© 2000 American Heart Association, Inc.
Cellular Biology |
Tie2 Receptor Expression Is Stimulated by Hypoxia and Proinflammatory Cytokines in Human Endothelial Cells
From the Departments of Nephrology and Medical Intensive Care (C.W., J.S.J., U.F., K.-U.E.), Neonatology (P.K.), Physiology (K.D.W.), and Anatomy (S.B.), Charité, Humboldt University Berlin, and Department of Cardiology (M.G.), German Heart Center, Berlin, Germany.
Correspondence to K.-U. Eckardt, MD, Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail kai-uwe.eckardt{at}charite.de
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Abstract |
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Abstract—The tyrosine kinase receptor Tie2 (also known as Tek) plays an important role in the development of the embryonic vasculature and persists in adult endothelial cells (ECs). Tie2 was shown to be upregulated in tumors and skin wounds, and its ligands angiopoietin-1 and -2, although they are not directly mitogenic, modulate neovascularization. To gain further insight into the regulation of Tie2, we have studied the effect of hypoxia and inflammatory cytokines, two conditions frequently associated with neoangiogenic processes, on Tie2 expression in human ECs. Exposure to 1% O2 led to a time-dependent significant rise of Tie2 protein levels in human coronary microvascular endothelial cells (HCMECs) and dermal microvascular ECs (HMEC-1) (3.2- and 2.5-fold within 24 hours), which was reversible after reoxygenation, and induced a less marked increase in human umbilical vein ECs (HUVECs; 1.7-fold). Hypoxia-conditioned medium and D-deoxyglucose did not change Tie2 expression, but desferrioxamine and cobalt, which are known to mimic hypoxia-sensing mechanisms, induced Tie2 at ambient oxygen tensions. Tumor necrosis factor-
induced Tie2 in a
time- and dose-dependent fashion in all 3 EC types (HUVEC, 2.3-fold;
HMEC-1, 2.8-fold; and HCMEC, 3.0-fold; 10 ng/mL, 24 hours). Enhanced
expression was also found after exposure to interleukin-1ß (1 ng/mL).
Changes in Tie2 protein levels were paralleled by changes in mRNA
expression. In accordance with these in vitro findings,
immunohistochemistry revealed focal upregulation of Tie2 in capillaries
at the border of infarcted human and rat myocardium. In
conclusion, the data show that hypoxia and inflammatory
cytokines upregulate Tie2, which may contribute to the
angiogenic response in ischemic tissues.
Key Words: receptor, tyrosine kinase • Tie2 • hypoxia • cytokines • endothelium
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Introduction |
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Endothelial receptor tyrosine kinases (RTKs) play a central role in establishment, maintenance, and structural adaptation of the vasculature.1 2 On the basis of their structure, the 5 RTKs so far identified can be divided into 2 families. One consists of the RTKs Flk-1 and Flt-1, which, together with their cognate ligand vascular endothelial growth factor (VEGF), are required for blood vessel development during both embryogenesis and conditions of postnatal neovascularization.3 4
Two structurally related tyrosine kinases with immunoglobulin and epidermal growth factor homology domains (Tie1 and Tie2) have more recently been described and constitute a second group of RTKs restricted mainly to endothelial cells (ECs) and their progenitors (for review, see References 2 and 5 ). Although signaling partners for Tie1 are yet unknown, angiopoietin (Ang)-1 and -2 have been identified as ligands of the Tie2 receptor.6 7 During embryonic development, the expression of Tie2 is transcriptionally activated in EC precursors and maintained throughout embryonic endothelium. Unlike the VEGF receptor system, Tie receptors are not required for vasculogenesis but appear essential to support functions of the more mature endothelium.1 2 5 Tie2–/– mice lack proper hierarchical organization of the vasculature and die during embryogenesis.8 9 Although the expression of Tie2 is downregulated postnatally, it persists in quiescent adult ECs, and tyrosyl phosphorylation in the normal vasculature suggests an active role in the maintenance of blood vessels.10 In addition, Tie2 is upregulated in capillaries during neovascularization processes, including skin wounds10 and tumors.11 12
Although the Tie2 ligands Ang-1 and Ang-2 do not produce a mitogenic response in cultured ECs,6 they were shown to modulate VEGF-induced postnatal neovascularization.13 Blocking Tie2 activation using recombinant soluble receptor antagonists prevents tumor growth,14 which supports an important role of Tie2 in tumor angiogenesis. Transcriptional regulatory regions of the Tie2 gene that drive angioblast- and endothelium-specific expression have been characterized.15 16 17 However, despite growing evidence of the functional relevance of Tie2, very little is known about the signals regulating the expression of this receptor.
A system of hypoxia-inducible transcriptional gene activation, which operates through the accumulation of hypoxia-inducible transcription factors (HIFs), has been found to control the activity of several growth factors, including VEGF (for review, see References 18 and 19 ). Transfection of 293 cells with an expression vector for the hypoxia-inducible transcription factor endothelial PAS protein-1 (EPAS-1, also known as HIF-2)19 stimulated transcription from a reporter gene composed of the promotor and intron 1 enhancer of the Tie2 gene.20 Although this observation raises the intriguing possibility that EPAS-1 or related factors mediate hypoxia-induced activation of the Tie2 gene, this hypothesis has not yet been confirmed.21 22
Apart from hypoxia, the release of inflammatory mediators is a
further characteristic of tissues undergoing neovascularization, such
as wounds, infarctions, or tumors.23 Cytokines,
including tumor necrosis factor- (TNF-) and interleukin-1ß
(IL-1ß), promote angiogenesis in animal models, although they
do not directly stimulate EC growth.24 TNF- and IL-1ß
were recently reported to downregulate Ang-1,25 which
provided the first evidence for their influence on the Tie2 receptor
pathway.
To further explore mechanisms of Tie2 regulation, we have studied its
expression in bovine aortic and human umbilical vein and small-vessel
ECs, including primary cultures of cardiac microvascular ECs, and
report here that hypoxia, TNF-, and IL-1ß upregulate Tie2
in human ECs in a time-dependent fashion. We propose that these
mechanisms contribute to the angiogenic response in ischemic
myocardium and other organs.
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Materials and Methods |
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Cell Culture
Human coronary microvascular endothelial cells (HCMECs) were obtained from explanted human hearts as described previously.26 Written consent was obtained from all patients, and tissue sampling was approved by the local ethics committee. Human umbilical vein endothelial cells (HUVECs) were prepared as described.27 Human dermal microvascular ECs transfected with the SV-40 large T promotor (HMEC-1), originally prepared from human neonatal foreskins,28 were kindly provided by R. Bicknell. Bovine aortic endothelial cells (BAECs) that were studied for comparison were gained by treating bovine aortas with collagenase and dispase as described.29
ECs were grown on 0.25% gelatin-coated dishes in medium 199 (HCMECs, HUVECs, and BAECs) or DMEM (HMEC-1), supplemented with 10% (HMEC-1) or 20% (HCMECs, HUVECs, and BAECs) FCS (GIBCO), penicillin (100 U/mL), streptomycin (100 µg/mL), glutamine (2 mmol/L), and 10 ng/mL EC growth factor (HCMECs, HUVECs, and BAECs) or 0.5 ng/mL epidermal growth factor and 1 mg/mL hydrocortisone (HMEC-1).
Cells were routinely incubated at 37°C in 5%
CO2 balance room air. For experiments, the second
to third passage of HUVECs, BAECs and HCMECs was used. Sixteen to 24
hours before exposing cells to different oxygen tensions, as described
previously,27 ECs were washed and incubated with medium
containing reduced FCS concentrations (1% for HMEC-1 and 10% for
HUVECs, BAECs, and HCMECs) and no further supplementation with
endothelial or epidermal growth factor and cortisone.
Immediately before starting the experiments, reduced FCS medium was
changed again. In some experiments, the following agents were added
with this final change of medium: TNF- (1 to 100 ng/mL), IL-1ß (1
ng/mL), desferrioxamine (DFO; 75 µmol/L), cobaltous chloride
(75 µmol/L), D-deoxyglucose (1 to 5 mmol/L),
cycloheximide (10 µg/mL), and actinomycin D (0.1 µg/mL) for 4 to 48
hours.
Cellular viability was tested microscopically using trypan blue exclusion and by measuring the lactate dehydrogenase release using a kit from Sigma.
Myocardial Infarction Model in Rats
The left anterior descending coronary artery was
occluded in male 10- to 15-week-old Wistar rats as
described.30 Animals were euthanized after 3 weeks, and
blocks of heart tissue were frozen in isopentane precooled in liquid
nitrogen and stored at -80°C.
Determination of Tie2 mRNA
Expression of Tie2 mRNA was assessed by RNase protection using a
189-bp fragment corresponding to the human mRNA coding region between
897 and 1085 bp of the Tie2 gene and normalized for expression of U6
small nuclear RNA (see online-only data supplement for details,
available at http://www.circresaha.org).
Immunoblot Analysis
Immunoblotting for Tie2 was performed after
electrophoresis of cell extracts using a polyclonal rabbit antibody
against Tie2 (Santa Cruz Biotechnologies) (see online-only data
supplement for details, available at http://www.circresaha.org).
Immunohistochemistry
Immunohistochemistry for Tie2 and von Willebrand
factor (vWF) was performed on cryosections of rat
myocardium and paraffin-embedded myocardium
from a patient who died 8 days after myocardial infarction (see
online-only data supplement for details, available at
http://www.circresaha.org).
Statistics
Results are expressed as mean±SEM. Mann-Whitney U
test and Kruskal-Wallis test were used for 2-group and multiple-group
comparisons between experimental and control groups. P<0.05
was considered significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Hypoxia, Cobalt, and DFO Stimulate Tie2 Protein Levels
In accordance with previous work,2 5 Tie2 was detectable by immunoblotting under baseline conditions in BAECs and all 3 adult human EC types investigated. To test for the effect of hypoxia on Tie2, EC cultures were incubated at 1% O2 for up to 48 hours. As shown in Figure 1
, exposure to low oxygen as compared
with continued exposure under normoxia resulted in a time-dependent,
significant increase of Tie2 protein levels in HCMECs that was on
average 3.1- and 3.9-fold after 24 and 48 hours, respectively. Hypoxic
exposure of different human EC types revealed a slightly less
pronounced increase in HMEC-1 and no statistically significant increase
in HUVECs (Figure 2A). In contrast to the
results with human ECs, hypoxia did not, however, change Tie2
expression in BAECs (Figure 3).
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The hypoxia-induced stimulation in human microvascular ECs was
found to be reversible, as shown in Figure 2C
; Tie2 expression
in HCMECs exposed to 1% O2 for 24 hours declined
gradually after reexposure to 20% O2 and
returned to baseline after 24 hours.
In view of the evidence indicating that cobalt and DFO can mimic
hypoxic signaling in other systems,18 19 cells were also
incubated in the presence of 75 µmol/L cobalt or DFO for 24
hours at 20% O2. As illustrated on
representative blots in Figure 2B (lanes 5 and
6), both DFO and cobalt led to a stimulation of Tie2 protein expression
in HCEMCs and HMEC-1 that was similar to the hypoxic response and on
average 2.3±0.4- and 2.1±0.3-fold (DFO) and 2.6±0.4- and
2.3±0.3-fold (cobalt) in the 2 microvascular EC types, respectively
(mean±SE; n=3; P<0.05). In HUVECs, similar to
hypoxia, both DFO and cobalt led to a less marked increase in
Tie2 protein that was not statistically significant (DFO,
1.4±0.2-fold; cobalt, 1.7±0.4-fold; mean±SE, n=3). In BAECs, DFO and
cobalt did not change Tie2 levels (Figure 3).
To further investigate whether the effect of hypoxia on Tie2 expression is a direct cellular one or is mediated by substances released into the medium, HMEC-1 were incubated for 24 hours at 20% O2 in cell culture medium harvested from cells exposed to 1% O2 for 24 hours. This exposure to hypoxia-conditioned medium did not change Tie2 protein levels (data not shown).
We also studied the effect of the antimetabolite D-deoxyglucose to assess the influence of substrate deprivation during hypoxia. Substrate deprivation, mimicked by addition of 1 to 5 mmol/L D-deoxyglucose to HMEC-1 incubated at 20% O2, did not affect Tie2 protein levels (data not shown).
TNF- and IL-1ß Stimulate Tie2 Protein Levels
To investigate the effect of proinflammatory cytokines on
Tie2 expression, cells were incubated in the presence of 1 to 100 ng/mL
TNF- under normoxic standard cell culture conditions. All 3 human EC
types responded to TNF- with an increase in Tie2 protein levels
(Figure 4). As shown in Figure 4B, the time course of TNF- stimulation resembled the hypoxic response,
with a slight increase detectable after 4 hours and a further increase
until 24 hours. Further experiments revealed that the stimulation by
TNF- was dose-dependent; whereas 1 ng/mL enhanced Tie2 signals in
some but not all experiments, 10 ng/mL led to a significant increase in
all 3 EC types (Figures 4A and 4C). As illustrated in Figure 4D, exposure to IL-1ß (1 ng/mL) also increased Tie2 levels. In
BAECs, in contrast to human ECs, Tie2 levels were not enhanced by
TNF- (Figure 3).
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Hypoxia- and Cytokine-Induced Increases in Tie2
Protein Expression Are Paralleled by Changes at the mRNA
Level
Figure 5B shows an
autoradiogram of a representative RNase
protection assay, and Figure 5A summarizes the results of RNA
analysis that were performed to test for changes in Tie2 gene
expression in response to cytokines, hypoxia, and
cobalt. Hypoxia, cobalt, and TNF- increased steady-state
mRNA levels of Tie2 in human ECs, and changes at the mRNA level roughly
paralleled the observed changes in protein expression, although
they were somewhat less marked. The protein synthesis
inhibitor cycloheximide had no significant effect on
hypoxia-induced Tie2 mRNA levels (Figure 6B).
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Addition of actinomycin D (0.1 µg/mL) abrogated the induction of Tie2
mRNA (Figure 6B) and protein (Figure 6A) during a
subsequent incubation at 1% O2 or at 20%
O2 in the presence of TNF-. Lactate
dehydrogenase release in cells treated with actinomycin D or
cycloheximide increased from 15% to 30% and 24% (normoxia) and from
29% to 38% and 31% (hypoxia), respectively.
Stimulation of Tie2 by TNF- and Hypoxia Is Not
Additive
To test for a potential interaction of cytokines and
hypoxia on Tie2 regulation, human ECs were exposed to 1%
O2 in the presence of TNF-. Although in some
experiments the presence of TNF- tended to enhance Tie2 mRNA
expression under hypoxic conditions (lane 4 versus lane 2 in Figure 7B), there was overall no
consistent and significant additive effect of hypoxia
and 1 ng/mL TNF- (Figure 7A) or 10 ng/mL TNF- (data not
shown). Similar results were obtained for protein expression (data not
shown).
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Tie2 Expression Is Focally Enhanced After Myocardial Infarction
In Vivo
To address the question of an in vivo correlate of the observed
changes in Tie2 in response to hypoxia and cytokines,
we have investigated Tie2 expression by immunohistochemistry in rat and
human heart tissue after myocardial infarction. In accordance with
previous work,10 31 Tie2 was detectable in
arterial and venous vessels of different diameter
throughout normal myocardium (Figures 8A, 8C, and 8E), and costaining for vWF
confirmed the endothelial localization in capillaries
and larger vessels (Figure 8A). In peri-infarctious tissue from
rat and human infarcted myocardium, staining for Tie2 was
far less homogenous and focally appeared more intense in the vicinity
of tissue necrosis (Figures 8D and 8F). As expected, these areas
also showed a marked inflammatory cell infiltrate. Costaining for Tie2
and vWF in peri-infarctious rat myocardium (Figures 8G and 8H) also revealed a predominately vascular pattern of
Tie2 expression (Figure 8H), but some isolated, possibly
mononuclear infiltrating cells were also positive.
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Angiogenesis is required for embryonic development, wound healing, and other repair processes. Hypoxia and inflammation are 2 frequent characteristics of tissues undergoing neovascularization, and we show in this study that both are independently capable of inducing the expression of the Ang receptor Tie2. The underlying mechanisms seem to be widely preserved in the human vasculature. Thus, hypoxia and the proinflammatory cytokines TNF-
and IL-1ß tended to
influence Tie2 expression in all 3 types of human ECs investigated,
although the amplitude of modulation in response to hypoxia was
more marked in dermal and cardiac microvascular cells and did not reach
statistical significance in HUVECs (Figure 2). In contrast, the
cytokine response was more uniform in microvascular and
umbilical vein ECs (Figure 4). In all 3 cell types, changes in
Tie2 protein expression roughly paralleled changes at the mRNA
level (Figure 5), indicating that the latter is an important
determinant of the observed regulation.
The effect of hypoxia on Tie2 expression appears to depend,
however, on the origin of the ECs. In previous studies, Oh et
al22 did not find hypoxia to influence Tie2 mRNA
expression in bovine retinal or aortic ECs, and Mandriota and
Pepper21 described a downregulation of Tie2 mRNA to 62%
in bovine adrenal microvascular ECs exposed to hypoxia. In view
of these discrepancies with our results in human ECs, we have also
studied BAECs for comparison. As reported by Oh et al22
and Mandriota and Pepper,21 in these bovine cells we did
not find a hypoxic stimulation (Figure 3). Whether these
differences in response are related to species or reflect differences
in the oxygen sensitivity of different vascular beds remains to be
determined.
The effect of hypoxia observed in human ECs appears to be a
direct cellular one rather than one mediated through auto- or paracrine
signals, because we found no effect of hypoxia-conditioned
medium on Tie2 expression. Several characteristics of this response
were determined that have previously been described for a widely
operative system of cellular oxygen sensing involving the accumulation
of a family of HIFs.18 19 32 As described for several
other genes regulated through this system, the effect of
hypoxia on Tie2 expression appeared not to be due to substrate
deprivation and could be mimicked by the addition of cobalt
and DFO (Figure 2). Both are believed to interfere with the role
of iron in the hypoxia-sensing mechanism, although the
underlying reactions have not been clarified.18 19
Actinomycin D abrogated the hypoxic response, indicating that the
induction is at least partially due to transcriptional activation.
These data complement the findings by Tian et al20 and
Schlaeger et al,16 who reported that overexpression of
EPAS-1/HIF-2 can activate transcription of a Tie2-driven
reporter gene. In contrast, however, to several other genes regulated
by HIF, experiments in the presence of cycloheximide suggested that
hypoxic induction of Tie2 mRNA does not require ongoing protein
synthesis (Figure 6).
With respect to the effects of cytokines on Tie2 expression,
Ristimäki et al25 have reported no change of Tie2
expression on HUVECs after exposure to IL-1ß and TNF- for 6 hours,
which does not necessarily contradict our findings, showing stimulation
of Tie2 in response to more prolonged exposure to these
cytokines (Figure 4). Mandriota and
Pepper21 have previously detected a moderate, 1.6-fold
induction by basic fibroblast growth factor, which is somewhat less
than the response to TNF- and IL-1ß observed in this study.
Interestingly, although the mechanisms by which hypoxia and
TNF- stimulate Tie2 are likely to be dissimilar, we were unable to
demonstrate an additive effect by exposing cells to TNF- under
hypoxic conditions (Figure 7). In ischemic tissues, in
vivo effects of hypoxia and inflammation cannot be dissected in
immunohistochemical studies, and both mechanisms may have contributed
to focal upregulation of Tie2 in peri-infarctious
myocardium, illustrated in Figure 8.
The pathophysiological consequences of upregulation of Tie2 certainly depend on the function of Ang signaling. Although both Ang-1 and Ang-2 bind to Tie2 with high affinity, only Ang-1 causes receptor autophosphorylation in ECs.7 Because of its paracrine expression pattern and the observed defects in genetically altered mice, it has been proposed that Ang-1 maintains vessel structures and influences the assembly of perivascular cells in vascular remodeling.2 5 Recent studies on the stabilizing effects of Ang-1 on the HUVEC network support this notion.33 Because Tie2 phosphorylation by Ang-1 can be inhibited by an excess of Ang-2 and the phenotype of mice overexpressing Ang-2 resembles Ang-1 or Tie2 knockouts,7 Ang-2 is believed to serve as a physiological antagonist for Ang-1.
Interestingly, 2 factors reported here to stimulate Tie2, hypoxia and IL-1ß, have recently been found to downregulate Ang-1,25 34 whereas Ang-2 is stimulated by hypoxia.21 22 In concert, these effects of hypoxia and cytokines on the local balance of Ang-1/Ang-2 may disrupt the interactions between ECs and their microenvironment by inhibiting Ang-1–induced Tie2 signaling and thereby make ECs more responsive to angiogenesis initiators.2 7 13 In this situation, enhancing Tie2 signaling by upregulation of the receptor may in fact counterbalance the proangiogenic stimuli exerted by hypoxia and cytokines. However, somewhat contradicting this hypothesis, Ang-1 but not Ang-2 gene transfer was found to enhance collateral vessel formation in the rabbit ischemic hind limb model,35 and such an effect would be promoted by the upregulation of Tie2 in response to ischemia that is demonstrated in this study. In addition, it is also possible that Ang-1 and Ang-2 activate Tie2 in fundamentally different ways, stimulating different signaling pathways and resulting in different biological responses.36
Further potential interaction has to be considered between the Tie2 and the VEGF pathway. Although Ang-1 and Ang-2 are not directly mitogenic, both augment the formation of neovessels when coadministered with VEGF.13 Hypoxia induces VEGF expression by transcriptional activation via HIF-1 and by post-transcriptional stabilization of its mRNA.4 Several cytokines, including IL-1ß, stimulate VEGF expression in vitro.4 The VEGF receptor Flk-1 is also induced by hypoxia, although possibly indirectly,37 38 and by IL-1ß.25 Furthermore, VEGF induces endothelial Ang-2 mRNA levels.21 Existing evidence therefore suggests that both hypoxia and inflammatory mediators invoke an orchestration of several endothelial growth factors and receptors that may play different, but interacting roles to achieve an adaptation of the vasculature under conditions of impaired perfusion, including wound healing and tumor growth.
The relevance of hypoxia signaling alone for tumor angiogenesis has been demonstrated in experimental models of tumors lacking a functional HIF complex, which are less vascularized and grow significantly slower.39 40 In view of our findings and work showing that inhibition of Tie2 signaling prevents tumor growth,14 it is tempting to speculate that reduced expression of Tie2 may contribute to growth inhibition in HIF-deficient tumors.
Moreover, the question arises whether the effect of hypoxia on Tie2 expression may also be relevant during embryonic development. Mice lacking HIF-1, EPAS (HIF-2), or their heterodimeric binding partner aryl hydrocarbon nuclear receptor translocator (ARNT) die during gestation, indicating that hypoxia is an important determinant of embryonic development.20 41 42 EPAS–/– embryos revealed no evidence for an essential role of this transcription factor in either vasculogenesis or angiogenesis,20 but deficiency of HIF-1 or ARNT leads to impaired vascularization.41 42
In summary, this study has identified hypoxia and inflammatory cytokines as stimuli of Tie2 expression in human ECs. Because both factors are closely associated with active angiogenesis in pathological conditions and developmental vascularization, they may be important regulators of the Ang receptor pathway.
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Acknowledgments |
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This work was supported by university research funds, the German Academic Exchange Service, and the German Research Foundation (DFG, EC 87 3-1). The technical assistance of S. Schulz is gratefully acknowledged.
Received February 28, 2000; revision received July 3, 2000; accepted July 24, 2000.
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