Differential expression of Tie-2 receptors and angiopoietins in response to in vivo hypoxia in rats

Kefeya Abdulmalek, Fathia Ashur, Nadine Ezer, Fengchun Ye, Sheldon Magder, and Sabah N. A. Hussain

Critical Care and Respiratory Divisions, Royal Victoria Hospital, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec H3A 1A1, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha 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-1alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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-1alpha , 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-1alpha , 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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Table 1.   Molecular sequences and expected lengths of RT-PCR products for angiopoietins, Tie-2 receptors, HIF-1alpha , and 18S

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-alpha -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-1alpha , 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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Changes in angiopoietin (Ang)-1, Ang-2, Ang-3, Tie-2 receptor, and ribosomal 18S mRNA expression as detected by RT-PCR and Southern blotting in various organs of normoxic (N) and hypoxic (12H and 48H) rats. Cereb, cerebellum.



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Fig. 2.   Mean optical densities of Ang-1 (A), Ang-2 (B), Ang-3 (D), and Tie-2 (C) mRNA levels in various organs of normoxic and hypoxic rats. mRNA levels are expressed in relative units (relative to optical density of ribosomal 18S subunit). Note the significant decline in Ang-1 and Tie-2 mRNA in liver, cerebellum, heart, and lung in response to 12 and 48 h of hypoxia. Also note the significant decline in Ang-2 mRNA in heart, lung, and diaphragm (Diaph) as opposed to the significant rise in cerebellum in response to hypoxia. Ang-2 mRNA was not detectable in the liver, whereas Ang-3 mRNA was not detectable in the heart. *P < 0.05 compared with normoxic values.

As in the case of Ang-1, Tie-2 mRNA expression was relatively more abundant in the lung (mean relative units of 3.93) compared with that in other organs (Fig. 1). Prolonged hypoxia (48 h) reduced Tie-2 mRNA expression in the lung, liver, cerebellum, and heart (average of 32, 60, 60, and 58%, respectively; P < 0.05 compared with normoxic rats), whereas that of the kidney and diaphragm remained unchanged (Fig. 2). Similarly, a shorter period of hypoxia (12 h) reduced Tie-2 mRNA in the lungs, with no significant changes in receptor mRNA in the diaphragm and kidney (Fig. 2). There was a significant positive correlation between Tie-2 and Ang-1 mRNA expression in various tissues (r = 0.965; P < 0.001).

Figure 1 also illustrates the changes in Ang-2 mRNA expression in various tissues in normoxic and hypoxic rats. In normoxic tissues, Ang-2 mRNA was relatively more abundant in the lung (mean relative units of 4.3) compared with levels in other organs. Very weak Ang-2 mRNA expression was detected in the cerebellum, whereas no transcript was amplified in the normoxic liver (Fig. 1). After 48 h of hypoxia, Ang-2 mRNA levels rose more than sevenfold in the cerebellum (Fig. 2), whereas those of the heart, lung, and diaphragm declined to 2, 16, and 76%, respectively, of normoxic values (P < 0.05; Fig. 2). By comparison, Ang-2 mRNA expression in the kidney was not influenced by hypoxia (Fig. 2). Decline in lung and diaphragmatic Ang-2 mRNA expression was detected after only 12 h of hypoxia (Fig. 2).

Hypoxia (48 h) elicited a substantial induction of Ang-3 mRNA expression in the lung, kidney, and diaphragm (11-, 14-, and 40-fold rises, respectively; Figs. 1 and 2). By comparison, Ang-3 mRNA expression declined significantly in the liver and cerebellum of hypoxic rats (P < 0.05 compared with normoxia; Figs. 1 and 2). No detectable Ang-3 mRNA transcripts were detected in the hearts of normoxic and hypoxic animals. Figure 3 illustrates the changes in HIF-1alpha mRNA expression in various tissues. Hypoxia elicited a significant rise in HIF-1alpha mRNA expression in the kidney and the diaphragm, especially after 48 h of hypoxia (317 and 299%, respectively, of normoxic values), whereas no alterations in HIF-1alpha expression were observed in other organs of hypoxic rats (Fig. 3).


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Fig. 3.   Changes in hypoxia-inducible factor (HIF)-1alpha and ribosomal 18S mRNA expression as detected by RT-PCR in various organs of normoxic and hypoxic rats. Note the increase in HIF-1alpha mRNA in the kidney and diaphragm of hypoxic rats.

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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Table 2.   Effects of hypoxia on Tie-2 receptor protein expression



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Fig. 4.   Representative immunoblot of Tie-2 protein expression in the lungs of normoxic and hypoxic rats. Three samples are shown in each experimental group. Note that 48 but not 12 h of hypoxia resulted in a significant decline in Tie-2 protein expression. Nos. at right, molecular mass.



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Fig. 5.   Influence of 48 h of hypoxia on Tie-2 receptor tyrosine autophosphorylation in the diaphragms of rats. A: immunoblot of Tie-2 receptor protein in 2 normoxic and 2 hypoxic (48H) diaphragmatic samples. Tie-2 receptors were immunoprecipitated with anti (alpha )-Tie-2 receptor antibody, and the immunoprecipitates (P) were then probed with anti-phosphotyrosine antibody (P-Tyr; B). Note that even though hypoxia resulted in small changes in Tie-2 protein level (A), tyrosine phosphorylation of Tie-2 receptors declined significantly in hypoxic diaphragms. Nos. at left, molecular mass.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-1alpha 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-1alpha , 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-1alpha and endothelial PAS domain protein 1 (5, 31). We believe that activation of these two factors during hypoxia is not a likely explanation of our observations because these factors enhance rather than inhibit the Tie-2 transcription rate. A 48-bp portion of murine Tie-2 receptor promoter has recently been found to contain binding elements for a transcription repressor. This could be involved in the downregulation of Tie-2 receptors under hypoxic conditions (7). Clearly, more research is needed to elucidate the molecular mechanisms responsible for inhibition of Ang-1 and Tie-2 mRNA expression in response to hypoxia.

Regulation 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-1alpha is known to bind a hypoxia-responsive element in the 5'-flanking region of the VEGF genes and promotes significant induction of VEGF mRNA transcription in response to in vivo or in vitro hypoxia (24). Our current study indicates that 12 or 48 h of hypoxia elicited a significant increase in HIF-1alpha mRNA expression, mainly in the kidney and diaphragm (Fig. 3). Interestingly, these two organs showed a significant induction of Ang-3 mRNA in response to hypoxia, suggesting that HIF-1 may be involved in the regulation of Ang-3 mRNA transcription. We should emphasize, however, that tissue levels of HIF-1 are regulated mainly through protein stabilization and prevention of HIF-1alpha protein degradation rather than through transcription (23). Thus measurement of HIF-1alpha protein levels and the binding of this factor to Ang-3 promoters are better indexes of the importance of HIF-1alpha in the regulation of Ang-3 mRNA expression in response to hypoxia than measurement of HIF-1alpha mRNA expression.

As to the factors regulating Ang-2 expression, Mandriota et al. (16) recently explored the mechanisms responsible for Ang-2 mRNA expression in cultured endothelial cells. These authors found that the flavoprotein oxidoreductase inhibitor diphenyleneiodonium and the related compound iodonium diphenyl mimicked the induction of Ang-2 mRNA by hypoxia in cultured endothelial cells. By comparison, these compounds had no effect on VEGF expression in normoxic cells. Cycloheximide, which inhibits the activity of HIF-1alpha activity (23), reduced basal Ang-2 mRNA expression in cultured endothelial cells, whereas a significant induction of VEGF mRNA was noticed in these cells in response to cycloheximide (16). Finally, removal of serum for 15 h from the culture medium resulted in a twofold increase in Ang-2 mRNA levels, whereas VEGF mRNA expression was reduced by ~30% in response to serum withdrawal. These results suggest that Ang-2 expression is regulated in a different fashion than VEGF and that Ang-2 expression is under the control of diphenyleneiodonium-sensitive oxidoreductase flavoproteins. Clearly, further research is warranted to identify the molecular regulators of Ang-2 expression under normoxic and hypoxic conditions.

Comparison 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

We are grateful to J. Nicolac for technical expertise.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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