 minutes and maximal release was reached only
after about 1 hour. Figure 2B demonstrates the dose-response curve for
soluble tie-1 release in the presence of TNF and VEGF. For TNF,
soluble tie-1 release was evident at 0.1 ng/mL and peaked at 10 ng/mL. VEGF-mediated release occurred within a narrower dose range; only minimal tie-1 release was seen at 10 ng/mL VEGF, with maximal release
seen at 100 ng/mL. These results suggested that the release of soluble
tie-1 receptor by TNF and VEGF was an immediate-early response and
occurred at physiologically relevant concentrations.
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| Fig 2.
Kinetics and dose-response of TNF- and VEGF-stimulated
soluble tie-1 release in HUVECs. (A) HUVECs were incubated in the
absence () or presence of 10 ng/mL TNF ( ) or 100 ng/mL VEGF
( ) for the indicated times. (B) HUVECs were incubated for 1 hour
with the indicated concentrations of TNF () or VEGF (). After
treatment, supernatants were collected and assayed for soluble tie-1
release as described in Materials and Methods. Results are shown as the
mean absorbance at 450 nm ± SD.
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Previous work demonstrated that PMA-mediated tie-1 release is inhibited
by GF109203X, indicating that activation of protein kinase C is
critical in this pathway.20 To determine if protein kinase
C was similarly involved in cytokine-mediated release, HUVECs were
treated with TNF or VEGF in the presence or absence of GF109203X and
assayed for soluble tie-1 release (Fig 3).
GF109203X did not inhibit tie-1 release in response to TNF or VEGF,
although, as expected, GF109203X completely inhibited PMA-dependent
tie-1 release. In addition, soluble tie-1 release was evaluated in the presence of the MEK inhibitor, PD098059. As shown in Fig 3, inhibition of MAPK signaling did not inhibit the release of soluble tie-1 in
response to PMA, TNF, or VEGF. These results indicated that, although activation of protein kinase C modulates tie-1 release in
response to PMA, tie-1 release in response to inflammatory cytokines or
VEGF is modulated by a distinct set of signaling pathways.
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| Fig 3.
PMA-mediated, but not TNF- or VEGF-mediated soluble
tie-1 release is regulated by protein kinase C activation. HUVECs were
incubated with 200 nmol/L GF109203X or 20 µmol/L PD098059 for 10 minutes before the addition of 10 ng/mL PMA, TNF, or 20 ng/mL VEGF
for 2 hours. Supernatants were collected and assayed for soluble tie-1
release by sandwich ELISA as described in Materials and Methods.
Results are expressed as the mean percentage of control ± SD, where
100% control represents soluble tie release in the absence of
inhibitor.
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The specificity of soluble tie-1 release with regard to endothelial
cell surface molecules and to hematopoietic cells expressing tie-1 was
addressed by FACS analysis. First, HUVECs were treated with
either TNF or PMA for 1 hour and the expression of several cell
surface markers was evaluated. Figure 4
shows the expected loss of tie-1 expression in response to TNF and
PMA. In contrast, expression of the EGF-R, like tie an RTK, and CD31
were not affected by TNF or PMA. The expression of several other
cell surface molecules, including the IGF-1R, CD54, erbB2/Her2, and
VLA-4, was also unaffected by TNF or PMA (data not shown).
Interestingly, the expression of axl, an RTK associated with myeloid
leukemia cells,24 was also downregulated in response to
PMA, but not TNF. In addition, the expression of FGF-R1 decreased
slightly in response to PMA and TNF, although soluble FGF-R1 could
not be detected in the media (data not shown). The upregulation of
CD62E (E-selectin) expression after TNF stimulation served as a
positive control. Tie-1 expression was also evaluated on UT-7
megakaryoblastic leukemia cells stimulated with PMA, TNF, IL-1,
or VEGF (Fig 5). In contrast to endothelial
cells, UT-7 cells demonstrated no significant decrease in tie-1
expression in response to inflammatory cytokines, VEGF, or PMA. Control
experiments confirmed that IL-1 and TNF receptors were expressed on
UT-7 cells (data not shown). These experiments demonstrated that the
release of soluble receptors from endothelial cells was not a universal
response to TNF or PMA stimulation. Moreover, soluble tie-1 release
was also not a general response in hematopoietic cells expressing
tie-1. These data suggested that soluble tie-1 is specifically released
from endothelial cells in response to TNF and PMA.
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| Fig 4.
Specificity of tie-1 release in endothelial cells. HUVECs
were incubated in the absence (blue) or presence of 10 ng/mL TNF
(green) or PMA (red) for 1 hour before harvesting. Aliquots of cells
were stained for 45 minutes on ice with Abs specific for tie-1, EGF-R,
CD31 (PECAM), axl, CD62E (E-selectin), or FGF-R1. Control samples were
stained with isotype-matched Abs (black). FACS analysis was performed
as described in Materials and Methods.
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| Fig 5.
Soluble tie receptor is not released from megakaryocytic
UT-7 cells. UT-7 cells were incubated in the absence (blue) or presence
of 10 ng/mL PMA (red), TNF (green), IL-1 (pink), or 100 ng/mL
VEGF (orange) for 1 hour. After treatment, cells were stained for 45 minutes on ice with phycoerythrin-coupled anti-tie-1 MoAb 42G10. An
isotype matched IgG (black) was used as the negative control. FACS
analysis was performed as described in Materials and Methods.
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The specificity of soluble tie-1 release from endothelial cells in
response to inflammatory cytokines suggested that tie-1 cleavage
occurred at a discrete site. To determine the cleavage site, amino acid
sequencing of the cleaved receptor was performed. The C-terminal
fragment of tie-1, which remains cell-associated after cytokine
treatment,20 was isolated by affinity chromatography and
subjected to N-terminal microsequencing. Fifteen N-terminal amino acids
were identified with the sequence
Ser-Arg-Ala-Ala-Glu-Glu-Gly-Leu-Asp-Gln-Gln-Leu-Ile-Leu-Ala. As shown
in Table 1, these data indicated that tie-1
was cleaved between amino acids E749 and S750
in the juxtamembrane domain. The same amino acid sequence was obtained
from HUVECs treated with either TNF or PMA or from stably transfected CHO-tie-1 cells treated with PMA. The sequence surrounding the tie-1 cleavage site was compared with the consensus cleavage sites
of several proteases in the hopes of identifying the tie-1 protease.
However, convincing homology between these sequences was not readily
apparent. The closest match was to human neutrophil collagenase, which
shows a threefold increase in protease activity when the P1
amino acid is changed to Glu, yielding a consensus cleavage sequence of
GPQE/IAGQ.25 Thus, identification of the tie-1 cleavage
site did not unequivocally identify the tie-1 protease.
Although tie-1 protease activity is unaffected by serine-, cysteine-,
aspartate-, or traditional metallo-protease inhibitors, activity is
inhibited by EGTA.20 These data, along with recent evidence
that PMA-stimulated shedding of TNF-R is inhibited by the hydroxamic
acid derivative TAPI,26 suggested that the tie-1 protease
might be a variant metalloprotease. Release of soluble tie from HUVECs
was evaluated in the presence of BB-24, a hydroxamic acid-derived
protease inhibitor.27,28 BB-24 inhibited the
TNF-mediated release of soluble tie-1 from HUVECs in a
dose-dependent manner (Fig 6A). Similarly,
BB-24 inhibited the low levels of basal tie-1 release from unstimulated
cells. The IC50 for soluble tie-1 release was approximately
0.5 µmol/L for both TNF-dependent and basal release. BB-24 was also
tested in endothelial cells stimulated with PMA, VEGF, IL-1, or
LPS+CD14 (Fig 6B). Inhibition of soluble tie-1 release was
dose-dependent, but the extent of inhibition depended on the stimulus.
The IC50s for VEGF-, IL-1-, and LPS+CD14-mediated release were in the range of 0.3 to 0.5 µmol/L, similar to the IC50 for TNF. However, the IC50 for
PMA-mediated tie-1 release was approximately 2.5 µmol/L, which is
about 10-fold higher. Interestingly, these results correlated with the
extent of soluble tie-1 release in cells; PMA-mediated tie-1 release
was approximately twice that seen with other stimuli, suggesting that
PMA might also activate a second protease capable of cleaving tie-1.
These data indicated that the release of soluble tie-1 was mediated by
an endothelial cell metalloprotease sensitive to hydroxamic
acid-derived inhibitors.
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| Fig 6.
Inhibition of soluble tie-1 release by the hydroxamic
acid-based metalloprotease inhibitor BB-24. HUVECs were preincubated
with the indicated concentrations of BB-24 for 15 minutes before the
stimulation of tie-1 release by (A) 10 ng/mL TNF () or (B) 10 ng/mL PMA (), 100 ng/mL VEGF (), 10 ng/mL IL-1 ( ), or 10 ng/mL LPS+100 ng/mL soluble CD14 ( ). After 1 hour of incubation,
supernatants were collected and analyzed for soluble tie-1 release by
sandwich ELISA as described in Materials and Methods. Basal soluble
tie-1 release in the presence of media alone ( ) was also inhibited
by BB-24. Results are expressed as the percentage control (±SD),
where 100% control represents soluble tie released by HUVECs in the
absence of inhibitor.
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The modulation of tie-1 cell surface expression by BB-24 was also
evaluated by FACS (Fig 7). As previously
shown, cell surface expression of tie-1 decreased dramatically in
HUVECs treated with TNF. However, in the presence of BB-24, tie-1
expression was essentially unchanged, demonstrating that inhibition of
soluble tie-1 release leads to the retention of full-length tie-1 on
the cell surface. To rule out the possibility that BB-24
nonspecifically inhibited cytokine signaling, expression of E-selectin
on HUVECs was evaluated in the presence and absence of BB-24. Although
resting HUVECs did not express detectable levels of E-selectin (Fig 7), cell activation by TNF significantly upregulated E-selectin
expression. Moreover, upregulation of E-selectin expression by TNF
was unaffected by BB-24. These results indicated that cell surface
levels of tie-1 remained stable in the presence of TNF when shedding
was inhibited by BB-24 and that BB-24 did not interfere with cytokine signaling in general. In addition, BB-24 did not exhibit any
nonspecific cytotoxic effects on HUVECs (data not shown).
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| Fig 7.
Modulation of tie-1, but not E-selectin, cell surface
expression by BB-24. HUVECs were incubated in the presence or absence
of 5 µmol/L BB-24 for 10 minutes before the addition of TNF
(green) or DMSO (blue) for 1 hour. After harvesting with versine-EDTA,
cells were stained on ice with Abs specific for tie-1 or CD62E
(E-selectin). Control samples were stained with isotype matched Abs
(black). FACS analysis was performed as described in Materials and
Methods.
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To more thoroughly characterize the endothelial cell tie-1 protease, a
tie-1 peptide assay was developed. HUVEC plasma membranes were prepared
by cell fractionation and differential centrifugation and incubated
with a DNP-labeled peptide substrate spanning the tie-1 cleavage point.
Cleavage of the substrate was determined by HPLC analysis and compared
with a control intact peptide or a peptide truncated at the appropriate
Glu residue. In Fig 8A, tie-1
protease activity was evaluated in the cytosol (a) and membrane fractions (b) of TNF-treated HUVECs. Protease activity was associated primarily with the membrane fraction. The low level of activity in the
cytosol could be attributed to spillover or the presence of a second
protease. As shown in Fig 8B, membranes prepared from untreated cells
(a) did not cleave the intact peptide substrate into a product of the
correct size and mobility. The appearance of a peak with intermediate
mobility (slower than the truncated control peptide but faster than the
intact control) suggested that HUVEC membranes may contain a second
TNF-insensitive protease. More significantly, when membranes from
TNF-treated cells were incubated with the intact substrate (b), a
cleaved peptide of the correct size and mobility was generated.
Furthermore, generation of the TNF-dependent cleavage product was
inhibited in the presence of BB-24 (c). These data demonstrated that
tie-1 protease activity was membrane-associated and specifically
activated by TNF. In addition, TNF-dependent peptide cleavage was
inhibited by BB-24, thus establishing specificity and correlating
protease activity in intact endothelial cells with activity in cell
membranes. These experiments also established a suitable assay system
for the characterization and identification of the tie-1 protease.
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| Fig 8.
The tie-1 protease is membrane-associated,
activated specifically by TNF, and inhibited by BB-24. HUVECs
incubated in the presence or absence of TNF were harvested, and cell
fractionation was performed as described in Materials and Methods. (A)
Cytosol (a) or membrane (b) fractions prepared from TNF-treated
HUVECs were incubated with 500 µg/mL tie-1a peptide for 1 hour at
37°C. (B) HUVEC membrane fractions from untreated (a) or
TNF-treated (b and c) cells were incubated with 500 µg/mL tie-1a
peptide in the presence (c) or absence (b) of BB-24 for 1 hour at
37°C. Samples quenched with EDTA/EGTA were separated by HPLC and
peptide elution followed by absorbance at 350 nm. The control
full-length tie-1a and truncated tie-1b peptides are shown in the
control chromatogram (d). The arrows indicate the peaks that correspond
to peptide fragments with the same size and mobility as the truncated
control peptide tie-1b.
|
|
Finally, we investigated whether inhibition of soluble tie-1 release
had an effect on endothelial cell proliferation. HUVECs were incubated
with either bFGF or VEGF in the presence or absence of increasing
concentrations of BB-24. As shown in Fig 9,
BB-24 dose-dependently inhibited endothelial cell proliferation in
response to VEGF, but not to bFGF. In fact, bFGF-mediated proliferation was modestly increased over controls in the presence of BB-24. Considering that VEGF, but not bFGF, stimulated soluble tie-1 release
in cell culture (Fig 1A), these data suggested that release of soluble
tie-1 is important in the regulation of endothelial cell proliferation.
These data also provided further evidence that bFGF and VEGF use
distinct signaling pathways in the modulation of endothelial cell
proliferation.
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| Fig 9.
BB-24 inhibits VEGF-mediated endothelial cell
proliferation. HUVECs were incubated with increasing concentrations of
BB-24 for 1 hour before the addition of 1 ng/mL bFGF or 20 ng/mL VEGF.
After overnight incubation, BrdU was added for an additional 24 hours
before measuring incorporation as described in Materials and Methods.
Plates were read at 450 nm and results are expressed as mean percentage
of control ± SD, where 100% control represents BrdU incorporation in
the absence of inhibitor.
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|
| |
DISCUSSION |
The regulation of receptor expression in endothelial cells plays an
important role in their ability to respond to external stimuli. Whether
that response encompasses endothelial cell proliferation, as seen in
angiogenesis, or activation, as seen in inflammation, receptor
expression provides a mechanism to propagate and modulate alterations
in endothelial cell function. In this study, we focused on the
regulation of expression of tie-1, an orphan endothelial cell receptor
likely to be involved in cell-cell interactions. Inflammatory cytokines
and VEGF stimulated the release of soluble tie-1 from endothelial
cells, effectively decreasing full-length receptor expression on the
cell surface. Soluble tie-1 was not released from cells treated with
bFGF, TGF, or TGF. FACS analysis demonstrated the specificity of
this response; the release of soluble receptors from endothelial cells
was not a universal response to TNF or PMA, and soluble tie-1 was
not released from megakaryocytic UT-7 cells under any conditions.
Furthermore, whereas activation of protein kinase C mediated
PMA-dependent soluble tie-1 release, TNF- and VEGF-dependent release
signaled through an alternative pathway(s). Preliminary
experiments have suggested that activation of p38 may play a role in
soluble tie-1 release in response to TNF and VEGF (data not shown).
Although microsequencing demonstrated that tie-1 was cleaved between
E749 and S750 in the sequence PVQESRAA, this
information did not clarify the identity of the tie-1 protease.
However, inhibition of soluble tie-1 release by BB-24 suggested that
the protease was a metalloprotease sensitive to hydroxamic acid-based
inhibitors. Cell fractionation studies indicated that the tie-1
protease was membrane-associated, inhibited by BB-24, and activated
specifically in TNF-treated cells. In addition, we found that
inhibition of soluble tie-1 release inhibited endothelial cell
proliferation in response to VEGF, but not bFGF. These results
demonstrated that inflammatory cytokines and VEGF regulate tie-1
expression on endothelial cells through the activation of a
membrane-associated metalloprotease that releases soluble tie from the
cell surface.
The release of soluble forms of membrane-associated growth factors and
receptors by PMA has been documented in many systems, including tumor
cells, transfected CHO cells, and monocytes.29,30 However,
PMA is such a pluripotent biological response modifier that
investigators have questioned the specificity of the response as
exemplified by Arribas et al,31 who showed that the
PMA-dependent release of soluble L-selectin, TGF, and IL-6R in
transfected CHO cells occurs through a common proteolytic pathway. In
contrast, shedding of the type II IL-1R in polymorphonuclear cells
(PMNs) is stimulated by TNF or GM-CSF, but not by
several other cytokines, including IL-1, even though TNF and
IL-1 share considerable similarity in their signal transduction
pathways.32 Moreover, TNF does not induce shedding of class
I MHC, Fc R, or 2 integrins in PMNs. These data
suggest that the release of soluble receptors can be tightly regulated
by signaling pathways that are cell-type specific. We have shown that
the release of soluble tie-1 from endothelial cells is regulated by
proinflammatory cytokines, including TNF, IL-1, and LPS, and the
angiogenic growth factor VEGF. Interestingly, another angiogenic growth
factor bFGF did not stimulate soluble tie-1 release, suggesting that
activating endothelial cell mitogenesis per se is not sufficient to
induce tie-1 shedding. This differential response to VEGF and bFGF was
further underscored by the finding that inhibition of tie-1 shedding by
BB-24 inhibited endothelial cell proliferation in response to VEGF, but
not to bFGF. These results suggested that release of soluble tie-1 may
play a role in VEGF-mediated proliferation.
Although the stimulation of tie-1 release by an inflammatory cytokine
and an angiogenic growth factor, TNF and VEGF, respectively, might
appear to be unrelated, these factors do share the ability to increase
vascular permeability. Because the ligand for tie-1 remains
unidentified, direct examination of the role of tie-1 in vascular
permeability is difficult. However, the death of tie-1 knock-out mice
during embryogenesis due to extensive hemorrhaging suggests that tie-1
function is critical for vascular integrity.11,17 Recent
data on the tie-2 receptor, and its ligands angiopoietin-1 and -2, strongly suggest a role for the tie receptor family in cell-cell
interactions.16,18,33 Moreover, increased tie-1 expression
has been associated with the leaky tumor vasculature in glioblastoma
and melanoma, but not with the adjacent normal vasculature.12,13 These diverse pieces of evidence suggest that inflammatory cytokines and VEGF may modulate vascular permeability partly by regulating the expression of tie-1 on endothelial cells.
Although our results demonstrated that soluble tie-1 release was
induced by a specific subset of cytokines and growth factors, it was
unclear whether receptor shedding in response to cytokines was a
general endothelial cell phenomenon. For example, LPS induces shedding
of both TNF and the p60 TNF-R from human peripheral blood
monocytes.26 In this study, FACS analysis was used to demonstrate that loss of endothelial cell receptors was not a universal
response to TNF or PMA. The expression of CD31, a cell-cell adhesion
molecule, was stable in the presence of both TNF and PMA, as was the
expression of several RTKs associated with endothelial cell
proliferation, including the EGF-R and IGF-1R. In contrast, axl
receptor expression decreased in the presence of PMA, but not TNF.
Loss of tie-1 expression in response to inflammatory cytokines or PMA
was not observed in megakaryocytic UT-7 cells, even though these
cells express tie-1, TNF, and IL-1 receptors. Similarly,
angiopoietin-2, the tie-2 receptor antagonist, stimulates receptor
phosphorylation in transfected fibroblasts but not in endothelial cells
expressing endogenous receptor.33 Thus, as yet undefined
endothelial cell components may confer a level of specificity on these
signaling pathways beyond receptor-ligand binding. These results
demonstrated that activation of the tie-1 protease did not result in
the universal cleavage of endothelial cell receptors and that
soluble tie-1 release likely depends on endothelial cell-specific
signal transduction pathways.
To further characterize the tie-1 protease, the cleavage site was
determined by microsequencing. The amino acid sequence surrounding the
cleavage site, PVQE/SRAA, did not match any known cleavage site
consensus sequences and thus did not reveal the nature of the tie-1
protease. Comparison of the tie-1 cleavage site with the cleavage sites
of other receptors or membrane-associated growth factors also failed to
identify any significant homology.30 Currently, despite
extensive research in this area, no consensus sequence for cleavage of
these molecules has been established. Indeed, an alternative approach
using mutagenized angiotensin-converting enzyme has generated evidence
that cleavage may be a positional effect occurring at a minimum
distance from both the membrane and the proximal extracellular
domain.34 Clearly, the identification of the protease(s)
involved in the release of soluble receptors and growth factors will
answer many of these questions.
Initial attempts to categorize the tie-1 protease through the use of
protease inhibitors were unsuccessful.20 Except for EGTA,
none of the other inhibitors affected soluble tie-1 release, suggesting
that the protease was Ca2+-dependent but little else. In
this report, the hydroxamic acid-based inhibitor BB-24 was shown to
dose-dependently inhibit soluble tie-1 release in response to
inflammatory cytokines, VEGF and PMA, and inhibit cleavage of a tie-1
peptide by membranes prepared from TNF-treated cells. The
IC50 for TNF-mediated release in the cell-based assay
was approximately 500 nmol/L, similar to the activity of hydroxamic
acid-based compounds in the inhibition of endotoxin-mediated release of
TNF from whole blood.35 These data suggested that the
tie-1 protease was likely a metalloprotease, although the lack of
inhibition by phosphoramidon20 and TIMP-2 (data not shown)
suggested that it was not MMP-1, MMP-2, etc, or another
well-characterized metalloprotease.35,36 However, the tie-1
protease may be a member of the disintegrin metalloprotease family that
includes the TNF convertase.27,37 These proteases are
insensitive to phosphoramidon but susceptible to inhibition by
hydroxamic acid-based compounds with IC50s in cell-based
assays similar to that described for tie-1
proteolysis.21,35,36 Preliminary results demonstrated that
the putative TNF convertase ADAM 10 did not cleave tie-1 in a peptide
cleavage assay (D. Lyons, personal communication, June 1997) and
expression of ADAM 10 was not detectable in HUVECs by immunoblot (data
not shown). Furthermore, recent reports have suggested that the TNF
convertase may be constitutively active,21,37 whereas in
HUVECs, TNF stimulation was necessary for protease
activity. However, we cannot rule out the possibility that the tie-1
protease may be another member of the ADAM family of
metalloproteases.38
The data presented in this study highlight an alternative pathway for
the regulation of endothelial cell function. Although signaling can be
directly initiated through receptor-ligand interactions, secondary
control systems are clearly critical for fine tuning responses. Among
these secondary pathways are the expression of endogenous antagonists
such as angiopoietin-2,33 the downregulation of receptors
through inhibition of transcription such as that reported for
VEGF-R,39 and the levels of sequestering proteins in
circulation such as the IGF-1 binding proteins.40 More
recently, the regulation of receptor/growth factor activity through
proteolysis of membrane-bound forms has emerged as an intriguing
control mechanism. Even though the soluble receptors, eg, IL-2R or
TNF-R, often retain binding activity, the change in localization has a
dramatic effect on function. Thus, soluble IL-2R may act as a sink for
IL-2 and suppress immune system activation,41 and soluble
TNF-R may stabilize serum TNF and act as a slow release reservoir
exacerbating systemic inflammation in septic shock.42
Similarly, the release of soluble tie-1 in response to inflammatory
cytokines or VEGF could lead to the modulation of proliferation,
inflammatory responses, or vascular permeability. The identification of
the tie-1 ligand(s) and the tie-1 protease described in this report
will help clarify these issues not only to illuminate the role of tie-1
in endothelial cell biology, but also to place the tie receptor family
into the larger context of vascular physiology.
| |
ACKNOWLEDGMENT |
The authors thank D. Lyons, M. Rosendahl, D. Thomson, and R. Wahl for
their excellent advice regarding ADAM proteases and inhibitors; the
Amgen Protein Synthesis group for their expertise; and T. Burgess, S. Hu, B. Ratzkin, and K. Farrell for continuing support, interest, and
critical review of the manuscript.
| |
FOOTNOTES |
Submitted February 4, 1998; accepted November 6, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rachel Yabkowitz, PhD, Hoechst Marion
Roussel, Inc, Department of Oncology, M/S G203A, Route 202-206, PO Box 6800, Bridgewater, NJ 08807-0800; e-mail:
rachel.yabkowitz{at}hmrag.com.
| |
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ABSTRACT
INTRODUCTION