Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado
Submitted 9 June 2005 ; accepted in final form 20 July 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
hypoxia inducible factor-1; pulmonary edema; vascular permeability; vascular endothelial cell growth factor
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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; -actin, Sigma). Membranes were reprobed for
-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
-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 manufacturers 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, -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
-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 manufacturers 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 mRNA.
To determine whether changes in the expression of HIF-1
mRNA are involved in ET-mediated upregulation of VEGF in the lung, we measured by HIF-1
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
was constructed by ligation of a PCR amplified fragment of rat HIF-1
mRNA (bp 19772265, 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 78 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 manufacturers 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 Tukeys posttesting for multiple comparisons between groups. Two group comparisons were made by an unpaired Students t-test. Differences were considered statistically significant for P values < 0.05. Results are expressed as means ± SE unless otherwise noted.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
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.
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 mRNA expression is highly suggestive that ET-driven increases in HIF-1
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
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
levels in the lung. Whereas some previous studies have demonstrated increased HIF-1
protein or activity in the lung at very low oxygen concentrations (40), other studies have been unable to detect increases in lung HIF-1
expression or activity at inhaled oxygen concentrations >6% (34). Similarly, our data suggest that, while HIF-1
likely contributes to increased VEGF transcription in the lung in the setting of moderate hypoxia following recent infection, HIF-1
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
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 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.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |