Ann Thorac Surg 2004;77:1751-1755
© 2004 The Society of Thoracic Surgeons
Original article: general thoracic
Upregulated hypoxia-inducible factor-1 DNA binding activity to the vascular endothelial growth factor-A promoter mediates increased vascular permeability in donor lung grafts
Dietmar Abraham, PhDa,
Katharina Krenn, MDa,
Gernot Seebacher, MDb,
Patrick Paulus, MSa,
Walter Klepetko, MDb,
Seyedhossein Aharinejad, MDa,b*
a Laboratory for Cardiovascular Research, Department of Anatomy, University of Vienna, Vienna, Austria
b Department of Cardiothoracic Surgery, University of Vienna, Vienna, Austria
Accepted for publication October 10, 2003.
* Address reprint requests to Dr Aharinejad, Laboratory for Cardiovascular Research, Department of Anatomy, University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria
e-mail: ahas{at}univie.ac.at
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Abstract
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BACKGROUND: Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts.
METHODS: We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs.
RESULTS: While HIF-1
mRNA expression was unchanged, HIF-1ß was downregulated (p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1 and -ß was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p < 0.05).
CONCLUSIONS: These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 mRNA expression did not affect HIF-1 protein levels. Endothelin-1 increases HIF-1 accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.
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Introduction
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Maintenance of homeostatic mechanisms in response to hypoxia is essential for survival. The lung exhibits unique mechanisms for adaptation to conditions of low oxygen tension. For example, acute exposure to hypoxia results in vasoconstriction of pulmonary arteries and increased pulmonary arterial pressure [1]. These changes are designed to correct imbalances between ventilation and perfusion. Several agents, eg, vascular endothelial growth factor (VEGF), platelet-derived growth factor, endothelin (ET)-1, angiotensin-converting enzyme, and nitric oxide have been reported to play a role in mediating the pathophysiologic changes associated with hypoxia [2–4].
In lung transplantation, donor grafts become hypoxic due to blood flow cessation and lack of ventilation. Hypoxia upregulates VEGF gene transcription, and pro-moter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF [5, 6]. Vascular endothelial growth factor is also a major factor in regulating lung vascular permeability [7, 8]. The contribution of VEGF and kinase insert domain protein receptor (KDR) to acute lung injury is well established in animal models [9]; we have shown that VEGF-A expression increases in donor lung grafts associated with ET-1 overexpression and increased tissue fluid content [10, 11]. Recent studies have demonstrated increased HIF-1 and -ß mRNA levels in the lungs of mice exposed to acute hypoxia [12], and others have reported that ET-1 potently stimulates VEGF-A production [13].
Thus we hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in donor lung grafts. In this study, we found unchanged HIF-1 mRNA but upregulated HIF-1 protein expression, and enhanced HIF-1 binding to the HBS of the VEGF promoter in donor lung grafts.
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Material and methods
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Patients
A total of 18 lung donors were included in the study. Multiple biopsies were taken from lung grafts. Pulmonary tissue was excised to reduce the graft volume shortly before transplantation, or from single lung lobes that were preserved but not used for transplantation due to surgical reasons. Grafts were preserved using Perfadex solution [14] and stored on ice until transplanted. The mean ischemia time of the grafts was 5 ± 1 hour. Ten nonsmoking, otherwise healthy, age- and sex-matched patients who were admitted for endoscopic pleurectomy because of repeated spontaneous pneumothorax and gave informed consent to be enrolled served as controls. Biopsies were taken shortly after single-lung ventilation started. In each lung, two biopsies were immediately processed to evaluate wet-to-dry lung weight ratio. Other biopsies were snap-frozen in liquid nitrogen until further processed as described next.
Real-time reverse transcription-polymerase chain reaction
Total RNA was isolated from lung tissue by guanidinium thiocyanate-phenol-chloroform extraction [15]. Complementary DNA (cDNA) was synthesized using avian myeloblastosis virus reverse transcriptase and 2 µg of total RNA primed with oligo dT-primer. The resulting cDNA was used in real-time RT-PCR to monitor gene expression (Light Cycler Instrument; Roche, Mannheim, Germany) as described previously [10, 16]. The specificity of the amplification reaction was determined by melting curve analysis. Standard curves for expression of each gene were generated by serial dilution of known quantities of the respective cDNA gene template. Relative quantification of the signals was performed by normalizing the signals of the different genes with the ß-actin signal. Primers for PCR were designed using Prime software (Genetics Computer Group Package, Madison, WI). The primer pair sequences for the oligonucleotides used are as follows (sense/antisense): HIF-1: 5'-CCTGAGCCTAATAGTCCC-3'/5'-GGTGGCATTAGCAGTAGG-3'; HIF-1ß: 5'-GGTACCAGGAAGAGATGG-3'/5'-GGGAGAATAGCTGTTGGG-3'; ß-Actin: 5'-TGCCATCCTAAAAGCCAC-3'/5'-TCAACTGGTCTCAAGTCAGTG-3'. The annealing temperature used was 60°C. PCR was performed using Hot Start reaction mix (Light Cycler–FastStart DNA Master SYBR Green I; Roche) [16].
Western Blotting
Biopsies were lysed in solubilization buffer (10 mmol/L Tris-Cl, 50 mmol/L NaCl, 1% Triton X-100, 30 mmol/L sodium pyrophosphate, 100 µmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1x Complete EDTA-free protease inhibitor cocktail [Roche]). Tissue lysates (50 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were immunodetected on Hybond C super membrane using chemiluminescence (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK). The blots were probed with monoclonal antibodies against HIF-1 (Alexis, Lausen, Switzerland), HIF-1ß (Neomarkers, Union City, CA), and actin (Santa Cruz Biotechnology, Santa Cruz, CA) before incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech) and exposure to the chemiluminescence substrate. Protein bands were quantified [10, 16] by normalizing the signals of different proteins to the actin signal using Easy plus Win 32 software (Herolab, Wiesloch, Germany).
Electrophoretic mobility shift assay
Biopsies were lysed in solubilization buffer (10 mmol/L Tris-Cl, 50 mmol/L NaCl, 1% Triton X-100, 30 mmol/L sodium pyrophosphate, 100 µmol/L Na3VO4, 1 mmol/L phenylmethyl-sulfonyl fluoride, 1x Complete EDTA-free protease inhibitor cocktail). Insoluble material was removed by centrifugation (15,000 rpm, 10 minutes, 4°C). Then EMSA probes were prepared by labeling a single-stranded oligonucleotide with [
-32P] adenosine triphosphate (ATP; Applied Biosystems, Boston, MA), using a T4 polynucleotide kinase (New England BioLabs Inc, Beverly, MA) and by annealing a complementary single stranded oligonucleotide. Labeled probes were purified using Sephadex-G50 spun columns. The sequence of the EMSA probe was for HIF 5'-CAGTGCATACGTGGGCTCCAACAGGTCCTCTTCC-3' sense, and 5'-GGAAGAGGACCTGTTGGAGCCCACGTATGCACTG-3' antisense. The probes were prepared by mixing 10 µL 2x binding buffer (40 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 mmol/L MgCl2, 8% Ficoll, 80 mmol/L KCl, 0.2 mmol/L ethylene-diamine-tetra-acetic acid (EDTA), 0.2% NP-40), 1 µL salmon sperm DNA (1 µg/µL), 0.3 µL labeled double-stranded oligonucleotide, and 20 µg protein extract. DNA binding complexes were separated by a 5% polyacrylamide tris-borate-EDTA (TBE) gel run at 4°C for 4 hours at 180V. Gels were dried, exposed to a PhosphoImager cassette and quantified using the ImageQuant (Molecular Dynamics) software.
Wet-to-dry lung weight
As a measure of pulmonary edema formation, wet-to-dry lung weight ratio was determined as described earlier [10]. The wet-to-dry lung weight ratio would be expected to increase with an increase in extravascular lung water.
Data analysis
The mRNA and protein expression levels of all molecules as well as wet-to-dry lung weight ratios were compared between biopsies of donor grafts and controls, using analysis of variance. Data are expressed as mean values ± standard deviation (SD) and statistical significance was set at p < 0.05.
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Results
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The two forms of HIF-1 showed a differential expression pattern at mRNA level. Whereas HIF-1 mRNA expression was not changed, HIF-1ß expression was downregulated in donor lung tissues as compared with controls (p < 0.05; Fig 1A).
In contrast to mRNA, protein expression of both HIF-1 and -ß was upregulated in donor lung tissues versus controls (p < 0.05; Fig 1B). To analyze whether the DNA-binding activity of HIF-1 was affected, we examined the ability of HIF-1 to bind to the HBS in the VEGF promoter by EMSA. We found that HIF-1 binding to the VEGF gene was dramatically increased in donor lung biopsies versus controls (p < 0.001; Fig 2A).
To confirm the binding specificity, excess of unlabeled HBS probe was used as a competitor. Figure 2A shows that there is complex formation with the HBS probe in donor lungs and that this complex can be partially inhibited by 50x excess of the cold HBS probe, suggesting that this complex is specific for the HBS probe. The wet-to-dry weight ratio was increased in donor lungs as compared with control lungs (p < 0.05; Fig 2B).
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Fig 2. (A) Hypoxia-inducible factor-1 (HIF-1) DNA binding activity to the HIF-1 binding site of the vascular endothelial growth factor-A (VEGF-A) gene promoter. Representative electrophoretic mobility shift assay image of control biopsy, donor lung biopsy, and a donor lung biopsy preincubated with 50x excess of nonlabeled cold HIF-1 probe to assess the specificity of complex formation. The arrow indicates the complex formation. Quantification of DNA binding activity shows increased HIF-1 DNA binding activity to VEGF promoter in donor lungs. (B) The wet-to-dry lung weight ratio is significantly increased in donor versus control lungs. Results are expressed as mean ± SD. *Significantly different from control (p < 0.001 in A and p < 0.05 in B).
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Comment
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Ischemia and subsequent reperfusion are a major cause for tissue injury, and pulmonary edema is the main clinical manifestation of this injury after lung transplantation [17]. We have shown that increased vascular permeability and edema formation are associated with upregulated VEGF-A in preserved donor grafts [16]. The present data indicate that HIF-1 binding to the HBS of the VEGF-A promoter is significantly increased in donor lungs, suggesting an important function of HIF-1 in mediating upregulated VEGF-A levels in donor lung tissue.
Hypoxia-inducible factor-1 was originally identified by its binding to the hypoxia response element located in the 3'-flanking region of the erythropoietin gene [18]. Expression of HIF-1 is tightly regulated by O2 availability because both HIF-1 and HIF-1 DNA binding activity increased exponentially when cultured human cancer (HeLa) cells were exposed to decreasing O2 concentrations [19]. The HIF-1 expression is the limiting factor for HIF-1 DNA binding activity and transcriptional activity in cultured cells [19], and it regulates the expression of genes encoding VEGF [20], inducible nitric oxide synthase [21], and several other factors.
Studies in lungs of mice exposed to acute or chronic hypoxia have demonstrated increased HIF-1 and HIF-1ß mRNA levels [21, 22]. In donor lungs, however, we found unchanged HIF-1 or in the case of HIF-1ß even decreased mRNA levels, whereas both HIF-1 and HIF-1ß protein levels were significantly upregulated. These findings indicate that the regulation of HIF-1 expression in donor lungs occurs at the level of protein stabilization. In line with this finding, HIF-1 has been shown to be regulated by ubiquitination and proteasomal degradation depending on the cellular O2 concentration, resulting in increased HIF-1 protein expression under hypoxic conditions [23–25]. High levels of HIF-1 then lead to accumulation of HIF-1 in the nucleus [26], subsequent dimerization with HIF-1ß [27], and finally to transcriptional activation of VEGF through binding of HIF-1 to the HBS of VEGF-A in donor lungs, as shown in this study.
Hypoxia-inducible factor-1 might play multiple roles in donor lung grafts, because HIF-1 also controls the expression of the vasoconstrictor ET-1 in response to hypoxia [28]. Endothelin-1 expression increases in human donor lungs [11], and hypoxia induces ET-1 expression within the pulmonary vasculature of rats [29]. A HBS in the ET-1 gene promoter is required for hypoxia-induced transcription [28], suggesting that ET-1 mRNA expression by hypoxic pulmonary vascular endothelial cells is mediated by HIF-1. In addition, reports in cancer cells suggest that ET-1 in turn is able to induce stabilization of HIF-1 protein and can induce higher levels of HIF-1 DNA binding activity, thereby promoting VEGF synthesis [30]. Therefore, ET-1-mediated increased HIF-1 protein stability most likely contributes to transcriptional activation of VEGF during lung graft preservation.
Control lung biopsies were shortly taken after single-lung ventilation was started, whereas samples of donor lung grafts had an ischemia time of 5 ± 1 hours. Therefore, the effect of ischemia time on HIF-1 binding activity to VEGF promoter deserves further study to elucidate the specific role of HIF-1 in unventilated, unperfused ischemia in donor lungs.
In summary, our findings suggest that increased HIF-1 protein stability and enhanced HIF-1 transcriptional activity promote VEGF-A- and ET-1-mediated increase of vascular permeability in donor lung grafts. Targeting HIF might be of benefit to counteract edema formation in lung grafts.
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Acknowledgments
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This study was supported by a grant from the Austrian Heart Foundation to Dr Aharinejad.
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Abstract
Introduction
