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Am J Physiol Lung Cell Mol Physiol 293: L497-L504, 2007. First published May 18, 2007; doi:10.1152/ajplung.00393.2006
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Postpneumonectomy lung expansion elicits hypoxia-inducible factor-1{alpha} signaling

Quiyang Zhang,1 Dennis J. Bellotto,1 Priya Ravikumar,1 Orson W. Moe,1,3 Richard T. Hogg,1 Deborah C. Hogg,1 Aaron S. Estrera,2 Robert L. Johnson, Jr.,1 and Connie C. W. Hsia1

1Dept. of Internal Medicine, 2Dept. of Cardiovascular and Thoracic Surgery, and 3The Charles and Jane Pak Center of Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 3 October 2006 ; accepted in final form 11 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We (42) previously reported differential regulation of hypoxia-inducible factors (HIF-1{alpha}, -2{alpha}, and -3{alpha}) mRNA in canine lungs during normal maturation and postpneumonectomy (PNX) compensatory growth in the absence of overt hypoxia. To test the hypothesis that lung expansion activates HIF signaling, we replaced the right lung of six adult foxhounds with inflated custom-shaped silicone prosthesis to keep the mediastinum in the midline and minimize lateral expansion of the remaining lung. After 3 wk of recovery and stabilization of perfusion, the prosthesis was acutely deflated in three animals, causing the remaining lung to expand by 114%. In three other animals, the prosthesis remained inflated. Three days following deflation, we observed significant elevation in the mRNA and nuclear protein levels of HIF-1{alpha} (~60%) as well as activation of its transcriptional regulator, the serine/threonine protein kinase B (phospho-Akt-to-total Akt ratio, 124%), and the mRNA and protein levels of its downstream targets, erythropoietin receptor (71–183%) as well as VEGF (33–58%) compared with the pre-PNX control lung from the same animal. The mRNA of HIF-2{alpha}, HIF-3{alpha}, and VEGF receptors did not change with acute deflation. We conclude that in vivo lung expansion by post-PNX deflation of space-occupying prosthesis elicits coordinated activation of HIF-1{alpha} signaling in adult lungs. This pathway could play an important role in mediating lung growth and remodeling during maturation and post-PNX compensation.

lung volume; compensatory lung growth; dog; lung resection; erythropoietin receptor; vascular endothelial growth factor; serine/threonine protein kinase B

THE HYPOXIA-INDUCIBLE FACTORS (HIFs) regulate transcription of genes involved in a wide array of functions including glycolysis, erythropoiesis, apoptosis, and angiogenesis. The HIFs are activated in response to oxidative stress, anemia, injury, and physical forces. Most of these stressors are directly or indirectly associated with a change of intracellular O2 tension, which leads to stabilization of the HIF-{alpha} protein and increases its transcriptional activity on target genes (35). Biochemical mediators such as insulin and insulin-like growth factor can also stimulate nascent HIF-{alpha} mRNA and protein synthesis through receptor-mediated signaling pathways under normoxic conditions (6, 38). In addition, physical forces can regulate cellular HIF activity through O2-independent pathways. Under normoxic conditions, mechanical stretch activates HIF-1{alpha} mRNA expression in capillaries of skeletal muscle (29) and myocardium (24), associated with upstream regulation by phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation or MAP kinase pathway (5, 24) as well as induction of target genes such as VEGF (24) among others.

The opposing elastic recoil of the lung and thorax as well as cyclic respiratory movements create chronic mechanical stresses on lung tissue and provide an important stimulus for mammalian lung growth and maturation. Mechanical feedback interactions between the lungs and thorax stabilize on reaching somatic maturity, but alveolar growth could be reinitiated after pneumonectomy (PNX), associated with asymmetric expansion and gross distortion of the remaining lung (13, 32). We (42) previously found elevated HIF-1{alpha} mRNA and protein expression in the lungs of actively growing dogs compared with that in adult lungs, and the elevated expression was further enhanced in the remaining lung 3 wk after PNX. However, our previous study was not designed to examine the relationship between lung expansion and HIF activation, and there are no reports in the literature that directly addressed the effect of mechanical lung strain on HIF signaling. Based on the above observations, we hypothesized that post-PNX expansion and distortion of the remaining lung activates HIF signaling. To test this hypothesis, we replaced the right lung of adult foxhound with inflated custom-shaped silicone prosthesis that kept the mediastinum in the midline and minimized lateral expansion and distortion of the remaining lung (16, 17, 41). After 3 wk of recovery, we acutely deflated the prosthesis in half of the animals, causing the remaining lung to expand into the right hemithorax. In this fashion, the signals associated with lung expansion were temporally separated from signals associated with surgical trauma and wound healing. Three days following prosthesis deflation, lung tissue was obtained and analyzed for mRNA and/or protein expression of HIF-{alpha} subunits, its transcriptional regulation by the serine/threonine protein kinase B (Akt and phospho-Akt) pathway, as well as its downstream targets, erythropoietin receptor (EPO-R) and VEGF and its receptors (VEGF-R1 and VEGF-R2). In simultaneous control animals, the prosthesis remained inflated for comparison of pre- to post-PNX changes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal surgery and prosthesis implantation. The Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center approved all the protocols. Six litter-matched adult male purpose-bred foxhounds (average body wt 22.2 ± 4.0 kg, SD) underwent right PNX at ~1 yr of age. The methods of surgery, prosthesis construction, and management had been described in detail elsewhere (16, 17, 41). Briefly, under general anesthesia and via a right lateral thoracotomy through the fifth intercostals space, the right upper lobe was doubled-ligated with silk suture at end-inspiration and removed; tissue blocks were sampled from different regions of the lobe and processed (described below) as pre-PNX control samples. The vessels to each lobe of the right lung were double-ligated and cut between ligatures. The right main-stem bronchus was stapled and cut, and the remaining lobes of the right lung were removed. A space-occupying inflatable silicone prosthesis (McGhan Medical, Santa Barbara, CA) manufactured in the shape of the normal right lung of an adult foxhound (41) was placed in the empty right hemithorax; the prosthesis was inflated with SF6-air (50:50) to a volume of 32 ml/kg (equivalent to ~20% above the functional residual capacity of the right lung measured under anesthesia in the supine position). Mixing air with SF6 retarded gas absorption and minimized the rate of volume loss from the prosthesis. A filling tube attached to the dorsolateral aspect of the prosthesis was brought out through the intercostal space, tunneled subcutaneously to the nape of the neck, connected to an injection port, and buried. The chest wall was closed in five layers, and residual air in the thoracic cavity aspirated.

In all animals, the inflated prosthesis successfully maintained a midline position of the mediastinum evidenced by postoperative chest X-rays and was easily tolerated without causing respiratory discomfort. Gas volume of the prosthesis was checked weekly via the subcutaneous injection port using helium dilution measured with a mass spectrometer (MGA 1100, PerkinElmer); volume of the prosthesis was readjusted if necessary to keep the mediastinum in the midline. Mediastinal position was verified by chest X-ray.

Three weeks after surgery, the prosthesis was acutely deflated in three animals (DEF group), allowing the remaining lung to expand and the mediastinum to shift into the right hemithorax. In their three littermates, the prosthesis was kept inflated (INF group). Three days later, the animal was deeply anesthetized, intubated via a tracheostomy, and mechanically ventilated. Through a left lateral thoracotomy in the fifth intercostal space, the remaining lung was exposed. The left upper lobe was double-ligated with silk suture at end-inspiration and removed while a simultaneous overdose of pentobarbital and phenytoin (Euthasol) was given intravenously to stop the heart.

Lung sampling. The removed upper lobe was placed in a standard orientation. Multiple samples were taken from peripheral and central locations within the lobe, immediately frozen in liquid nitrogen, and stored at –70°C until use. As much as possible, the sample locations were matched among animals. In addition to pre- and post-PNX tissue samples from animals with INF or DEF prosthesis, tissue samples were also collected from the left upper lobe of three normal adult male foxhounds that were euthanized in a separate project; these unoperated control samples were processed and analyzed for HIF-1{alpha} nuclear protein and VEGF mRNA in the same way as those from the experimental groups.

Lung volume. Lung volume was measured by high resolution computerized tomographic (CT) scan (General Electric high-speed CTi) 3 wk post-PNX using methods previously described by us (32). Each animal in the INF group was scanned once; each animal in the DEF group was scanned before and ~20 min following deflation of prosthesis. The animal was premedicated, anesthetized, intubated, mechanically ventilated, and scanned in the supine position using 3 x 3 mm collimation, 120 kV, 250 mA, a pitch of 1.0, and a rotation time of 0.8 s. Before each imaging sequence, a scout image was obtained. Then, the lungs were hyperinflated with 3 tidal breaths followed by passive expiration to functional residual capacity. The endotracheal tube was connected to a calibrated syringe set to deliver a volume of air that inflated the lung to 20 cmH2O of transpulmonary pressure to prevent atelectasis and permit easy identification of structural interfaces. The breath was held for ~40 s during scanning. The images were reconstructed at consecutive 1-mm intervals using a 512 x 512 "standard" algorithm resulting in ~300 images per animal. Images were analyzed using Object-Image v.1.6.2 with customized modification. The area occupied by lung was outlined on each image using attenuation thresholding, which excluded conducting structures larger than 1–2 mm in diameter. The trachea and next three generations of large conducting airways were excluded manually by marking them with the background color. Lung volume of each image was the product of its area and thickness (1 mm); total lung volume was calculated from the sum of the volume of all images.

Cloning and sequencing of cDNA probe. The mRNA expression of HIF-1{alpha}, HIF-2{alpha}, VEGF164, and VEGF-R2 were measured by RNA blot. The primer sequences for amplifying dog HIF-1{alpha}, -2{alpha}, and -3{alpha} cDNA probe have been published (42). The primers for amplifying VEGF cDNA probe was based on a published dog sequence and for VEGF-R2 based on the conserved regions of human and mouse (Table 1). The probes were amplified by standard RT-PCR; PCR fragments were cloned into pGEM-T Easy Vector (Promega, Madison, WI), and after verifying the sequences, the inserts were cut and used as probes in RNA blot.


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  		Table 1. Oligonucleotide primers

 
RNA extraction and RNA blots. Total RNA was isolated from ~300 mg of lung tissue by acid guanidinium thiocyanate-phenol-chloroform extraction and treated with DNA-free DNase (Ambion, Austin, TX) to remove any genomic DNA before use in real-time PCR. Aliquots of total RNA (20 µg) were fractionated by agarose gel electrophoresis, transferred to a nylon membrane by capillary action, and cross-linked to the membrane by ultraviolet irradiation. The cDNA probes were generated by Rediprime II random prime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ). The nylon membrane was incubated in QuickHyb (Stratagene, La Jolla, CA) containing 32P-labeled cDNA probe (>109 cpm/µg DNA) at 68°C for 2 h. The blots were washed in 2x saline-sodium citrate (SSC; 1x is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% SDS three times for 15 min each at room temperature and twice in 0.2x SSC for 10 min at 68°C and then exposed to autoradiography. For loading control, the membranes were stripped and rehybridized with a mouse 18S rRNA probe.

Quantitative real-time RT-PCR. For genes of low abundance or where RNA blot failed to detect sufficient signal intensity (HIF-3{alpha}, VEGF-R1, and EPO-R), the mRNA level was measured by real-time RT-PCR using an established protocol (42) and cyclophilin A as the reference gene (Table 1). VEGF mRNA level was measured by both RNA blot and real-time PCR and targeted the predominant isoform (VEGF164) in the mature canine lung (21). We evaluated three candidate genes for use as reference for real-time PCR, including beta-actin, 18S rRNA, and cyclophilin. Among these, the abundance of cyclophilin A mRNA was the closest to our target genes, and its level did not change in canine lungs following PNX (42). The primers were designed using Primer Express (Applied BioSystems, Foster City, CA) and manufactured by Integrated DNA Technologies (Coralville, IA). Pooled cDNA from four normal adult dog lungs was used as a calibration standard.

Immunoblot. For total protein, lung tissue (50–100 mg wet wt) was minced on ice and homogenized by Polytron (PT 10/35, Brinkmann Instruments, Westbury, NY) in an isolation buffer. The homogenates were incubated at 4°C for 30 min and cleared by centrifugation (16,000 g for 20 min, 4°C). For nuclear protein, ~100 mg of lung tissue was minced in ice-cold harvesting buffer (50 mM Tris, pH 7.4; 500 mM NaCl; 2 mM EDTA, pH 7.9; 0.5% Nonidet P-40; 100 µM CoCl2; 1 mM DTT) supplemented with protease inhibitor cocktail (Complete Mini, cat. no. 1 836 153; Roche, Indianapolis, IN). The tissue was transferred to 10 volumes (vol/wt) of ice-cold homogenization buffer (25 mM Tris, pH 7.4; 1 mM EDTA, pH 7.9; 10% glycerol; 100 µM CoCl2; 1 mM DTT, protease inhibitor cocktail) and grounded with a Dounce homogenizer. Total tissue extracts were centrifuged briefly at 200 g, and the supernatant was collected into a fresh tube. Nuclear extracts were prepared by centrifuging the above supernatants at 800 g for 5 min at 4°C. The pellets were gently resuspended in ice-cold wash buffer (0.25 M sucrose; 20 mM Tris, pH 7.8; 1.5 mM MgCl2; 8.5% glycerol; 0.5% Triton X-100; 100 µM CoCl2; 1 mM DTT, protease inhibitor cocktail). After centrifugation at 1,500 g for 10 min at 4°C, the pellets were washed again with ice-cold buffer without Triton X-100 followed by centrifugation. The pellets were homogenized in ice-cold lysis buffer (20 mM Tris, pH 7.8; 450 mM NaCl; 5 mM EDTA, pH 7.9; 10 mM EGTA; 25% glycerol; 100 µM CoCl2; 1 mM DTT, protease inhibitor cocktail) and incubated for 1 h at 4°C on a shaking station. Nuclear debris was pelleted by centrifugation at 13,000 g at 4°C for 30 min, and the supernatants were collected as nuclear extract.

The protein fractions were quantified by Bradford assay (Bio-Rad, Hercules, CA). Aliquots (~30 µg for EPO-R, VEGF, Akt, and p-Akt or 80 µg for HIF-1{alpha}) were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blots were blocked in Blotto-Tween solution (5% nonfat dry milk, 0.05% Tween in PBS) for 1 h and then incubated in Blotto-Tween with the primary antibody for 2 h. Primary antibodies (0.4 µg/ml for HIF-1{alpha} and 1 µg/ml for others) included rabbit anti-human HIF-1{alpha} polyclonal antibody (H206; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse EPO-R carboxyl terminal polyclonal antibody (M-20, Santa Cruz), rabbit anti-human VEGF polyclonal antibody (A20, Santa Cruz), rabbit anti-mouse Akt (no. 9272; Cell Signaling Technology, Danvers, MA), and phospho-Akt (Ser473, no. 9271; Cell Signaling Technology) polyclonal antibodies. For loading control, the membranes were stripped and reprobed with mouse anti-beta-actin monoclonal antibody (1 µg/ml; A1978; Sigma, St. Louis, MO). Labeled protein was visualized using a chemiluminescence detection system (ECL; Amersham, Piscataway, NJ) and quantified by densitometry.

Data analysis. For RNA blots, three to four replicate assays were performed using RNA from separate tissue samples. The autoradiographic intensity of each mRNA band was normalized to that of the corresponding 18S rRNA in the same sample and expressed as a ratio to the mean signal intensity in the corresponding pre-PNX control samples obtained from the same blot. For real-time PCR, triplicate assays were performed using RNA prepared from separate tissue samples. The relative quantity of mRNA with respect to the standard was calculated as described previously (42), and the result was expressed as a ratio to the mean value in the corresponding pre-PNX control samples. For immunoblot, triplicate assays were performed; the chemiluminescence signal was normalized to that of beta-actin and expressed as a ratio to the respective mean signal intensity in the corresponding pre-PNX control samples. Replicate results from each animal were averaged. The pre- and post-PNX results from INF and DEF groups were compared by paired t-test using each animal as its own control. Levels of VEGF mRNA and HIF-1{alpha} nuclear protein were compared with that from unoperated control lungs by unpaired t-test. Differences were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lung volume. In the INF group, volume of the remaining left lung 3 wk post-PNX (46 ± 7 ml/kg) was not different from that in a normal left lung (40 ± 5 ml/kg; Ref. 32). In the DEF group, volume of the left lung before deflation of prosthesis (35 ± 4 ml/kg) was not significantly different from that of a normal left lung but was lower than in the INF group (P < 0.05); lung volume increased by 115% to 76 ± 4 ml/kg immediately after deflation (P < 0.0001 vs. before deflation; Fig. 1). Thus the INF prosthesis minimized post-PNX lung expansion consistent with our previous findings (41).


Figure 1
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  		Fig. 1. Volume of the left lung was measured by high resolution computerized tomographic scan in normal control canines (n = 3), in canines 3 wk postpneumonectomy (post-PNX) with continuously inflated prosthesis (n = 3), or before and after prosthesis deflation (n = 3). Means ± SD, *P < 0.05 vs. normal, {dagger}P < 0.05 vs. inflated group, {ddagger}P < 0.05 vs. before inflation by one-way ANOVA.

 
Expression of HIF mRNA. In the DEF group 3 days after prosthesis deflation, HIF-1{alpha} mRNA in the remaining lung was 63% higher post-PNX relative to that pre-PNX (P < 0.05). On the other hand, no significant pre- to post-PNX change was seen in the INF group where the prosthesis remained inflated (Fig. 2). HIF-2{alpha} mRNA level tended to be lower post-PNX compared with pre-PNX in the INF group at a borderline significance (P = 0.06) but was unchanged in the DEF group (data not shown). HIF-3{alpha} mRNA did not change significantly post-PNX in either DEF or INF group (data not shown). There were no significant changes in the expression of reference genes among groups.


Figure 2
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  		Fig. 2. Hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) mRNA expression. Triplicate RNA blots were performed using separate tissue samples from each lung. Signal intensity was normalized to 18S rRNA and expressed as a ratio to the average intensity in pre-PNX tissue within each assay; replicate results from each animal were averaged, and the group averages were expressed as means ± SE. INF, the prosthesis remained inflated (n = 3); DEF, the prosthesis was deflated 3 days before tissue sampling (n = 3). Pre- vs. post-PNX comparison was by paired t-test.

 
Nuclear translocation of HIF-1{alpha}. The activated HIF-1{alpha} protein is translocated to the nucleus. There was no difference in baseline HIF-1{alpha} nuclear protein level between right and left upper lobes (pre-PNX vs. unoperated control). In the INF group, HIF-1{alpha} nuclear protein level in the remaining left lung was slightly but not significantly higher than in the corresponding pre-PNX or unoperated control lungs (Fig. 3). In DEF group, HIF-1{alpha} nuclear protein level in the post-PNX remaining left lung was 61% higher than in the corresponding pre-PNX right lung (P = 0.01) and 63% higher than in the unoperated control lung (P = 0.02; Fig. 3).


Figure 3
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  		Fig. 3. HIF-1{alpha} nuclear protein expression. Quadruplicate immunoblots were performed using separate samples from each lung. Signal intensity was normalized to beta-actin. Replicate results from each animal were averaged, and the group averages were expressed as means ± SE. Separate unoperated animals (n = 3) provided additional control lung samples. Comparison of pre- vs. post-PNX was by paired t-test. Comparison of post-PNX (INF or DEF) vs. unoperated control was by unpaired t-test.

 
Expression of VEGF, VEGF-R1, and VEGF-R2 mRNA. In the DEF group, post-PNX VEGF mRNA was 33% higher compared with that in the corresponding pre-PNX lung (P = 0.04) when measured by RNA blot and 58% higher when measured by real-time PCR (P = 0.002); in addition, post-PNX VEGF mRNA was 77% higher (P = 0.047) compared with the unoperated control lung by real-time PCR. In contrast, in the INF group, there was no significant pre- to post-PNX change in VEGF mRNA level (Fig. 4, A and B). There was no significant pre- to post-PNX change in VEGF-R1 or VEGF-R2 mRNA level in either the DEF or the INF group (data not shown).


Figure 4
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  		Fig. 4. A: VEGF mRNA expression measured by RNA blot. Triplicate RNA blots were performed using separate tissue samples. Signal intensity was normalized to 18S rRNA and expressed as a ratio to the average intensity in pre-PNX tissue within each assay. Replicate assays in each animal were averaged, and the group averages were expressed as means ± SE. Pre- to post-PNX comparison was by paired t-test. B: VEGF mRNA expression measured by real-time PCR. Triplicate assays were performed using separate tissue samples. Results were normalized to that of cyclophilin and a standard calibrator (pooled normal adult dog lung tissue, n = 4). Data from each animal were averaged, and the group averages were expressed as means ± SE. Separate unoperated animals provided additional control samples (n = 3). Pre- vs. post-PNX comparison was by paired t-test, post-PNX (INF or DEF) vs. unoperated control by unpaired t-test.

 
Expression of VEGF protein. Multiple bands were detected in canine lung by immunoblot; all were blocked by a blocking peptide (sc-152P, Santa Cruz; Fig. 5A). Data in the literature regarding canine VEGF mobility on SDS-PAGE immunoblot are extremely limited. The only two previous reports showed either a selectively cropped ~44-kDa band in canine myocardial interstitial fluid (28) or three bands between 22–46 kDa in canine muscle (11) consistent with our findings. Human VEGF also exhibits multiple bands within the same size range on immunoblot (2, 9). We detected an additional band (~70 kDa) that had not been shown previously, which may be a dimer, but the possibility of other proteins bearing similar epitope cannot be ruled out. The weak bands may represent partially processed or degraded products. We have shown all detected bands in this study. In the DEF group, expression of the dominant bands below 50 kDa was consistently elevated (by 350%) compared with the corresponding bands pre-PNX (P = 0.006). Overall VEGF protein expression based on all detected bands was 45% higher than that of pre-PNX (P = 0.02; Fig. 5B). In contrast, in the INF group, there was no significant change in corresponding VEGF protein expression from pre- to post-PNX regardless of the method of quantification (Fig. 5B). These results showing upregulated VEGF protein expression following post-PNX lung expansion are consistent with the corresponding elevation in VEGF mRNA expression shown in Fig. 4.


Figure 5
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  		Fig. 5. VEGF was measured by immunoblot. A: multiple bands were detected; all were blocked by 5x excess of a blocking peptide. B: triplicate immunoblots were performed using separate tissue samples from each lung. Signal intensity was normalized to beta-actin and expressed as a ratio to the average intensity in the pre-PNX sample within an assay. Replicate results from each animal were averaged, and the group averages were expressed as means ± SE. Pre- to post-PNX comparison was by paired t-test. In the DEF group, overall VEGF expression was 45% higher post-PNX compared with pre-PNX (P = 0.02). In the INF group, there was no significant pre- to post-PNX change in VEGF expression (P > 0.05).

 
Expression of EPO-R mRNA and protein. In the DEF group, EPO-R mRNA in the remaining lung post-PNX was markedly elevated (by 183%) compared with pre-PNX (P = 0.02; Fig. 6), associated with correspondingly higher (by 71%) EPO-R protein level (P = 0.04; Fig. 7). In contrast, in the INF group, neither EPO-R mRNA nor protein expression changed after PNX.


Figure 6
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  		Fig. 6. Erythropoietin receptor (EPO-R) mRNA expression. Triplicate real-time RT-PCR assays were performed using separate tissue samples from each lung. The target signal was normalized by that of cyclophilin and a standard calibrator (pooled normal adult dog lung tissue, n = 4) and expressed as a ratio to the average pre-PNX value within each assay. Replicate results from each animal were averaged. The group averages were expressed as means ± SE and compared pre- vs. post-PNX by paired t-test.

 

Figure 7
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  		Fig. 7. Immunoblot expression of EPO-R. Triplicate assays were performed using separate tissue samples from each lung. The band intensity was normalized to that of corresponding beta-actin and expressed as a ratio to the average intensity in pre-PNX samples within an assay. Replicate assays in each animal were averaged. The group averages were expressed as means ± SE and compared pre- vs. post-PNX by paired t-test.

 
Expression of total Akt and phospho-Akt protein. In the DEF group, the phospho-Akt-to-total Akt ratio in the remaining lung post-PNX was 124% higher than pre-PNX (P < 0.02; Fig. 8), indicative of activated Akt signaling. In addition, total Akt level was 92% higher post-PNX than pre-PNX (P < 0.05), associated with a 288% higher phospho-Akt level (P < 0.02). In contrast, in the INF group, there were no significant changes in total or phospho-Akt level or in phospho-to-total Akt ratio after PNX.


Figure 8
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  		Fig. 8. Immunoblot expression of total Akt and phospho-Akt (p-Akt). Triplicate assays were performed using separate tissue samples from each lung. After normalizing the band intensity to that of beta-actin, the phospho-to-total Akt ratio was calculated and expressed as a fraction of the mean intensity in pre-PNX samples within an assay. Data from replicate assays were averaged. The group averages were expressed as means ± SE and compared pre- vs. post-PNX by paired t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Summary of results. This is the first study to examine the effect of in vivo lung expansion on HIF-related signaling pathway. By keeping the prosthesis inflated for 3 wk after PNX, we minimized lung expansion while allowing cardiac output to be redirected to the remaining lung as well as allowing inflammation and wound healing to subside. Three days after imposing acute lung expansion by deflating the prosthesis and increasing volume of the remaining lung by 114%, we observed coherent upregulation of HIF-1{alpha} signaling in the remaining lung, associated with corresponding upregulation of its transcriptional regulator, total and phospho-Akt, as well as its target molecules, VEGF and EPO-R, relative to the respective pre-PNX normal lung using each animal as its own control. In contrast, post-PNX expression of these molecules was unchanged in the presence of inflated prosthesis. Regardless of the degree of expansion, PNX had no significant effect on mRNA expression of HIF-3{alpha}, VEGF-R1, or VEGF-R2. These data support our hypothesis that post-PNX lung expansion leads to coordinated activation of the HIF-1{alpha}-EPO-VEGF signaling pathway.

Critique of the methods. Each animal served as its own control for pre- to post-PNX comparisons. Given that lung volume normally fluctuates with posture and activity, the inflated prosthesis minimized but did not completely eliminate post-PNX lung expansion. Mediastinal position of the remaining lung was assessed on chest X-ray with respect to the midpoint along the left-right axis of the rib cage. The pre-/post-PNX paired design minimized the inherent intersubject variability and allowed very conservative use of animals. Even with the small number of animals, a consistent pattern of upregulation in key parameters of the HIF pathway (Akt/phospho-Akt, HIF-1{alpha}, EPO-R, VEGF) clearly emerged following institution of lung expansion. Tissue samples were taken from the upper lobe, which was the first lobe removed during surgery and hence subjected to the least manipulation. There were no significant differences in the expression of multiple mediators between left and right upper lobes of control dog lungs examined in the present study or in our previous studies (42). There were no significant differences in cyclophilin A mRNA or beta-actin levels among groups. In light of our previous data (42) that HIF-1{alpha} mRNA and EPO-R protein remain elevated for at least 3 wk following PNX in growing dogs without prosthesis, we chose 3 days as a reasonable time point for examining the early responses following lung expansion. Further studies will be required to define the temporal evolution of expansion-induced response.

Lung expansion and the HIF pathway. Mechanical stress is likely the major signal imposed by lung expansion, causing parenchymal strain that in turn distorts multiple ion channels, increases reactive oxygen species (ROS; Refs. 1, 34), and induces early response transcriptional regulators including Akt (24), which lead to activation of the HIF pathway. Both mechanical forces and hypoxia promote mitochondrial ROS generation (10). Mechanical forces may also exaggerate intracellular oxygen tension gradients, thereby activating HIF-1{alpha} signaling in an oxygen-dependent manner even in the absence of overt organ hypoxia. Further investigation is required to understand the mechanisms by which lung expansion activates the HIF pathway. Pulmonary blood flow may have changed following deflation of prosthesis and was not measured in this study. However, in our previous studies, resting pulmonary blood flow following PNX was similar in animals with INF or DEF prosthesis (41). In addition, perfusion to the remaining lung doubles immediately after PNX independent of the presence of prosthesis. The lack of upregulation in the INF group from pre- to post-PNX argues against perfusion alone as a major signal for HIF activation.

Hypoxia upregulates HIF-1{alpha} activity mainly via posttranslational stabilization of the protein, promoting dimerization of HIF-1{alpha} to the constitutive HIF-1beta subunit and enhancing the transcriptional activities of the heterodimer. In contrast, mechanical stretch enhances HIF-1{alpha} expression at transcriptional as well as posttranscriptional levels by promoting nuclear translocation of HIF-1{alpha} or enhancing its translational efficiency (5, 24, 29). In cultured vascular smooth muscle cells, cyclic stretch increases both HIF-1{alpha} mRNA and protein levels (5) similar to our present findings in response to lung expansion. The oxygen-independent regulation of HIF-1{alpha} requires upstream signaling via PI3K-Akt pathway (43), which may act on HIF-1{alpha} either directly or indirectly via coactivators such as heat shock proteins Hsp90 and Hsp70; the latter physically binds and stabilizes HIF-1{alpha}.

Targets of HIF signaling. Both VEGF and EPO are targets of HIF-1{alpha} signaling. VEGF is a mitogen as well as a mediator for the survival, differentiation, and barrier function of endothelial cells. The VEGF receptors, VEGF-R1 and VEGF-R2, are widely expressed in pulmonary vascular smooth muscle cells, type II alveolar epithelial cells, endothelial cells, and alveolar macrophages (39). HIF-1{alpha} signaling of VEGF and its receptors regulates pulmonary vascular remodeling (36). In addition to its induction by hypoxia (7), VEGF is inducible in normoxia by mechanical stretch, shown in the myocardium (25) and in primary pulmonary cell cultures (30) linked to stretch-activated ion channels (24). Fetal sheep with experimental diaphragmatic hernia subjected to lung distention via tracheal ligation show higher VEGF expression compared with sheep with diaphragmatic hernia alone (30). In our study, post-PNX expansion-induced HIF-1{alpha} signaling is associated with upregulated VEGF expression without significant regulation of VEGF receptors.

Paracrine EPO signaling is widespread in nonhematopoietic tissues; EPO signaling inhibits apoptosis, promotes cell differentiation and angiogenesis, and protects against ischemic and hypoxic injury (4, 22, 23). In the lung, EPO signaling is essential for vascular maintenance and remodeling. In rescued EPO-R–/– mice where EPO-R is absent in vascular tissue but present in hematopoietic tissue, pulmonary hypertension develops with impaired recruitment of endothelial progenitor cells to the pulmonary endothelium (33). EPO signaling is involved in sustaining lung growth; EPO-R expression in dog lung is upregulated during postnatal development and post-PNX compensatory lung growth (8, 42). Overexpressing HIF-1{alpha} in cultured cells enhances endogenous EPO-R expression (42), demonstrating a direct relationship between these two molecules. There may also be cross talk between VEGF and EPO signaling (37).

HIF signaling post-PNX. In adult animals, post-PNX compensatory lung growth is initiated only when more than ~50% of lung tissue is removed (14); below this threshold of resection, compensation occurs primarily via recruitment of the remaining physiological reserves, including unfolding of alveolar surfaces as well as microvascular recruitment and distention, without generation of new gas exchange tissue (15, 18). In separate cohorts of adult dogs, we have shown that minimizing lung expansion post-PNX using the inflated prosthesis significantly blunts compensatory alveolar growth and impairs lung function (16, 17, 41); ~70% of the compensatory increase in alveolar tissue volume and lung diffusing capacity could be attributed to lung expansion. The aggregate data from this canine model suggest that mechanical distortion of the remaining lung constitutes the primary signal for initiating post-PNX adaptation via dose-dependent activation of the PI3K-Akt, HIF-1{alpha}, EPO, and VEGF cascade.

HIF-2{alpha} is less hypoxia-dependent than HIF-1{alpha} and is reported to mediate pulmonary vascular development in normoxia (31). In a previous canine study, we (42) observed no change in HIF-2{alpha} mRNA during postnatal maturation but a significantly lower level post-PNX with respect to the control lung. Since HIF-2{alpha} is closely associated with hypoxia-induced mitogenic factor, a potent cytokine for pulmonary vasoconstriction, proliferation, and angiogenesis (40), and heterozygous HIF-2{alpha} deficiency ameliorates hypoxia-induced pulmonary hypertension and right ventricular hypertrophy (3), we suspect that post-PNX HIF-2{alpha} downregulation may mitigate the increase in pulmonary vascular resistance and the associated vascular remodeling. HIF-3{alpha} and its splice variants are believed to oppose HIF-1{alpha} action (12, 26, 27) by competing with HIF-1{alpha} for binding to the beta-subunit, forming an abortive complex incapable of binding the HIF-responsive element of target genes (12). In our previous canine study (42), HIF-3{alpha} mRNA expression was reduced during postnatal maturation, and the reduction further accentuated after PNX. In the present adult dogs, we did not find a significant change in HIF-3{alpha} mRNA expression from pre- to post-PNX regardless of inflation state of the prosthesis. These data are consistent with maturity-dependent post-PNX regulation of the HIF pathway.

In summary, in adult dogs after PNX delayed expansion of the remaining lung via deflation of a space-occupying prosthesis elicited coordinated upregulation of HIF-1{alpha} signaling pathway, including its upstream regulator, phospho-Akt, as well as its downstream targets, EPO-R and VEGF, without significant change in the expression of HIF-2{alpha}, HIF-3{alpha}, or VEGF receptors. These findings support our hypothesis that HIF-1{alpha} signaling in adult lungs is sensitive to mechanical forces. The HIF pathway likely plays an important role in mediating lung growth and/or remodeling during maturation and post-PNX compensation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Institutes of Health Grants R01-HL-040070, HL-054060, HL-045716, HL-062873, DK-048482, and DK-020543 and the Simmons Family Foundation.


   ACKNOWLEDGMENTS
 
We thank the staff of the Animal Resources Center for veterinary assistance and Jue Yang for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. C. W. Hsia, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9034

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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