Critical Care and Respiratory Divisions, Royal Victoria Hospital, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec H3A 1A1, Canada
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ABSTRACT |
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In this study, we assessed
the effects of in vivo hypoxia on the expression of Tie-2 receptors and
angiopoietins in various organs of conscious rats and correlated these
effects with the expression of hypoxia-inducible factor-1 (HIF-1).
RT-PCR and Southern blotting were used to amplify mRNA expression of
angiopoietin-1, -2, and -3, Tie-2, and HIF-1 in tissues of normoxic
and hypoxic (fraction of inspired oxygen of 9-10% for either 12 or 48 h) rats. Hypoxia provoked a decline in angiopoietin-1 mRNA
and Tie-2 mRNA, protein, and phosphorylation levels in the lung, liver,
cerebellum, and heart but not in the kidney and diaphragm. In
comparison, hypoxia raised the levels of angiopoietin-2 mRNA in the
cerebellum and angiopoietin-3 mRNA in the lung, kidney, and diaphragm.
HIF-1
mRNA was abundant in most organs of normoxic rats but was
significantly induced in the kidney and diaphragm of hypoxic
rats. We conclude that in vivo hypoxia exerts inhibitory effects on the
activity of the angiopoietin-1/Tie-2 receptor pathway through reduction of angiopoietin-1 and upregulation of angiopoietin-2 and -3. Induction of angiopoietin-3 in the kidney and diaphragm of hypoxic rats could be
mediated through the HIF-1 transcription factor.
angiogenesis; TEK receptors
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INTRODUCTION |
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REDUCTION IN TISSUE OXYGEN concentration is usually associated with numerous cellular, local, and systemic adaptive responses. Enhanced erythropoiesis represents an example of a systemic adaptive response to hypoxia, whereas cellular adaptations to hypoxia include induction of glycolytic enzymes, enhanced glycolysis, and ATP formation from anaerobic pathways. At the local level, reduction in oxygen supply elicits neovascularization and formation of new blood vessels. Formation of new vessels requires a series of events that includes differentiation and migration of endothelial cells, tube formation, and vascular maturation. It has been well established that hypoxia-induced neovascularization is mediated by several growth factors, the most important being vascular endothelial growth factor (VEGF), which binds to two main endothelium-specific tyrosine kinase receptors, VEGF receptor-1 [VEGFR-1, also known as fms-like tyrosine kinase (Flt-1)] and VEGFR-2 [kinase insert domain-containing receptor/fetal liver kinase (Flk)-1] (17). Upregulation of both VEGF and VEGFRs, which has been observed in response to in vitro and in vivo hypoxia, has been attributed in part to the induction of hypoxia-inducible factor-1 (HIF-1) (17).
In addition to VEGFRs, endothelial cells specifically express another family of tyrosine kinase receptors known as Tie-1 and Tie-2 (tyrosine kinases that contain immunoglobulin-like loops and epidermal growth factor-similar domains) (21). Although the functional significance of Tie-1 receptors and the nature of their ligands remain under investigation, Tie-2 receptors are believed to play an important role in angiogenesis and stabilization of vascular integrity (22). Angiopoietin (Ang)-1 and Ang-2 have recently been identified as ligands for Tie-2 receptors (4, 14). Although both of these ligands bind to Tie-2 receptors with a similar affinity, Ang-1 causes Tie-2 receptor autophosphorylation and activation, whereas Ang-2 antagonizes this effect (4, 14). Targeted disruption of Ang-1 or Tie-2 genes causes embryonic lethality as a result of defective modeling of primitive vascular plexus and lack of perivascular cells (4, 22). These results suggest that the Ang-1/Tie-2 receptor pathway is essential for the maturation of blood vessels during embryonic development. Overexpression of Ang-2-produced phenotypic manifestation is very similar to that observed in Ang-1 or Tie-2 knockout animals (14). The functional significance of angiopoietins and Tie-2 receptors in sprouting angiogenesis in the adult vasculature is still being investigated. Recent evidence indicates that Ang-1 promotes endothelial cell network formation, migration, and sprouting; prevents endothelial cell apoptosis; and attenuates vascular leakage (10, 11, 20, 29, 33). Little is known about the functional significance of Ang-2 expression in mature vasculature. Recent studies (14, 25, 28) indicate that Ang-2 is abundantly expressed in the endothelial cells of sprouting microvessels of healthy mature vasculatures and vasculatures of solid tumors and that increased Ang-2 expression is accompanied by induction of VEGF expression. However, the observation that Ang-2 production is also elevated at the sites of vascular regression where VEGF is not abundant led to the hypothesis that the role of Ang-2 in sprouting angiogenesis in mature vasculature is highly dependent on the presence of other angiogenesis factors, particularly VEGF (8, 9). In the presence of VEGF, Ang-2, by antagonizing the stabilizing or antiapoptotic effects of Ang-1 on endothelial cells, promotes migration, proliferation, and sprouting of these cells. By comparison, in the absence of VEGF, antagonism of the effects of Ang-1 by Ang-2 will likely lead to endothelial cell apoptosis and vascular regression (8, 9).
Two new members of the angiopoietin family have recently been discovered: Ang-3, which is expressed in mice and appears to function as an antagonist to Ang-1 activation of the Tie-2 receptor in a fashion similar to Ang-2, and Ang-4, the human counterpart of Ang-3, which functions as an agonist of Tie-2 receptors (32). The nature of the cells responsible for Ang-3 and Ang-4 production and their biological significance remain unknown.
Little is known about the regulation of angiopoietins in response to hypoxia, particularly under in vivo conditions. In vitro experiments revealed that hypoxia elicits significant upregulation of Ang-2 expression in endothelial cells, with no alterations in Ang-1 and Tie-2 receptor expression (15, 18). By comparison, hypoxia produced downregulation of Ang-1 mRNA expression in rat glioma cells (6). In this study, we evaluated the influence of in vivo hypoxia on endogenous activity of the Ang-1/Tie-2 receptor pathway and on mRNA expression of Ang-1, Ang-2, Ang-3, and Tie-2 receptors in various rat tissues. We also assessed whether changes in angiopoietin and Tie-2 receptor expression correlate with the induction of HIF-1 in various hypoxic organs. We hypothesized, on the basis of the proposal by Holash and colleagues (8, 9), that in vivo hypoxia, which is a fundamental angiogenic stimulus and a powerful inducer of VEGF expression, elicits significant inhibition of the Ang-1/Tie-2 receptor pathway in various organs and that this inhibition is mediated by both inhibition of Ang-1 and Tie-2 expression and elevation of the endogenous antagonist (Ang-2). We also propose that in certain organs, Ang-3 rather than Ang-2 may function as the primary endogenous antagonist for the Ang-1/Tie-2 receptor system, both under normoxic conditions and in response to hypoxia.
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MATERIALS AND METHODS |
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Animal preparation.
The Animal Ethical Committee of McGill University approved this
project. Male Sprague-Dawley rats (250-350 g) were placed in a
sealed Plexiglas chamber (12 × 7 × 5 in.). In two groups of
rats, hypoxia was induced by mixing the outflow of two independently controlled cylinders, which contained either pressurized air or N2, inside the chamber. Gas samples were periodically drawn
from the chamber for analysis with a blood gas analyzer (model 995; AVL
Instruments, Graz, Austria) to ensure that appropriate ambient PO2 was maintained. A total flow rate of 6 l/min prevented CO2 accumulation. The normoxic group of
rats (n = 6) was placed in a similar chamber but was
exposed to room air (fraction of inspired oxygen 21%). All rats were
provided with rat chow and water ad libitum. In rats exposed to
hypoxia, the gas inflow consisted of air at a flow rate of 3 l/min and
N2 at 3 l/min, with minor adjustments as needed to maintain
the O2 concentration within the chamber at 9-10%. In
four hypoxic rats, carotid arterial blood was sampled and revealed
arterial PO2 values of 35-39 mmHg. The temperature in the chamber was monitored with a temperature probe (SSTI, Physitep Instrument, Clifton, NJ) and was maintained at the
surrounding room temperature throughout the exposure period. Hypoxic
rats were killed (cervical dislocation) either 12 (n = 6) or 48 (n = 6) h from the start of the experiment.
Various tissues (heart, cerebellum, kidney, liver, diaphragm, and lung)
were quickly excised, flash-frozen in liquid N2, and then
stored at 80°C until further analysis.
RT-PCR and Southern blotting.
Expression of mRNA of the various angiopoietins and Tie-2 receptors was
evaluated with semiquantitative RT-PCR. Total RNA was extracted
from tissue samples following the method described by Chomczynski and
Sacchi (1). Total RNA (1 µg) was reverse transcribed with random hexamers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD).
RT-generated cDNAs encoding Tie-2, Ang-1, Ang-2, Ang-3, HIF-1, and
18S (both as an internal standard and positive control) were amplified
with PCR. RNA with no clear 18S band in the RT-PCR products (35 cycles) was discarded from further studies. Oligonucleotide primers
(synthesized in the McGill University DNA Synthesis Facility) for these
transcripts are listed in Table 1. In
each PCR, 5 µl of RT product (250 ng of total RNA) were used.
The experimental conditions for PCRs were as follows: initial
denaturation at 95°C for 5 min followed by 95°C for 30 s,
55°C for 45 s, 72°C for 1 min, and a final extension reaction
for 10 min. The only exception to these conditions was the use of an
annealing temperature of 65°C during Ang-3 amplification. To use
RT-PCR semiquantitatively, we assessed the relationship between cycle
number and the optical density (OD) of the PCR product by varying the
cycle number and fixing the cDNA concentration (250 ng/tube). Our
preliminary experiments revealed that a total of 27, 39, 40, 37, 35, or
12 cycles produced PCR product intensities within the linear
range of PCR amplification curves for Tie-2, Ang-1, Ang-2, Ang-3,
HIF-1
, and 18S transcripts, respectively. Accordingly, we used the
above-mentioned cycle numbers in the PCRs of normoxic and hypoxic
tissues samples. To quantify PCR products, they were
electrophoretically separated with 1% agarose gels. The gels were then
denatured, cDNAs were transferred to a Hybond-N+ membrane (nylon
membrane), cross-linked ultravioletly (model FB-UVXL-100, Fisher
Scientific), and then hybridized overnight at 30°C with
32P-labeled internal oligonucleotide probes (Table 1).
Membranes were then washed with 6× saline-sodium citrate buffer,
dried, and exposed for 12 h to X-ray film with an intensifying
screen at
80°C. ODs of the DNA bands were scanned with a
densitometer and quantified with SigmaGel software (Jandel Scientific,
San Rafael, CA). To verify the accuracy of the amplified sequence, PCR
products were sequenced in the McGill University DNA Sequencing Facility.
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Immunoblotting.
Frozen tissues were homogenized in 6 volumes (wt/vol) of
homogenization buffer (pH 7.4, 10 mM HEPES buffer, 0.1 mM EDTA, 1 mM
dithiothreitol, 1 mg/ml of phenylmethylsulfonyl fluoride, 0.32 mM
sucrose, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin A).
The crude homogenate was centrifuged at 4°C for 15 min at 5,000 rpm.
The supernatant was then collected and used for immunoblotting. Tissue
proteins (80 µg) were heated for 5 min at 90°C and then loaded on
gradient (4-12%) Tris-glycine SDS-polyacrylamide gels, electrophoretically separated, transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk, and subsequently incubated overnight at 4°C with primary polyclonal anti-Tie-2 receptor antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Specific proteins were then detected with horseradish peroxidase-conjugated anti-rabbit secondary antibody and enhanced chemiluminescence reagents
provided with an ECL kit from Amersham Canada (Oakville, ON). Loading
of equal amounts of proteins was confirmed by stripping the membranes
and reprobing them with an anti--tubulin antibody (Sigma). The blots
were scanned with an imaging densitometer (model GS700, 12-bit
precision and 42-µm resolution; Bio-Rad), and protein band OD was
quantified with SigmaGel software (Jandel Scientific). Predetermined
molecular mass standards (Novex) were used as markers.
Immunoprecipitation. To investigate whether the Tie-2 receptors are tyrosine phosphorylated in normal rat tissues and whether hypoxia influences the degree of tyrosine phosphorylation, we immunoprecipitated Tie-2 receptors and probed them with anti-phosphotyrosine antibodies. Tissue homogenates (500 µg) were incubated with primary polyclonal anti-Tie-2 receptor antibody for 2 h at 4°C. Protein A-agarose conjugates were then added, and the samples were incubated for another hour. After centrifugation, the pellets were washed three times with a buffer containing 125 mM Tris · HCl, pH 8.1, 500 mM NaCl, 0.5% Triton X-100, 10 mM EDTA, and 0.02% NaN3. The final wash was performed with water. Proteins were eluted with electrophoresis sample buffer, and immunoblotting of the supernatant and eluted proteins was undertaken as described in Immunoblotting. Membranes were probed with monoclonal anti-phosphotyrosine antibody (4G10, Upstate Biotechnology, Lake Placid, NY). Proper negative controls included omission of primary antibody and omission of protein A-agarose conjugates.
Data analysis.
Six separate animals were studied in each group, and six different
organs were sampled in each animal. For each organ sample, a final OD
value of mRNA and protein expression was obtained by averaging three
independent RT-PCR and immunoblotting experiments for angiopoietins,
Tie-2, HIF-1, tubulin, and 18S. The ODs of RT-PCR products were
normalized (for 18S) and are expressed in arbitrary units. Within a
group of animals, these final organ OD values were used for statistical
analysis and for calculation of mean values. Differences between
normoxic and hypoxic animals were compared with two-way analysis
of variance, and P values < 0.05 were considered
significant. The correlation coefficients between the ODs of various
transcripts were calculated as the ratios of the sum of the
cross-products about the mean and the square root of the product of the
sum of squares of deviations about the mean (2).
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RESULTS |
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Regulation of mRNA expression.
In normoxic rats, Ang-1 mRNA was detected in most tissues, with
relatively higher expression in the lungs (mean relative units of 3.56)
compared with that in other organs (Fig.
1). Prolonged hypoxia (48 h) produced a
significant reduction of Ang-1 mRNA expression in the lung, liver,
cerebellum, and heart (mean values of 33, 46, 41, and 44%,
respectively, of corresponding normoxic tissue values), whereas no
significant changes were observed in the kidney and diaphragm (Fig. 1).
In rats examined after only 12 h of hypoxia, Ang-1 mRNA in the
lung, kidney, and diaphragm averaged 26, 98, and 68%, respectively, of
normoxic values (P < 0.05 compared with normoxic
animals; Figs. 1 and 2).
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Regulation of protein expression.
Short periods of hypoxia (12 h) elicited a significant reduction in
cerebellar and heart Tie-2 protein expression, whereas protein levels
in other organs remained unchanged (Table
2). However, 48 h of hypoxia was
associated with a substantial decline in lung, liver, cerebellum, and
heart Tie-2 expression, whereas expression in kidney and diaphragm was
not significantly altered (Table 2). Figure
4 shows a representative immunoblot of
lung Tie-2 receptor protein expression, indicating that 12 h of
hypoxia did not affect lung Tie-2 protein expression but that Tie-2
receptor protein levels declined significantly after 48 h of
hypoxia. In addition to the decline in Tie-2 receptor expression,
48 h of hypoxia produced a decline in Tie-2 receptor tyrosine
phosphorylation, even in tissues such as the diaphragm in which Tie-2
receptor protein expression did not change significantly (Fig.
5). Similar declines in Tie-2 receptor
tyrosine phosphorylation were observed in other organs of
hypoxic animals.
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DISCUSSION |
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The main finding of this study is that in vivo hypoxia elicits
differential and tissue-specific alterations in angiopoietins and in
Tie-2 expression. Hypoxia produced a significant decline in Ang-1,
Tie-2 mRNA expression, and Tie-2 protein expression in the lungs,
heart, liver, and cerebellum but not in the kidney and diaphragm.
Tyrosine phosphorylation of Tie-2 receptors, however, declined
significantly in various organs of hypoxic animals. Moreover, Ang-2
mRNA in the cerebellum and Ang-3 mRNA in the lung, kidney, and
diaphragm rose significantly in response to hypoxia. The rise in Ang-3
mRNA expression in hypoxic kidney and diaphragm was associated with a
significant induction of HIF-1 mRNA.
Critique of methodology.
A major criticism that can be leveled against this study is that the
changes in angiopoietin and Tie-2 mRNA expression were quantified with
RT-PCR. We chose this method because our initial attempts to detect
mRNA expression of angiopoietins with Northern blotting or a RNase
protection assay were unsuccessful, presumably because of the
relatively low abundance of these transcripts in rat tissues. Although
RT-PCR has many advantages over RNA blotting methods, quantitative
information is difficult to obtain with RT-PCR because of the enzymatic
nature of the PCR amplification and differences in the efficiency of
the RT process between various tubes (3). To deal with
these deficiencies, we performed several preliminary experiments to
assess the amplification curve of each mRNA transcript by using
different cycle numbers at a total cDNA concentration of 250 ng/tube.
We also used amplified 18S transcript as both an internal standard and
positive control. Our results revealed that a total of 27, 39, 40, 37, 35, or 12 cycles produced PCR product intensities within the linear
range of PCR amplification curves for Tie-2, Ang-1, Ang-2, Ang-3,
HIF-1, and 18S transcripts, respectively. These cycle numbers were
then used to amplify the specific mRNA transcripts in this study.
Another precaution that was employed in this study was the use of
Southern blotting instead of ethidium bromide to detect PCR products.
The use of specific radiolabeled internal oligonucleotides provided us
with linear ranges of radioactivity (and hence OD) and accurate
assessment of the sequence of RT-PCR products. The use of
semiquantitative RT-PCR to evaluate the changes in angiopoietin and
Tie-2 mRNA expression was recently evaluated by Mandriota et al.
(16), who reported an excellent agreement between this
method and the RNase protection assay performed on samples obtained
from cultured endothelial cells and rat and mouse tissues.
Inhibition of Ang-1 and Tie-2 receptor expression. Although the functional importance of the Ang-1/Tie-2 receptor pathway in vasculogenesis and maturation of the embryonic vascular system has been well established (21, 22, 26), the exact role played by this pathway in the mature adult vasculature remains unclear. Recent observations of autophosphorylation of Tie-2 receptors in the in vivo quiescent vasculature of adult animals (34) and the histological detection of Ang-1 protein in blood vessels of mature mammals (10) suggest that the Ang-1/Tie-2 receptor pathway is active in mature vasculatures. Recently published studies (12, 19, 20, 30) have shed more light on the involvement of this pathway in the prevention of vascular leakage, promotion of endothelial cell survival through activation of the phosphatidylinositol 3-kinase/Akt pathway, and stabilization of in vitro endothelial capillary networks. Despite these new findings, the involvement of the Ang-1/Tie-2 receptor pathway in in vivo sprouting angiogenesis remains unclear. Ang-2 has been identified recently as a ligand of Tie-2 receptors, with an affinity similar to that of Ang-1 and an ability to antagonize the influence of Ang-1 on Tie-2 receptor activation (14). In this respect, Ang-2 represents the only known endogenous antagonist of a tyrosine kinase receptor pathway. In adult human tissue, Ang-2 is abundantly expressed in the uterus, ovary, and placenta (14) and has recently been localized at the sites of neovascularization in solid tumors and in areas of vascular regression (25, 28). These new findings prompted Holash and colleagues (8, 9) to propose a new hypothesis regarding the involvement of Ang-2 and the Ang-1/Tie-2 receptor pathway in sprouting angiogenesis in the adult vasculature. They hypothesized that sprouting angiogenesis requires cooperative regulation of both Ang-2 and VEGF. In the absence of VEGF, increased production of Ang-2 in response to proangiogenic stimuli will lead to inhibition of the Ang-1/Tie-2 receptor pathways, eventually causing endothelial cell apoptosis and vessel regression. However, the destabilizing effects of Ang-2 on endothelial cells in the presence of high VEGF levels facilitate endothelial cell migration, proliferation, and network formation. Recent in vitro studies (6) in which cultured cells were exposed to proangiogenic stimuli such as hypoxia support the argument of Holash et al. (8, 9). Indeed, when C6 glioma cells were exposed to hypoxia, Ang-1 mRNA declined significantly (6). In comparison, hypoxia elicited significant augmentation of Ang-2 mRNA expression (15, 18). Whether similar changes in Ang-1 and Ang-2 expression occur in response to in vivo hypoxia is still under investigation.
In this study, we used in vivo hypoxia, a fundamental proangiogenic stimulus that plays an essential role in embryonic development and tumor growth, to evaluate organ-specific regulation of the Ang-1/Tie-2 receptor pathway in various organs of rats. Our results indicate that in vivo hypoxia elicits downregulation of Ang-1 and Tie-2 mRNA expression in an organ-specific fashion. Reduction of Ang-1 and Tie-2 receptor mRNA expression was more pronounced in the lungs, whereas that of the kidney remained unchanged. Despite those differences in mRNA expression of this pathway, autophosphorylation of Tie-2 receptors was attenuated in response to hypoxia in all organs studied, indicating that hypoxia causes functional inhibition of the Ang-1/Tie-2 receptor pathway. Our study also indicates that this inhibition is likely to have been achieved by two different strategies, namely, reduction of Ang-1 production and/or augmentation of endogenous antagonists of Ang-1 (Ang-2 and Ang-3; see below). The mechanisms behind the reduction in Ang-1 and Tie-2 receptor mRNA expression in hypoxic rats have not yet been identified. However, we speculate that reduction in mRNA transcription and/or reduced mRNA stability are involved. Although Tie-2 receptor expression is restricted to endothelial cells, many studies (10, 15, 16) have localized Ang-1 production in mesenchymal and smooth muscle cells but not in endothelial cells. Although Ang-1 and Tie-2 mRNA transcripts are localized in different cell types, we found a significant positive correlation between the changes in Ang-1 and Tie-2 mRNA levels in response to hypoxia. A similar relationship between these two mRNA transcripts has also been reported in lung carcinoma (27), suggesting that the transcription rate and/or stability of mRNA of both Ang-1 and Tie-2 is under the influence of similar but not yet identified regulatory factors. The Tie-2 receptor promoter sequence has been well characterized, and its activity is stimulated by two hypoxia-sensitive transcription factors, HIF-1Regulation of Ang-2 and Ang-3 mRNA expression. Recent studies (15, 18) in cultured endothelial cells suggest that in vitro hypoxia exerts strong stimulatory effects on Ang-2 expression and that these effects are not mediated by VEGF (18). Little is known about the influence of in vivo hypoxia on Ang-2 expression. In this study, we found that the in vivo response of Ang-2 mRNA to hypoxia differed quantitatively and qualitatively among various organs. Whereas Ang-2 mRNA was induced in the cerebellum, hypoxia elicited a significant downregulation of Ang-2 mRNA in the lung, heart, and, to a lesser extent, the diaphragm (Fig. 1). Although the reasons behind these differences among organs have not yet been identified, we propose that Ang-2 functions as an endogenous antagonist of Ang-1 in an organ-specific manner and that Ang-3 may replace Ang-2 in certain organs as the primary endogenous antagonist of Ang-1. This proposal is based on the following observations. First, the Ang-3 protein is capable of antagonizing the stimulatory effect of Ang-1 on Tie-2 receptors in cultured endothelial cells in a fashion similar to that of Ang-2 (32). Second, the relative abundance of Ang-3 mRNA in normoxic rat tissues is the reverse of Ang-2 (Figs. 1 and 2). For instance, Ang-3 mRNA is relatively more plentiful in the liver and cerebellum compared with other organs, whereas Ang-2 mRNA is relatively more abundant in the lung, kidney, and diaphragm. We were unable to detect Ang-3 mRNA in normoxic heart, whereas RT-PCR readily detected Ang-2 mRNA in this organ (Fig. 1). Finally, we found a significant negative correlation (r = 0.61; P < 0.05) between Ang-2 and Ang-3 mRNA levels in various organs of normoxic rats. Third, hypoxia elicited organ-specific changes in Ang-3 mRNA that were opposite to those of Ang-2 mRNA (Fig. 1). For instance, Ang-3 mRNA was induced in the lung, kidney, and diaphragm of hypoxic rats, whereas Ang-2 mRNA expression in these organs either remained unchanged or declined significantly. These observations strongly support our proposal that Ang-3 may serve in certain organs (i.e., lung, kidney, and diaphragm) as the primary inhibitor of the Ang-1/Tie-2 receptor pathways in hypoxic animals.
The factors responsible for the regulation of Ang-3 mRNA in certain organs as opposed to others in hypoxic rats remain to be determined. A recent study (23) suggests that HIF-1 is a very important modulator of transcription of a variety of genes in response to hypoxia. HIF-1Comparison with previous studies. Lin et al. (13) recently studied the temporal changes in angiopoietin mRNA expression in response to focal ischemia-reperfusion in the rat cerebral cortex. These authors described a biphasic rise in Ang-2 mRNA that occurred within 24 h (6.4-fold) and 2 wk (4.6-fold) after ischemia, whereas Ang-1 mRNA rose significantly only after 1 wk of ischemia (13). While our current study was being evaluated for publication, Mandriota et al. (16) published a study in which in vivo hypoxia (6% O2) in rats reportedly caused a greater than sevenfold increase in brain Ang-2 mRNA expression. Interestingly, our finding of an approximate sevenfold rise in cerebellar Ang-2 mRNA expression after 48 h of hypoxia (Fig. 1) is qualitatively and quantitatively in agreement with the results of Mandriota et al. (16) as well as with the results of Lin et al. (13) and strongly supports the notion that brain Ang-2 mRNA is elevated within relatively short periods (12-24 h) of tissue hypoxia. However, the decline in cerebellar Ang-1 and Tie-2 mRNA expression observed in hypoxic animals in our study was not observed in the two above-mentioned studies. We speculate that this minor contradiction in the responses of Ang-1 and Tie-2 expression to hypoxia is attributable to regional differences among rat organs and to a divergence in the degree of tissue hypoxia in the three studies. We should point out that our study extends the studies of Mandriota et al. (16) and Lin et al. (13) in three aspects. First, unlike these two studies in which the functional status of the Ang-1/Tie-2 receptor pathway was not assessed, our study indicates for the first time that hypoxia results in a significant reduction of autophosphorylation of Tie-2 receptors in various organs, suggesting that this receptor pathway is inhibited by hypoxia. Second, we also found that the influence of in vivo hypoxia on Ang-2 mRNA expression is organ specific to the extent that certain organs such as the lung, heart, and diaphragm could even reduce their expression of Ang-2 mRNA in response to hypoxia (Fig. 1). Third, our study indicates for the first time that Ang-3 mRNA expression is also sensitive to tissue hypoxia and is regulated in an organ-specific fashion opposite to that of Ang-2. We propose, on the basis of these findings, that Ang-3 may replace Ang-2 as the primary endogenous antagonist of Ang-1 in certain organs both under normoxic conditions and in response to hypoxia.
In summary, our study indicates that in vivo hypoxia is associated with a reduction in tissue Ang-1 and Tie-2 receptor expression as well as a decline in Tie-2 autophosphorylation. Hypoxia, by comparison, elicits significant tissue-specific changes in Ang-2 and Ang-3 mRNA expression. The fact that these changes in angiopoietin expression and Tie-2 receptor activation occurred at time points (12 and 48 h) that preceded the formation of new blood vessels is supportive of the proposal that hypoxia-induced neovascularization requires an initial inhibition of the Ang-1/Tie-2 receptor pathway to allow VEGF to promote endothelial cell proliferation and migration. ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to J. Nicolac for technical expertise.
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FOOTNOTES |
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This study was funded by grants from the Heart and Stroke Foundation of Canada and the Canadian Institute of Health Research.
Address for reprint requests and other correspondence: S. Hussain, Rm. L3.05, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada (E-mail: sabah.hussain{at}muhc.mcgill.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2000; accepted in final form 9 April 2001.
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