Endothelin-mediated increases in lung VEGF content promote vascular leak in young rats exposed to viral infection and hypoxia

Todd C. Carpenter, Stacey Schomberg, and Kurt R. Stenmark

Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado

Submitted 9 June 2005 ; accepted in final form 20 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Viral respiratory infections increase the susceptibility of young animals to hypoxia-induced pulmonary edema formation. Previous work has shown that increased lung levels of endothelin (ET) contribute to this effect, though the mechanisms by which ET promotes vascular leak remain uncertain. Both in vitro and in vivo evidence suggests that ET can upregulate the production of VEGF, which is known to increase vascular permeability. We hypothesized that increases in lung ET promote increases in lung VEGF, which in turn increases vascular leak in the lung. Weanling rats were exposed to moderate hypoxia for 24 h while recovering from a mild viral respiratory infection, to hypoxia alone, or to viral infection alone. Lung VEGF mRNA and protein content were measured by RT-PCR and Western blotting, respectively. Animals exposed to hypoxia + virus demonstrated significant increases in lung VEGF mRNA and protein content. Immunohistochemical studies showed increased VEGF expression in alveolar septa and small pulmonary vessels in those animals. ET receptor blockade with bosentan prevented this increase in lung VEGF content, suggesting that ET promotes VEGF accumulation in the lung in this setting. Animals exposed to hypoxia + virus also demonstrated substantial increases in lung albumin extravasation, and those increases were blocked by both ET receptor blockade and VEGF antagonism. These findings suggest that ET-driven increases in lung VEGF content can contribute to the formation of pulmonary edema.

hypoxia inducible factor-1; pulmonary edema; vascular permeability; vascular endothelial cell growth factor


PULMONARY EDEMA, THE LEAKAGE of vascular fluids and proteins into the air spaces, is a component of many serious illnesses. Hypoxia can lead to pulmonary edema formation in some species, as occurs in the development of high altitude pulmonary edema (HAPE) in humans, and hypoxia may also exaggerate the effects of a prior inflammatory insult to the lung. For example, both viral respiratory infections and endotoxin exposure can increase the susceptibility of animals to hypoxia-induced pulmonary edema formation (6, 27). In the case of viral respiratory infection, previous work has demonstrated that those effects are mediated in large part via the effects of increased levels of endothelin (ET) in the lung.

ET is a peptide that is widely expressed in the lung and that has been implicated in the pathogenesis of not only HAPE but also acute lung injury (9, 11, 21, 31). ET antagonism has been shown to ameliorate pulmonary edema formation in several animal models, including oleic acid lung injury and endotoxin-associated lung injury as well as the combination of recent viral infection and exposure to moderate hypoxia (6, 13, 30). The mechanisms by which ET promotes pulmonary edema formation, however, remain uncertain. Several in vitro studies have suggested that ET can act in some cell types to promote the production of vascular endothelial cell growth factor (VEGF) (24, 28, 33). VEGF is well known to increase vascular permeability and has also been implicated in both experimental and clinical acute lung injuries (2, 8, 18, 25, 36). Recent studies using the ET-B receptor-deficient rat, an animal characterized by elevated levels of ET in the lung, demonstrated an exaggerated vascular leak in response to hypoxia produced via ET-mediated induction of lung VEGF expression and subsequent increases in lung vascular permeability (4).

We sought to determine whether a similar effect could be demonstrated in the setting of increased lung levels of ET in a genetically unaltered animal. We studied this question in normal weanling rats exposed to moderate hypoxia while recovering from a mild viral respiratory infection, a setting that we have previously demonstrated leads to increased pulmonary vascular leak and increased lung ET levels. VEGF and preproendothelin (ppET) mRNA levels were measured by RT-PCR, and VEGF, VEGF receptor (R)-1, VEGFR-2, and hypoxia-inducible factor (HIF)-1{alpha} protein levels were measured by Western blotting and gel mobility shift assay. Pulmonary vascular leak was measured as Evans blue-labeled albumin extravasation, and the roles of ET and VEGF in promoting that leak were assessed with ET receptor and VEGF antagonists.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
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Animals. Experimental animals were pathogen-free weanling male Sprague-Dawley rats purchased from a commercial vendor (Harlan Sprague-Dawley, Indianapolis, IN). Animals in the infected experimental groups were inoculated with virus 3–4 days after arrival and then housed in a separate facility from the noninfected animals. All animals were allowed free access to food and water and were subjected to a similar day and night light cycle. Animals were housed at Denver altitude (1,600 m) at all times. The University of Colorado Health Sciences Center Institutional Animal Care and Use Committee approved all procedures and animal use.

Inoculation with Sendai virus. Infection with Sendai virus was performed as previously described (6). Briefly, animals were lightly anesthetized with halothane, and 50 µl of virus stock solution (5 x 105 TCID50) were then instilled intranasally. Successful viral infection was confirmed by serologic testing or by PCR. For serologic testing, plasma obtained at death was tested for seroconversion to Sendai virus with a commercial ELISA kit (Charles River Laboratories, Cambridge, MA). For PCR testing, Sendai virus RNA was amplified from whole lung RNA isolated with a commercial kit (RNEasy Mini, Qiagen). PCR amplification was done by a one-tube RT-PCR system (Invitrogen, Carlsbad, CA) and primers designed from the published sequence of the Sendai virus HN gene. Animals were considered positive for Sendai infection if either serology or PCR testing were positive. No animal in the uninfected test groups tested positive by either serology or PCR (data not shown).

Experimental design. Animals were studied in four experimental groups: normal controls, viral infection alone, hypoxic exposure alone, and viral infection combined with hypoxic exposure. Animals in the hypoxic test groups were exposed to normobaric hypoxia (FIO2 = 0.1) for 24 h. Exposure to hypoxia was timed so that all animals were killed on day 7 postinoculation to compare animals at the same age and time point in the course of their Sendai infection.

In some studies, the combined ETA/ETB receptor antagonist bosentan (Tracleer, Actelion) was used to block the effects of endogenous ET. Bosentan was prepared as a microsuspension in 5% gum arabic (Sigma, St. Louis, MO) at a concentration of 25 mg/ml. Animals received a single oral dose of either gum arabic vehicle or bosentan (150 mg/kg) by gavage feeding 1 h before the start of hypoxic exposure. In other studies, the effect of VEGF was antagonized with the soluble decoy receptor VEGF-Trap (generously provided by Regeneron Pharmaceuticals, Tarrytown, NY) (14, 15). We administered VEGF-Trap as a single dose of 25 mg/kg injected subcutaneously 4 h before exposure to hypoxia was begun.

Measurement of vascular protein leak. We assessed the leakage of protein from the pulmonary vasculature by measuring the accumulation of Evans blue dye in the peripheral lung. After exposure to room air or to 24 h of hypoxia, animals were anesthetized with ketamine (80 mg/kg ip) and xylazine (20 mg/kg ip) and then injected via a tail vein with 30 mg/kg of Evans blue dye in sterile saline. After Evans blue injection, normoxic animals remained in room air for 10 min; hypoxic animals were returned to hypoxia for 10 min. After that time, the heart-lung block was excised, the lungs were perfused free of blood via the pulmonary artery with phosphate-buffered saline, and the extrapulmonary airways and tissues were removed. A portion of the distal right lung was weighed, minced, and then incubated in formamide for 18 h at 37°C to extract the Evans blue dye. The left lung was weighed and then dried in an oven at 55°C, and the wet-to-dry weight ratio from that lung used to estimate the dry weight of the right lung. The extracted dye was quantitated in a spectrophotometer by measurement of absorbance at 620 nm with standards of Evans blue dissolved in formamide, and Evans blue dye extravasation was then expressed as ng Evans blue/mg dry tissue.

Immunohistochemical studies. To determine which cells within the lung were expressing higher levels of VEGF in the hypoxic infected animals, we conducted immunohistochemical studies of VEGF protein expression. Lungs from normoxic control or hypoxia + virus animals were inflation-fixed via the trachea with zinc formalin at 20 cmH2O pressure for 18 h before paraffin-embedding and sectioning. Sections were probed with either a polyclonal antibody specific for VEGF (Labvision, Fremont, CA) or preimmune rabbit serum and a commercial ABC-AP kit (Vector Labs, Burlingame, CA).

Western blotting studies. To determine whether the changes in lung albumin extravasation were associated with changes in lung VEGF, VEGFR-1, or VEGFR-2 protein levels, we studied lung homogenates by standard Western blotting techniques using commercially available antibodies (VEGF, Labvision; VEGFR-1, Labvision; VEGFR-2, Santa Cruz; {beta}-actin, Sigma). Membranes were reprobed for {beta}-actin to confirm equal protein loading and transfer. The resulting images were scanned into a computer and analyzed by densitometry with NIH Image software. Results were expressed as arbitrary units, representing the ratio of VEGF, VEGFR-1, or VEGFR-2 to {beta}-actin in each lane.

RT-PCR studies. The expression of ppET and VEGF mRNAs were assessed by relative RT-PCR. Frozen lung tissue was homogenized in Tri reagent (Sigma), and total RNA was isolated following the manufacturer’s instructions. We accomplished reverse transcription to cDNA by priming 5 µg of total RNA per sample with oligo dT and then using the Superscript II reverse transcriptase kit (Invitrogen, Carlsbad, CA). PCR reactions were performed on 1 µl of cDNA from each sample.

Relative RT-PCR of ppET mRNA was performed as previously described (6). Relative RT-PCR of VEGF mRNA was performed with previously published primers and cycling conditions (3). These primers amplify four splice variant mRNAs for rat (r) VEGF, rVEGF188, rVEGF164, rVEGF144, and rVEGF120. As an internal control for each PCR reaction, {beta}-actin mRNA expression was also assessed by RT-PCR as previously described, with primers that span an intron as a control for genomic DNA contamination of the samples (6). No genomic DNA contamination was found in any of the cDNA samples used in these experiments (data not shown). For each set of primers, an initial set of reactions was performed to confirm that amplification remained in the linear range under the conditions used for these experiments (data not shown). PCR products were loaded onto a 1.5% agarose/tris-acetate-EDTA gel, separated by electrophoresis, and visualized with ethidium bromide. Gels were photographed under UV transillumination with a digital camera, and the images were transferred to a computer for densitometric analysis. Final results were expressed as the ratio of VEGF or ppET PCR product to {beta}-actin PCR product for each sample. All experiments were repeated three times with similar results.

Nuclear extract preparation and gel mobility shift assays. To determine whether the transcription factor HIF-1 could be involved in ET-mediated upregulation of VEGF in the lung, we measured HIF-1 binding activity on lung nuclear extracts by gel mobility shift assay. Nuclear extracts were isolated from the lungs of an additional series of animals (normoxic controls, n = 3; hypoxia + virus, n = 4; hypoxia + virus + bosentan, n = 4). To minimize exposure to room air during nuclear protein isolation, we treated animals as described above with the exception that exposure to normobaric hypoxia was performed in a modified glove box, allowing the samples to be collected and initially processed under hypoxic conditions. After excision of the heart-lung block in hypoxia, peripheral lung tissue was immediately Dounce homogenized under hypoxic conditions in buffer with protease inhibitors with a commercial kit for nuclear protein extraction (NE-PER, Pierce Biotechnology). The remainder of the nuclear protein extraction was then done in room air following the manufacturer’s recommendations. Nuclear extracts were aliquotted and stored at –70°C until assayed.

To assess nuclear HIF-1 binding activity, we performed gel mobility shift assays. Binding reactions were performed in a buffer consisting of 10 mM Tris·HCl, pH 7.4, 50 mM NaCl, 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5 mM DTT, and 70 µg/ml poly(dI-dC). We incubated 5 µg of nuclear extract on ice for 10 min in binding buffer before adding 10 ng of biotinylated HIF-1 probe (Panomics). After the addition of labeled probe, the samples were incubated at room temperature for an additional 30 min. Binding reactions were separated by electrophoresis in a 6% acrylamide DNA retardation gel (Invitrogen, Carlsbad, CA) and then electroblotted to positively charged nylon membranes. Biotinylated DNA/protein complexes were detected with peroxidase-conjugated streptavidin and chemiluminescent substrate followed by exposure to film. We performed control reactions by adding unlabeled competitor HIF-1 probe to the reaction mix, as well as positive control reactions using hypoxia-exposed COS-7 cell extract.

Northern blotting for HIF-1{alpha} mRNA. To determine whether changes in the expression of HIF-1{alpha} mRNA are involved in ET-mediated upregulation of VEGF in the lung, we measured by HIF-1{alpha} mRNA levels Northern blotting. Total lung RNA was denatured with glyoxal, separated on an agarose gel, and transferred to positively charged nylon membranes by downward alkaline transfer using a commercial kit (Ambion, Austin, TX). A digoxigenin-labeled RNA probe specific for rat HIF-1{alpha} was constructed by ligation of a PCR amplified fragment of rat HIF-1{alpha} mRNA (bp 1977–2265, GenBank accession number AF057308) into the pCRII-TOPO vector followed by in vitro transcription. Membranes were probed overnight at 68°C, and hybridized probe was detected with antidigoxigenin antibody and chemiluminescent detection.

Bronchoalveolar lavage fluid VEGF content. VEGF content in the lung is thought to be highly compartmentalized. To determine whether changes in total lung VEGF content were reflected in the amount of VEGF in the air space compartment, bronchoalveolar lavage (BAL) was performed in 16 additional rats divided into the same four experimental groups (n = 4 per group). Tracheal cut downs were performed with the rats under anesthesia with pentobarbital sodium (80 mg/kg ip), and lung lavage was then performed with 9 ml of total of sterile phosphate-buffered saline, lavaged in 3-ml aliquots. Returned lavage fluid was pooled and immediately placed on ice. The volume of lavage fluid recovered from each animal was 7–8 ml. Lavage fluid was centrifuged briefly to remove cells and debris, and the supernatant was aliquotted and frozen at –70°C until assayed. The VEGF content of the supernatant was then measured with a commercial ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.

Statistical analysis. For statistical analysis we used Prism 4.0 (GraphPad Software, Durham, NC) on an Apple Macintosh computer. Comparisons between the four experimental groups were made by a one-way ANOVA with Tukey’s posttesting for multiple comparisons between groups. Two group comparisons were made by an unpaired Student’s t-test. Differences were considered statistically significant for P values < 0.05. Results are expressed as means ± SE unless otherwise noted.


    RESULTS
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Hypoxia following viral infection increases lung ppET mRNA expression. We have previously reported that the combination of recent viral respiratory infection and exposure to moderate hypoxia results in significant increases in lung ET peptide content as well as ppET mRNA expression (6). To confirm those results in this cohort of animals, we assessed lung ppET mRNA expression by relative RT-PCR in normal control (n = 3), virus only (n = 3), hypoxia only (n = 4), and hypoxia + virus (n = 4) animals. The combination of recent viral infection and exposure to hypoxia led to a significant increase in ppET mRNA compared with all other groups (Fig. 1, ANOVA P = 0.04; multiple-comparison posttesting, hypoxia + virus P < 0.03 vs. all other groups).



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Fig. 1. Increased lung preproendothelin (ppET) mRNA expression in animals exposed to viral infection combined with hypoxia. C, control; V, viral infection alone; H, hypoxia alone; HV, viral infection and hypoxia. *P < 0.03 vs. all other groups.

 
Hypoxia following viral infection increases lung VEGF expression. As we have previously reported in a different animal model that elevated lung ET levels can promote increases in lung VEGF content (4), we sought to determine whether the combination of recent viral infection and exposure to moderate hypoxia results in elevated lung VEGF content. The VEGF protein content of lung homogenates from animals in our four experimental groups was measured by Western blotting (Fig. 2). Although viral infection alone did not change lung VEGF expression and exposure to hypoxia alone led to a modest, statistically insignificant increase in VEGF expression, exposure to the combination of viral infection and hypoxia led to a significant increase in lung VEGF content (ANOVA P = 0.004; posttest, P < 0.05 hypoxia + virus vs. all other groups).



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Fig. 2. Increased lung VEGF protein expression in animals exposed to viral infection combined with hypoxia. A: representative Western blot of lung VEGF protein; B: densitometry of VEGF bands normalized to {beta}-actin, expressed as arbitrary units. *P < 0.05 vs. all other groups.

 
To determine whether the increase in lung VEGF protein content seen with the combination of viral infection and hypoxia was due to increased transcription of VEGF mRNA, we assessed expression of VEGF mRNA by relative RT-PCR using primers that amplified four alternatively transcribed VEGF transcripts: VEGF189, VEGF165, VEGF144, and VEGF121. Consistent with the results of the Western blotting studies, we found a significant increase in lung VEGF mRNA expression in the hypoxic infected animals compared with all three other groups (ANOVA P = 0.007; posttest, P < 0.05 hypoxia + virus vs. all others; Fig. 3). The detectable increase in VEGF mRNA expression was limited to the VEGF189 transcript. No differences were found in the expression of other VEGF mRNAs between the four experimental groups.



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Fig. 3. Increased lung VEGF mRNA expression in animals exposed to viral infection combined with hypoxia. A: representative VEGF and {beta}-actin PCR products on agarose gel; B: densitometry of VEGF189 bands normalized to {beta}-actin, expressed as arbitrary units. *P < 0.05 vs. all other groups.

 
To determine which cells in the lung accounted for the increase in lung VEGF content detected in the hypoxic infected animals, we performed immunohistochemical studies of VEGF expression in zinc formalin-fixed paraffin-embedded sections. Under the staining conditions used, normoxic control animals had weak VEGF staining detectable in the bronchiolar epithelium, in alveolar septa and presumed type II pneumocytes, and in the outer medial smooth muscle layer of small and medium-sized pulmonary arteries. As seen in Fig. 4, hypoxic infected animals demonstrated patchy but marked increases in VEGF staining most notably in alveolar septa. Many of the septa in these areas appeared thickened and edematous. Increased VEGF staining was also found in the medial smooth muscle layer of medium-sized pulmonary arteries and in the walls of small vessels unaccompanied by airways that were most consistent in appearance with pulmonary veins. Control sections stained with preimmune serum showed only occasional very weak staining of the bronchial epithelium. Together, these findings suggested that the combination of hypoxia and recent viral infection leads to an increase in VEGF expression in and around the distal pulmonary vasculature.



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Fig. 4. Increased VEGF immunoreactivity in sections of lung from animals exposed to viral infection combined with hypoxia. Note increased VEGF (red) in alveolar septa (arrows) and small pulmonary vessels (arrowhead). Original magnification x200.

 
To determine whether the increases in lung VEGF content were reflected in airway VEGF levels, BAL fluid (BALF) collected from animals in all four experimental groups was assayed for VEGF by ELISA (n = 4 per group). In contrast to our expectations and to our findings by Western blotting of lung homogenate, we found a significant decrease in BALF VEGF content in both the hypoxia and hypoxia + virus groups (50 ± 1 and 50 ± 5 pg/ml, respectively) compared with normal controls (77 ± 7 pg/ml, ANOVA P = 0.05).

To determine whether the changes in lung VEGF content were associated with a change in lung VEGF receptor expression, we also measured VEGFR-1 and VEGFR-2 protein expression by Western blotting of lung homogenates. We found no significant differences among the four experimental groups in either VEGFR-1 (P = 0.12) or VEGFR-2 (P = 0.46) protein expression, though there was a trend toward increased VEGFR-1 expression in the animals exposed to hypoxia alone compared with the other three groups.

Increases in lung VEGF and HIF-1 are mediated by ET in hypoxic infected animals. To determine whether the increases in lung VEGF content were caused by ET, we gave virus-infected animals bosentan to block both the ET-A and ET-B receptors before exposure to hypoxia. After 24 h of hypoxia, animals given bosentan had significantly less VEGF protein in their lungs than did untreated hypoxic infected animals (Fig. 5, P = 0.0006), suggesting that the increased lung ET levels directly contributed to the elevations in lung VEGF content in those animals.



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Fig. 5. ET receptor blockade reduces lung VEGF protein expression in animals exposed to viral infection combined with hypoxia. A: representative Western blot of lung VEGF protein; B: densitometry of VEGF bands normalized to {beta}-actin, expressed as arbitrary units. *P < 0.01 vs. all other groups.

 
VEGF transcription is known to be regulated by the transcription factor HIF-1. ET has been reported to increase VEGF transcription by promoting increased levels of HIF-1 in some cells (33). To determine whether increases in lung ET caused increases in lung HIF-1 activity, HIF-1 binding activity was measured by gel mobility shift assay of lung nuclear extracts from normoxic animals, hypoxic infected animals, and hypoxic infected animals given bosentan before exposure to hypoxia. As shown in Fig. 6, lung nuclear extracts from hypoxic infected animals had significantly more HIF-1 DNA binding activity than extracts from normoxic lungs (ANOVA P = 0.004). In addition, ET receptor blockade with bosentan markedly reduced lung HIF-1 DNA binding activity in hypoxic infected animals (P < 0.01), suggesting that the increase in HIF-1 activity in the hypoxic infected animals was driven at least in part by ET.



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Fig. 6. Increased lung hypoxia-inducible factor (HIF)-1{alpha} mRNA expression and DNA binding activity in animals exposed to viral infection combined with hypoxia is reduced by ET antagonism. A: Northern blot of lung RNA probed for HIF-1{alpha} and graph of densitometry of HIF-1{alpha} bands, expressed as arbitrary units; B: representative gel mobility shift assay showing HIF-1 DNA binding and graph of densitometry of HIF-1 bands, expressed as arbitrary units. *P < 0.01 vs. other groups.

 
To determine whether increased HIF-1{alpha} mRNA expression might contribute to the increase in HIF-1 DNA binding activity, we measured HIF-1{alpha} mRNA expression by Northern blotting. As shown in Fig. 6, significantly more HIF-1{alpha} mRNA was present in the lungs of the hypoxic infected animals than in control lungs (P < 0.01), and ET receptor blockade with bosentan also markedly reduced lung HIF-1{alpha} mRNA expression in the hypoxic infected animals (P < 0.05).

VEGF and ET antagonism reduce lung albumin leak following viral infection and hypoxia. We have previously reported that weanling rats exposed to hypoxia while recovering from a viral respiratory infection demonstrate increased levels of pulmonary vascular protein leak as measured using both radiolabeled and nonradioactive tracer molecules (5, 6). To confirm this finding in this cohort of rats, we measured lung Evans blue-labeled albumin extravasation in our four experimental groups. Similar to the results from our earlier study, compared with normoxic control animals (96 ± 10 ng Evans blue/mg dry lung, n = 4), we found no significant increase in albumin extravasation in animals exposed to viral infection alone (97 ± 8 ng Evans blue/mg dry lung, n = 4) and only a modest but statistically insignificant increase in animals exposed to hypoxia alone (132 ± 14 ng Evans blue/mg dry lung, n = 4). Animals exposed to the combination of recent viral infection and hypoxia (n = 6), however, exhibited a marked increase in lung albumin extravasation compared with all other groups (217 ± 27 ng Evans blue/mg dry lung; ANOVA P = 0.002, multiple-comparison posttest P < 0.05 hypoxia + virus vs. all other groups). We have also previously reported that ET receptor blockade with bosentan reduces lung albumin extravasation in weanling rats exposed to viral infection and hypoxia (6). To confirm this finding, we gave a group (n = 6) of virus-infected animals bosentan before exposure to hypoxia. As predicted, ET antagonism with bosentan led to a significant reduction in lung albumin extravasation (91 ± 12 ng Evans blue/mg dry lung; P = 0.002, Fig. 7). To determine whether the increases noted in lung VEGF content contribute to the increased albumin extravasation seen in the hypoxic infected animals, we injected a cohort (n = 6) of virus-infected animals with the soluble decoy VEGF receptor VEGF-Trap, before exposure to hypoxia. Hypoxic infected animals receiving VEGF-Trap demonstrated a marked reduction in lung albumin extravasation compared with animals not receiving VEGF-Trap (88 ± 10 ng Evans blue/mg dry lung, P = 0.001, Fig. 7), comparable in magnitude to the effect of bosentan. These results suggest that ET-mediated increases in lung VEGF content contribute to increased vascular leak in the animals exposed to viral infection and hypoxia.



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Fig. 7. VEGF antagonism with the soluble decoy receptor VEGF-Trap and ET receptor blockade with bosentan both reduce lung vascular albumin extravasation in hypoxic infected (HV) animals. *P < 0.01 vs. all other groups.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The major finding of this study was that the combination of a recent viral respiratory infection and subacute exposure to moderate hypoxia leads to increased VEGF expression in the lung. The increase in VEGF is mediated in large part by increased lung levels of ET and is accompanied by ET-induced increases in lung HIF-1 mRNA expression and HIF-1 DNA binding activity. Furthermore, the increase in lung VEGF content contributes to pulmonary vascular albumin extravasation, and this vascular leak can be reduced with both ET and VEGF antagonists. These findings suggest that VEGF may contribute to pulmonary edema formation in circumstances associated with elevated lung ET content.

ET has been implicated in the pathogenesis of acute lung injury in a number of models, including lung injury induced by endotoxin, leukotoxin, and oleic acid (13, 16, 30). ET has also been implicated in human HAPE (10, 31) and acute respiratory distress syndrome (ARDS) (11, 21). The mechanisms by which ET contributes to pulmonary edema formation in these settings remain uncertain but may include alterations in vascular reactivity, recruitment of inflammatory cells, or upregulation of other mediators. Previous work using ETB receptor-deficient rats has shown that excess lung ET can contribute to pulmonary edema formation by stimulating increases in lung VEGF content and that VEGF antagonism reduces albumin extravasation without reducing vascular pressures (4). On the basis of those findings, we sought to determine whether other stimuli known to increase lung ET content had similar effects in the lungs of genetically normal animals. We chose to study the combination of viral infection and hypoxia in weanling rats because that combined stimulus has been shown to increase vascular leak in the lung as a result of increased lung ET content (6). Our finding of increases in lung VEGF content in the hypoxic infected animals, which were prevented by an ET receptor antagonist, provides a second animal model in which ET in the lung appears to drive VEGF production and vascular leak. These results not only extend our previous studies in ETB receptor-deficient rats but also are consistent with in vitro data, suggesting that this mechanism may be important in a broader range of settings.

VEGF is well known to increase vascular permeability in a number of vascular beds, and, like ET, VEGF has been implicated in acute lung injury in a variety of experimental settings as well in human ARDS (19, 20, 22, 35, 36). Studies to date, however, have generated conflicting evidence about the precise role VEGF plays in lung injury, suggesting that lung VEGF levels may increase, decrease, or remain unchanged in acute lung injury. Our findings of increased lung VEGF content in the hypoxic infected animals and of reduced vascular leak following VEGF antagonism with VEGF-Trap support the argument that the increased lung levels of VEGF are physiologically relevant and contribute to edema formation. Some of the disparities in previous studies may result from the known compartmentalization of VEGF, leading to different outcomes depending on whether the airspace, plasma, or lung interstitium is sampled (17). Our results support this interpretation as well, showing a clear increase in lung tissue VEGF in the hypoxic infected animals despite a decrease in VEGF levels in lavage fluid. The reasons for the decrease in lavage fluid VEGF are not immediately clear, especially given that widespread alveolar damage is not evident histologically in these animals, though possible explanations might include an altered direction of VEGF secretion by alveolar epithelial cells from luminal to abluminal or increased epithelial permeability leading to a "leak" of VEGF out of the alveoli into the interstitium. At the least, this finding would seem to suggest that sampling the airway alone may not always provide accurate information about important physiological effects of a given mediator in the rest of the lung. In addition, our immunostaining results demonstrated increased expression of VEGF in alveolar septa and in the distal pulmonary vasculature. These results are not only consistent with previous studies of VEGF localization in the normal lung (12) but also suggest that in the hypoxic infected animals VEGF expression is increased in close proximity to the lung microvasculature, where it could act to alter vascular permeability and promote edema formation.

In the hypoxic infected animals, we found upregulation of VEGF in the lung at both the protein and the mRNA levels. Interestingly, the increase in VEGF mRNA expression was largely limited to the VEGF189 transcript. This splice variant has been reported to be the predominant VEGF transcript in the lung in several species, in contrast to extrapulmonary tissues where VEGF165 is generally the most highly expressed isoform (38). Although VEGF189 is thought to be largely a matrix-bound isoform that does not diffuse readily in tissues, it has nonetheless been shown to increase capillary permeability as effectively as does VEGF165 (1). These results suggest, then, that the production of VEGF189 in or near the lung microcirculation contributes substantially to the vascular leak found in the hypoxic infected animals.

To determine whether changes in VEGF receptor expression might contribute to these effects, we assessed VEGFR-1 and VEGFR-2 protein levels in the lung. The lack of change in VEGFR-2 expression is consistent with previous work showing that acute hypoxia does not alter VEGFR-2 expression and that the VEGFR-2 gene does not contain a hypoxia-response element (HRE) in its promoter. VEGFR-1, on the other hand, is a known hypoxia-regulated gene and its promoter does contain an HRE (23). Although our hypoxic animals displayed a tendency toward increased VEGFR-1 expression, that trend was reversed in the setting of a recent viral infection. VEGF receptor upregulation by cytokines has been described (7, 32), as has receptor downregulation by VEGF binding (37). The net zero change in receptor expression in the hypoxic infected lung, then, may represent a balance between hypoxia and/or cytokine-driven receptor upregulation and VEGF-mediated receptor downregulation.

Previous in vitro work has shown that ET can induce VEGF production in several cell types (26, 28), and, at least in ovarian carcinoma cells, this change occurs by stimulating the normoxic induction of the transcription factor HIF-1, which then promotes VEGF transcription (33). We investigated the possibility that this mechanism contributes to the upregulation of VEGF expression in our animals using gel mobility shift assays of HIF-1 binding activity in the lungs. The finding that ET receptor blockade with bosentan not only reduced lung VEGF content but also markedly reduced lung HIF-1 DNA binding activity and HIF-1{alpha} mRNA expression is highly suggestive that ET-driven increases in HIF-1{alpha} expression contribute to the upregulation of VEGF in the lung following viral infection combined with hypoxia. Although these findings may seem surprising given that HIF-1{alpha} is well known to be upregulated by hypoxia alone, there exists a paucity of data showing that moderate hypoxia alone (10% oxygen or greater) substantially increases HIF-1{alpha} levels in the lung. Whereas some previous studies have demonstrated increased HIF-1{alpha} protein or activity in the lung at very low oxygen concentrations (40), other studies have been unable to detect increases in lung HIF-1{alpha} expression or activity at inhaled oxygen concentrations >6% (34). Similarly, our data suggest that, while HIF-1{alpha} likely contributes to increased VEGF transcription in the lung in the setting of moderate hypoxia following recent infection, HIF-1{alpha} itself may be upregulated more strongly by mediators such as ET than by moderate hypoxia per se. This interpretation is consistent with previous work showing a greater upregulation of HIF-1{alpha} in vascular smooth muscle cells by vasoactive mediators such as angiotensin II than by hypoxia (29). Finally, the ET-1 gene itself is known to be regulated by HIF-1, suggesting a possible positive feedback loop in the production of ET and HIF-1. While possible, such a phenomenon seems unlikely given previous work demonstrating that even in hypoxic conditions HIF-1 binding alone is not sufficient to significantly increase ET gene transcription. Instead, a complex of transcription factors including HIF-1 appears to be required for significant induction of ET gene expression (39). Whether the "priming" effect of the viral infection in our system is a result of upregulation of additional transcription factors required for maximal ET or VEGF gene transcription is a subject of ongoing study in our laboratory.

In summary, we have shown that the combination of a mild recent viral infection and moderate hypoxia, a combined stimulus known to increase ET levels in the lung, leads to ET-mediated increases in lung VEGF content and HIF-1 binding activity. The excess VEGF, in turn, leads to increased vascular leak. These results suggest that ET-mediated increases in HIF-1{alpha} and VEGF expression may be an important component of some forms of lung injury and thus may represent potential points of intervention in the process of pulmonary edema formation.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-04484 and HL-07743 (T. C. Carpenter).


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Carpenter, Developmental Lung Biology Lab., Box B-131, Univ. of Colorado School of Medicine, 4200 E. 9th Ave., Denver, CO 80262 (e-mail: todd.carpenter{at}uchsc.edu)

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.


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 REFERENCES
 

  1. Ancelin M, Buteau-Lozano H, Meduri G, Osborne-Pellegrin M, Sordello S, Plouet J, and Perrot-Applanat M. A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus. Proc Natl Acad Sci USA 99: 6023–6028, 2002.[Abstract/Free Full Text]
  2. Becker PM, Alcasabas A, Yu AY, Semenza GL, and Bunton TE. Oxygen-independent upregulation of vascular endothelial growth factor and vascular barrier dysfunction during ventilated pulmonary ischemia in isolated ferret lungs. Am J Respir Cell Mol Biol 22: 272–279, 2000.[Abstract/Free Full Text]
  3. Burchardt M, Burchardt T, Chen MW, Shabsigh A, de la Taille A, Buttyan R, and Shabsigh R. Expression of messenger ribonucleic acid splice variants for vascular endothelial growth factor in the penis of adult rats and humans. Biol Reprod 60: 398–404, 1999.[Abstract/Free Full Text]
  4. Carpenter T, Schomberg S, Steudel W, Ozimek J, Colvin K, Stenmark K, and Ivy DD. Endothelin B receptor deficiency predisposes to pulmonary edema formation via increased lung vascular endothelial cell growth factor expression. Circ Res 93: 456–463, 2003.[Abstract/Free Full Text]
  5. Carpenter TC, Reeves JT, and Durmowicz AG. Viral respiratory infection increases susceptibility of young rats to hypoxia-induced pulmonary edema. J Appl Physiol 84: 1048–1054, 1998.[Abstract/Free Full Text]
  6. Carpenter TC and Stenmark KR. Endothelin receptor blockade decreases lung water in young rats exposed to viral infection and hypoxia. Am J Physiol Lung Cell Mol Physiol 279: L547–L554, 2000.[Abstract/Free Full Text]
  7. Chintalgattu V, Nair DM, and Katwa LC. Cardiac myofibroblasts: a novel source of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR. J Mol Cell Cardiol 35: 277–286, 2003.[CrossRef][ISI][Medline]
  8. Christou H, Yoshida A, Arthur V, Morita T, and Kourembanas S. Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol 18: 768–776, 1998.[Abstract/Free Full Text]
  9. Dobyns EL, Eells PL, Griebel JL, and Abman SH. Elevated plasma endothelin-1 and cytokine levels in children with severe acute respiratory distress syndrome. J Pediatr 135: 246–249, 1999.[ISI][Medline]
  10. Droma Y, Hayano T, Takabayashi Y, Koizumi T, Kubo K, Kobayashi T, and Sekiguchi M. Endothelin-1 and interleukin-8 in high altitude pulmonary oedema. Eur Respir J 9: 1947–1949, 1996.[Abstract/Free Full Text]
  11. Druml W, Steltzer H, Waldhausl W, Lenz K, Hammerle A, Vierhapper H, Gasic S, and Wagner OF. Endothelin-1 in adult respiratory distress syndrome. Am Rev Respir Dis 148: 1169–1173, 1993.[ISI][Medline]
  12. Fehrenbach H, Kasper M, Haase M, Schuh D, and Muller M. Differential immunolocalization of VEGF in rat and human adult lung, and in experimental rat lung fibrosis: light, fluorescence, and electron microscopy. Anat Rec 254: 61–73, 1999.[CrossRef][ISI][Medline]
  13. Guimaraes CL, Trentin PG, and Rae GA. Endothelin ET(B) receptor-mediated mechanisms involved in oleic acid-induced acute lung injury in mice. Clin Sci (Lond) 103, Suppl 48: 340S–344S, 2002.[Medline]
  14. Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, and Rudge JS. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 99: 11393–11398, 2002.[Abstract/Free Full Text]
  15. Huang J, Frischer JS, Serur A, Kadenhe A, Yokoi A, McCrudden KW, New T, O’Toole K, Zabski S, Rudge JS, Holash J, Yancopoulos GD, Yamashiro DJ, and Kandel JJ. Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci USA 100: 7785–7790, 2003.[Abstract/Free Full Text]
  16. Ishizaki T, Shigemori K, Ameshima S, Nakai T, Miyabo S, Hayakawa M, Ozawa T, and Voelkel NF. Protective effects of BQ-123, an ETA receptor antagonist, against leukotoxin-induced injury in rat lungs. Am J Physiol Lung Cell Mol Physiol 271: L459–L463, 1996.[Abstract/Free Full Text]
  17. Kaner RJ and Crystal RG. Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung. Mol Med 7: 240–246, 2001.[ISI][Medline]
  18. Kaner RJ and Crystal RG. Pathogenesis of high altitude pulmonary edema: does alveolar epithelial lining fluid vascular endothelial growth factor exacerbate capillary leak? High Alt Med Biol 5: 399–409, 2004.[CrossRef][ISI][Medline]
  19. Kaner RJ, Ladetto JV, Singh R, Fukuda N, Matthay MA, and Crystal RG. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema. Am J Respir Cell Mol Biol 22: 657–664, 2000.[Abstract/Free Full Text]
  20. Karmpaliotis D, Kosmidou I, Ingenito EP, Hong K, Malhotra A, Sunday ME, and Haley KJ. Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury. Am J Physiol Lung Cell Mol Physiol 283: L585–L595, 2002.[Abstract/Free Full Text]
  21. Langleben D, DeMarchie M, Laporta D, Spanier AH, Schlesinger RD, and Stewart DJ. Endothelin-1 in acute lung injury and the adult respiratory distress syndrome. Am Rev Respir Dis 148: 1646–1650, 1993.[ISI][Medline]
  22. Le Cras TD, Spitzmiller RE, Albertine KH, Greenberg JM, Whitsett JA, and Akeson AL. VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am J Physiol Lung Cell Mol Physiol 287: L134–L142, 2004.[Abstract/Free Full Text]
  23. Marti HH and Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA 95: 15809–15814, 1998.[Abstract/Free Full Text]
  24. Matsuura A, Yamochi W, Hirata K, Kawashima S, and Yokoyama M. Stimulatory interaction between vascular endothelial growth factor and endothelin-1 on each gene expression. Hypertension 32: 89–95, 1998.[Abstract/Free Full Text]
  25. Medford AR, Keen LJ, Bidwell JL, and Millar AB. Vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome. Thorax 60: 244–248, 2005.[Abstract/Free Full Text]
  26. Okuda Y, Tsurumaru K, Suzuki S, Miyauchi T, Asano M, Hong Y, Sone H, Fujita R, Mizutani M, Kawakami Y, Nakajima T, Soma M, Matsuo K, Suzuki H, and Yamashita K. Hypoxia and endothelin-1 induce VEGF production in human vascular smooth muscle cells. Life Sci 63: 477–484, 1998.[CrossRef][ISI][Medline]
  27. Ono S, Westcott JY, Chang SW, and Voelkel NF. Endotoxin priming followed by high altitude causes pulmonary edema in rats. J Appl Physiol 74: 1534–1542, 1993.[Abstract]
  28. Pedram A, Razandi M, Hu RM, and Levin ER. Vasoactive peptides modulate vascular endothelial cell growth factor production and endothelial cell proliferation and invasion. J Biol Chem 272: 17097–17103, 1997.[Abstract/Free Full Text]
  29. Richard DE, Berra E, and Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 275: 26765–26771, 2000.[Abstract/Free Full Text]
  30. Rossi P, Wanecek M, Konrad D, and Oldner A. Tezosentan counteracts endotoxin-induced pulmonary edema and improves gas exchange. Shock 21: 543–548, 2004.[ISI][Medline]
  31. Sartori C, Vollenweider L, Loffler BM, Delabays A, Nicod P, Bartsch P, and Scherrer U. Exaggerated endothelin release in high-altitude pulmonary edema. Circulation 99: 2665–2668, 1999.[Abstract/Free Full Text]
  32. Shih SC, Ju M, Liu N, Mo JR, Ney JJ, and Smith LE. Transforming growth factor beta1 induction of vascular endothelial growth factor receptor 1: mechanism of pericyte-induced vascular survival in vivo. Proc Natl Acad Sci USA 100: 15859–15864, 2003.[Abstract/Free Full Text]
  33. Spinella F, Rosano L, Di Castro V, Natali PG, and Bagnato A. Endothelin-1 induces vascular endothelial growth factor by increasing hypoxia-inducible factor-1alpha in ovarian carcinoma cells. J Biol Chem 277: 27850–27855, 2002.[Abstract/Free Full Text]
  34. Stroka DM, Burkhardt T, Desbaillets I, Wenger RH, Neil DA, Bauer C, Gassmann M, and Candinas D. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J 15: 2445–2453, 2001.[Abstract/Free Full Text]
  35. Thickett DR, Armstrong L, Christie SJ, and Millar AB. Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome. Am J Respir Crit Care Med 164: 1601–1605, 2001.[Abstract/Free Full Text]
  36. Thickett DR, Armstrong L, and Millar AB. A role for vascular endothelial growth factor in acute and resolving lung injury. Am J Respir Crit Care Med 166: 1332–1337, 2002.[Abstract/Free Full Text]
  37. Wang D, Donner DB, and Warren RS. Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF. J Biol Chem 275: 15905–15911, 2000.[Abstract/Free Full Text]
  38. Watkins RH, D’Angio CT, Ryan RM, Patel A, and Maniscalco WM. Differential expression of VEGF mRNA splice variants in newborn and adult hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol 276: L858–L867, 1999.[Abstract/Free Full Text]
  39. Yamashita K, Discher DJ, Hu J, Bishopric NH, and Webster KA. Molecular regulation of the endothelin-1 gene by hypoxia. Contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP. J Biol Chem 276: 12645–12653, 2001.[Abstract/Free Full Text]
  40. Yu AY, Frid MG, Shimoda LA, Wiener CM, Stenmark K, and Semenza GL. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am J Physiol Lung Cell Mol Physiol 275: L818–L826, 1998.[Abstract/Free Full Text]




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