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(The FASEB Journal. 2006;20:1519-1521.)
© 2006 FASEB

Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats

M. Godzich*,1, M. Hodnett*,1, J. A. Frank{dagger}, G. Su{dagger}, M. Pespeni*, A. Angel*, M. B. Howard*, M. A. Matthay{dagger} and J. F. Pittet{dagger},2

* Laboratory of Surgical Research, Departments of Anesthesia, Surgery, and

{dagger} Medicine and Cardiovascular Research Institute, University of California, San Francisco, California, USA

2Correspondence: Department of Anesthesia, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110, USA. E-mail: pittetj{at}anesthesia.ucsf.edu

ABSTRACT

Lung endothelial damage is a characteristic morphological feature of ischemia-reperfusion (I/R) injury, although the molecular steps involved in the loss of endothelial integrity are still poorly understood. We tested the hypothesis that the activation of vascular endothelial growth factor (VEGF) cell signaling would be responsible for the increase in lung vascular permeability seen early after the onset of I/R in rats. Furthermore, we hypothesized that the I/R-induced pulmonary edema would be significantly attenuated in rats by the activation of the stress protein response. Pretreatment with Ad Flk-1, an adenovirus encoding for the soluble VEGF receptor type II, prevented I/R-mediated increase in lung vascular permeability in rats. Furthermore, the I/R-induced lung injury was significantly decreased by prior activation of the stress protein response with geldanamycin or pyrrolidine dithiocarbamate. In vitro studies demonstrated that VEGF caused an increase in protein permeability across primary cultures of bovine macro- and microvascular lung endothelial cell monolayers that were associated with a phosphorylation of VE- and E-cadherin and the formation of actin stress fibers. Activation of the stress protein response prevented the VEGF-mediated changes in protein permeability across these cell monolayers and reduced the phosphorylation of VE-and E-cadherins, as well as the formation of actin stress fibers in these cells. .—Godzich, M., Hodnett, M., Frank, J. A., Su, G., Pespeni, M., Angel, A., Howard, M. B., Matthay, M. A., Pitte, J. F. Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats.

ISCHEMIA-REPERFUSION (I/R) LUNG injury is a common complication seen after hemorrhagic shock, lung transplantation, or pulmonary embolism (1) . It is characterized by the flooding of the alveolar spaces with a protein-rich edema that impairs pulmonary gas exchange leading to arterial hypoxemia and respiratory failure. Lung endothelial damage is a characteristic morphological feature in I/R injury. Several mediators, including early growth response 1 (EGR-1) and protein kinase C IIß (PKCIIß) have been shown to mediate I/R injury in the lung (2 , 3) , although the molecular steps causing the loss of endothelial integrity in patients with I/R lung injury are still poorly understood, and no specific therapies are currently available. Recent experimental studies have implicated vascular endothelial growth factor (VEGF) as an important factor that increases vascular permeability in the lung (4) . Overexpression of the VEGF gene in the lung-induced pulmonary edema (5) and increased expression of VEGF mRNA and protein in the lung was associated with ischemia–reperfusion and endotoxin-mediated lung injury (6) . Furthermore, thrombin, which induces lung hyperpermeability and pulmonary edema, up-regulated VEGF receptors on these cells (7) . Taken together, these results suggest that VEGF might play a role in the increase of vascular permeability associated with I/R lung injury.

The heat shock or stress protein response (SPR) is a highly conserved cellular defense mechanism characterized by the increased expression of stress proteins that allow the cell to withstand a subsequent insult that would otherwise be lethal, a phenomenon referred as "stress preconditioning" (8 , 9) . Recent studies indicate that SPR activation was associated with a decrease in lung endothelial permeability in experimental animal models of I/R injury, although the mechanisms explaining this protective effect are still unknown (10 , 11) . Therefore, we hypothesized that SPR activation could attenuate the activation of the VEGF cell signaling that is associated with the increase in lung vascular permeability caused by I/R in rats.

MATERIALS AND METHODS

Material and Reagents
Male Sprague-Dawley rats weighing 300–350 g were purchased from Charles River Laboratories. The chemiluminescence kit was obtained from Amersham (Piscataway, NJ). 14C-albumin was purchased from Perkin Elmer Life Sciences (Norwalk, CT). VEGF mouse monoclonal antibody and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1) rabbit polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphotyrosine antibody (Ab) was obtained from Cell Signaling Technology (Beverly, MA). Hsp72 Ab was obtained from Stressgen (San Diego, CA). Protein A- and G- Sepharose beads were purchased from Amersham. Recombinant human VEGF165 and ELISA kits were obtained from R&D. Protein concentration of cell lysates was determined using the Bio-Rad (Hercules, CA) protein assay kit with BSA as the standard. All other chemicals were purchased from Sigma (St. Louis, MO).

Lung Barrier Function Studies
The protocol for these studies was approved by the University of California, San Francisco, Animal Research Committee. Sprague-Dawley rats were anesthetized with pentobarbital sodium (60 mg/kg i.p.), then 30 mg/kg i.p. every 2 h. After placement of a tracheostomy, the rats were maintained in right lateral decubitus position and were mechanically ventilated with an inspired oxygen fraction of 1.0, and a peak airway pressure of 8–12 cm H2O supplemented with a positive end-expiratory pressure of 3 cm H2O for 30 min. The respiratory rate was adjusted to maintain a PaCO2 between 35 and 40 mM Hg, as previously reported (12 , 13) . Subsequently, a left thoracotomy was performed to expose the left pulmonary hilum. Sodium heparin (500 U/kg) was administered 10 min before clamping of the left hilum. Next, the left hilum was clamped 30 min with an umbilical tape placed around the left pulmonary hilum. To measure lung endothelial protein permeability, a vascular tracer, 125I-labeled human albumin, was administered to the animals 15 min before reperfusion (12 , 13) . Perfusion and ventilation of the left lung were resumed for 30 min to 3 h after ischemia. Then, the rats were exsanguinated, and the lungs were excised separately. The right and left lungs were homogenized separately for measurement of water-to-dry wt ratio and radioactivity counts (12 , 13) .

Specific Protocols
Group 1
Time course of I/R lung injury (n=16). The first series of experiments included rats that underwent a left thoracotomy and then had their left hilum clamped for 30 min and then had their left lung reperfused for 30 to 180 min. Controls included rats that underwent a left thoracotomy, had their left hilum exposed, but not clamped.

Group 2
Role of VEGF in I/R-mediated lung injury (n=10). The second series of experiments included rats that were pretreated with an adenovirus encoding the extracellular portion of the murine VEGF receptor type II fused to sequences encoding murine IgG2{alpha}-Fc (14) . The control adenovirus is encoding the green fluorescent protein and was obtained from Dr. G. E. Davis (Texas A&M University System Health Science Center, College Station, TX). Adenoviral injections were performed by administering 109 pfu of either control or VEGF receptor type II adenovirus in the tail vein of the rat. Forty-eight hours later, rats pretreated with adenovirus underwent a left thoracotomy, had the left hilum clamped for 30 min, and then had the left lung reperfused for 180 min.

Group 3
Stress preconditioning and I/R-mediated lung injury (n=20). The third series of experiments included rats that underwent SPR activation with geldanamycin (1 mg/kg i.p. 48 h and 24 h before onset of surgery) or PDTC (200 mg/kg i.v., 30 min before onset of the ischemia of the left lung). The first series of experiments included rats that underwent a left thoracotomy, had the left hilum clamped for 30 min and then had the left lung reperfused for 180 min. Control experiments included rats that were pretreated with geldanamycin or PDTC vehicle, had the left hilum clamped for 30 min and then had the left lung reperfused for 180 min.

Hemodynamics and pulmonary gas exchange
Systemic arterial, central venous, and airway pressures were continuously measured. Arterial blood gases were measured at 1-h intervals.

Albumin flux across endothelial barrier
To measure the flux of albumin across the lung endothelial barrier, 131I-albumin (a vascular protein tracer) was measured in the extravascular space of the lungs (12 , 13) . To determine 131I binding to albumin, trichloroacetic acid (20%) was added to all tubes, which were then centrifuged to obtain the supernatant for measurement of free 131I radioactivity in a gamma counter. The results are expressed as a percentage of the unbound 131I radioactivity to the total amount of 131I-albumin radioactivity instilled. These fluid samples always had less than 1% of unbound iodine present.

Western blot analysis for detecting Hsp 72
In order to detect the induction of Hsp72 protein expression in the lung tissue after SPR activation, lung tissue samples were solubilized by homogenization in a Tris buffer (50 mmol, pH 7.4) containing NaCl (20 mmol), KCl (10 mmol), DTT (0.1 mmol) EDTA (1 mmol), SDS (1%), sonicated for 30 s, boiled for 10 min and centrifuged at 14,000 g for 10 min at 4°C. Supernatants were analyzed by Western blot, as described previously (15) . Equal amounts of protein were separated by 10% SDS-PAGE and transferred to Immobilon-P membranes. Membranes were blocked with 5% dry milk and incubated with the primary Ab (1:1,000) for 1 h at room temperature and with HRP-conjugated goat antimouse (1:2,000) for 1 h at room temperature. Proteins were visualized using chemiluminescence. Quantification was done using a digital image analysis system (NIH Image).

VEGF ELISA
Protein extracted from the lung tissues as described for the Western blot analysis were also analyzed for VEGF protein concentrations using a Quantikine M VEGF immunoassay. Samples were diluted 1:5 and 1:10 in assay diluent, added to the wells of 96-well plate and incubated for 2 h at room temperature. The wells were washed 3 times, and mouse VEGF conjugate was incubated with the protein samples. Reactions were developed, and optical density was measured at 450 nM and 570 nM to quantitate VEGF protein spectrophotometrically from a standard curve in accordance with the manufacturer’s instructions. VEGF protein concentration in each sample was normalized to the total protein concentration in the lung tissue homogenate.

Immunoprecipitation for detecting phospho-KDR/flk-1 (VEGF receptor type II)
Protein extracted from the lung tissues, as described for the Western blot analysis, were also used for immunoprecipitation to detect phosphorylation of KDR/flk-1 in rats that underwent I/R lung injury. Immunoprecipitations were performed by incubating the precleared lysates with the primary Ab overnight at 4°C. Then, the cell lysate was incubated for 2 h at 4°C under continuous mixing with protein A- and G- Sepharose beads and the Sepharose-bound immune complexes were washed 4 times with lysis buffer and then boiled in 2x Laemmli sample buffer. As controls, immunoprecipitations were performed with irrelevant Ab of the same IgG subtype. Proteins were separated by SDS-PAGE in 10% gels, transferred onto nitrocellulose membranes, blocked with 3% BSA in PBS containing 0.1% Tween-20, and incubated with the appropriate antibodies (primary Ab 1:1,000 dilution overnight at 4°C; secondary Ab 1:2,000 dilution 1 h at room temperature). Immunoreactive bands were visualized using enhanced chemiluminescence. Quantification was done using a digital image analysis system (NIH Image).

Effect of SPR activation on VEGF-mediated increase in lung endothelial permeability
Bovine pulmonary arterial endothelial cells (BPAEC passage 5) were obtained from the American Type Culture Collection (CCL 209), cultured in Dulbecco’s modified Eagle medium with 20% FBS and 1% penicillin/streptomycin, and used at passages 9–11. Bovine lung microvascular endothelial cells (BLMVEC passage 5) were obtained from VEC Technologies (Rensselaer, NY) and were used at passages 7–9. BLMVECs were cultured in a complete medium (MCDB 131) obtained from VEC Technologies. For all studies, cells were plated on collagen-coated 6.5 mM transwells for permeability studies and 24-mM diameter transwells for immunoprecipitation studies. The cell monolayers were used at days 3–4 postseeding for all experiments.

Measurement of protein permeability across lung endothelial cell monolayers
Macro- and microvascular lung endothelial cells were seeded (2x105 cells) on collagen-treated polycarbonate membrane transwells (6.5 mM diameter, 0.4 µM pore size) and cultured for 72 h. The baseline barrier function of each monolayer is determined by applying 14C-BSA to the upper compartment (0.5 ml) for 1 h at 37°C, after which the cell medium from the lower compartment (1.5 ml) is counted for 14C activity. Only monolayers retaining ≥97% of the 14C-BSA were used in the permeability studies. Six hours later, VEGF165 (50 ng/ml) or its vehicle was added to the medium on both sides of the transwells. In some experiments, SPR was activated with heat (60 min at 43°C, then recovered overnight at 37°C) or pretreated with geldanamycin (10 ng/ml) for 6–8 h before the exposure to VEGF or its vehicle.

Immunoprecipitation for detecting phospho-VE- and E-cadherins
Lung macro- and microvascular endothelial cells were grown to confluence on filters, washed with serum-free medium and starved for 6 h in serum-free media. The cells were treated for 20 min with 1 mM vanadate, then stimulated with recombinant VEGF165 (50 ng/ml) for 5 min. In some experiments, SPR was activated with geldanamycin (10 ng/ml) for 8 h before exposure to VEGF165 or its vehicle. The cells were washed twice in PBS containing calcium and magnesium and solubilized on ice for 20 min with a lysis buffer (150 mM NaCl, 2 mM CaCl2, 10 mM Tris HCl, pH 7.5 [TBS], 1% Triton X-100) supplemented with a cocktail of phosphatase and proteinase inhibitors (1 mM vanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride) with occasional gentle agitation. The cells were scraped from the filters and the lysates processed for immunoprecipitation and Western blot analysis, as described above.

Immunocytochemistry for detecting actin stress fiber formation
Bovine macro- and microendothelial cells were grown on collagen-coated round glass coverslips to confluence, serum-starved for 6 h, stimulated with VEGF165 (50 ng/ml for 10 min), fixed with 3.7% formaldehyde, washed twice in PBS, permeabilized with 0.5% Triton X-100 for 5 min, incubated with rhodamine-phalloidin (Molecular Probes) for 15 min, and imaged (Leica DM5000B equipped for epifluorescence). Some of the coverslips were pretreated with geldanamycin (10 ng/ml) or its vehicle for 8 h before exposure to VEGF165 or its vehicle.

Statistics
All of the data are summarized as mean ± SEM. One-way ANOVA and the Fisher’s exact t test were used to compare experimental with control groups. A P value of <0.05 was considered statistically significant.

RESULTS

VEGF cell signaling mediates I/R lung injury in rats
Severe ischemia-reperfusion injury occurred in rats that had a 30-min ischemia of their left lung followed by a 3-h reperfusion period. The lung injury was characterized by an increase in lung endothelial permeability in the ischemic lung compared with the contralateral lung. As early as 30 min of reperfusion, there was a 6-fold increase in the quantity of plasma proteins that accumulated in the extravascular spaces of the lung compared with control rats (Fig. 1 A). There was also a 55% increase in the accumulation of extravascular lung water in rats that underwent a 60-min lung reperfusion period but did not further increase after 3 h of reperfusion (Fig. 1B ). The accumulation of pulmonary edema fluid was associated with a progressive decrease in the arterial PaO2 (Fig. 1C ).


Figure 1
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  		Figure 1. Role of vascular endothelial growth factor cell signaling in I/R-mediated lung injury in rats. A) Pretreatment with Ad Flk1-Fc, an adenovirus encoding the soluble vascular endothelial growth factor receptor type II, prevents I/R-mediated lung injury in rats. Extravascular plasma equivalents (µl/lung) are shown for control and ischemic lungs of rats that have been pretreated with Ad Flk1-Fc (109 pfu), or with Ad GFP (109 pfu), (30 min ischemia and 180 min reperfusion); results are shown as means ± SEM; *P < 0.05 from control lung. B) Effect of I/R injury on lung tissue vascular endothelial growth factor concentration in rats. Vascular endothelial growth factor concentrations were measured by ELISA in rat lungs that underwent I/R lung injury (30 min ischemia and 180 min reperfusion) or sham surgery; results are shown as means ± SEM. C) Effect of I/R injury and inhibition of VEGF cell signaling with Ad Flk1-Fc, on lung tissue VEGF receptor type II (KDR/flk-1) phosphorylation in rats. KDR/flk-1 phosphorylation was measured in the lungs of rats that underwent I/R lung injury or sham surgery; in some experiments, rats were pretreated with Ad Flk1-Fc (109 pfu) or with Ad GFP (109 pfu); lung tissues were harvested and subjected to immunoprecipitation with an Ab against VEGF receptor type II protein and immunoblotted with an Ab to phosphotyrosine. The same blots were then reprobed with an Ab to VEGF receptor type II protein. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of four experiments; *P < 0.05 from control experiments.

The molecular steps causing the loss of endothelial integrity secondary to I/R injury in the lungs are still poorly understood. However, recent experimental studies have implicated VEGF cell signaling as an important factor that increases lung vascular permeability in the lung (5) . To test for the role of VEGF cell signaling in I/R-mediated change in lung vascular permeability, rats were treated with an adenovirus encoding the extracellular portion of the murine VEGF receptor type II fused to sequences encoding murine IgG2{alpha}-Fc (Ad-Flk1-Fc). Treatment with this adenovirus has been shown to prevent activation of the VEGF cell signaling pathway (14) . Rats pretreated with this adenovirus showed a sixfold reduction in the quantity of plasma proteins that accumulated in the extravascular spaces of the ischemic lung compared with the rats that were treated with the control adenovirus encoding the GFP protein (Fig. 2 A). In addition, the decrease in the arterial PaO2 caused by the I/R lung injury was significantly less in the group of rats pretreated with the Ad-Flk1-Fc virus compared with those pretreated with the GFP adenovirus (data not shown). The next series of experiments were then designed to determine whether I/R would alter the concentrations of VEGF and KDR/flk-1 in the lung tissue. There was no difference in the amount of VEGF protein extracted from the lung tissue of ischemic and contralateral lungs (data not shown), as well as between sham rats and those that underwent I/R lung injury (Figure 2B ). In contrast, I/R was associated with a sustained phosphorylation of the VEGF receptor 2, still present 3 h after reperfusion that was not observed in the lungs of sham rats or when rats were pretreated with Ad-Flk1-Fc virus (Fig. 2C ).


Figure 2
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  		Figure 2. Time course of changes in lung vascular permeability formation of pulmonary edema and arterial PO2 induced by lung ischemia-reperfusion (I/R) injury in rats. The time of ischemia was 30 min and the time of reperfusion varied from 30 to 180 min. Results are shown as means ± SEM; *P < 0.05 from control lung. A) Time course of changes in vascular permeability induced by I/R in the ischemic (left) lung and contralateral (right) lung in rats. Extravascular plasma equivalents (µl/lung) are shown for control and ischemic lungs. B) Time course of changes in extravascular lung water induced by I/R in the ischemic (left) lung and contralateral (right) lung in rats. Extravascular lung water values (g of water/g of dry lung tissue) are shown for control and ischemic lungs. C) Time course of changes in arterial PO2 induced by I/R lung injury in rats. Arterial PaO2 values (mm Hg) are shown for control rats and rats that underwent I/R lung injury.

SPR activation protects lungs from I/R injury in rats
We have previously reported that SPR activation significantly decreased the accumulation of pulmonary edema in the lung of hemorrhaged and fluid-resuscitated rats, a model of whole body I/R injury (16) . Thus, the next series of experiments were designed to determine whether SPR activation with geldanamycin or PDTC would attenuate I/R-mediated increase in lung vascular permeability. The results indicated that pretreatment with geldanamycin or PDTC caused a stress protein response in the lungs, as shown by the increased expression of Hsp72 protein in the lung, a classical marker of SPR induction (Fig. 3 A, B). Furthermore, SPR activation was associated with greater than a twofold decrease in the amount of proteins that accumulated in the extravascular spaces of the lung after 30 min of ischemia and 3 h of reperfusion (Fig. 3C, D ). SPR activation was also associated with a significant decrease in the I/R-mediated accumulation of pulmonary edema fluid measured by the gravimetric method after 3 h of reperfusion (geldanamycin pretreatment: 4.6±0.3; PDTC pretreatment: 4.5±0.3 vs. 6.1±0.4 in ischemic nonpretreated rats) and a corresponding attenuation of the decrease in the arterial PaO2 caused by the I/R lung injury (geldanamycin pretreatment: 452±25 mmHg; PDTC pretreatment: 472±19 vs. 271±35 mmHg in ischemic nonpretreated rats). Finally, stress preconditioning with geldanamycin prevented the phosphorylation of the VEGF receptor 2, KDR/flk-1 in the lung (Fig. 3E ).


Figure 3
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  		Figure 3. SPR activation with geldanamycin or PDTC significantly attenuates I/R-mediated lung injury in rats. A and B) SPR activation with geldanamycin or PDTC leads to increased expression of Hsp72 protein in the lung. Rats were left untreated or pretreated with geldanamycin (1 mg/kg i.p., 48 and 24 h before sacrifice) or PDTC (200 mg/kg i.p., 4 h before sacrifice). Animals were sacrificed, the lungs removed, and then examined for their expression of Hsp72 by Western blot analysis. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments. C and D) SPR activation with geldanamycin or PDTC significantly attenuates I/R-mediated lung injury in rats. Extravascular plasma equivalents (µl/lung) are shown for control and ischemic lungs of rats that have been pretreated with geldanamycin or PDTC or their vehicle; (30 min ischemia and 180 min reperfusion); results are shown as means ± SEM; *P < 0.05 from control lung; **P < 0.05 from ischemic lung that did not undergo prior SPR activation. E) Effect of I/R injury and stress preconditioning with geldanamycin on lung tissue VEGF receptor type II (KDR/flk-1) phosphorylation in rats. KDR/flk-1 phosphorylation was measured in rat lungs that underwent I/R lung injury or sham surgery; in some experiments, rats were stress preconditioned with geldanamycin (1 mg/kg i.p., 48 and 24 h before sacrifice); lung tissues were harvested and subjected to immunoprecipitation with an Ab against vascular endothelial growth factor receptor type II protein and immunoblotted with an Ab to phosphotyrosine. The same blots were then reprobed with an Ab to vascular endothelial growth factor receptor type II protein. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments.

Effect of SPR activation on VEGF-mediated increase in lung endothelial permeability
The results of the in vivo studies demonstrated that the development of pulmonary edema observed in the early phase after I/R was mediated by the activation of VEGF cell signaling in the lung and was prevented by either expression of the soluble VEGF receptor type II or prior SPR activation. The next series of experiments were designed to test the hypothesis that SPR activation was directly inhibiting VEGF-dependent increase in protein permeability across lung endothelial cell monolayers. Bovine macrovascular lung endothelial cells were cultured on transwells until they formed confluent monolayers. The cell monolayers were exposed to VEGF (50 ng/ml) for 1 h, and protein permeability across these monolayers was measured with 14C-albumin. In some experiments, cells were stress preconditioned with heat (60 min at 43°C, then recovered overnight at 37°C) or pretreated with geldanamycin (10 ng/ml) for 6–8 h before the exposure to VEGF. VEGF induced a greater than twofold increase in the protein permeability across these cell monolayers (Fig. 4 A, B). SPR activation using either geldanamycin or heat, documented by a significant increase in the expression of Hsp72, (Fig. 4C, D ) prevented the VEGF-dependent increase in protein permeability across these macrovascular lung endothelial cell monolayers (Fig. 4A, B ). Lastly, the VEGF-mediated increase in protein permeability was associated with phosphorylation of the adherens junction protein VE-cadherin (Fig. 5 A) and the formation of actin stress fibers (Fig. 5B ). VEGF-dependent VE-cadherin phosphorylation and stress fibers formation were also inhibited in stress preconditioned cells (Fig. 5A, B ).


Figure 4
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  		Figure 4. SPR activation with geldanamycin or hyperthermia significantly attenuates vascular endothelial growth factor-mediated increase in protein permeability across primary cultures of macrovascular bovine lung endothelial cells. A and B) SPR activation with geldanamycin or hyperthermia significantly attenuates vascular endothelial growth factor-mediated increase in protein permeability. Bovine macrovascular lung endothelial cells were cultured for 4 d, as described in the Materials and Methods section. After 96 h, confluent cell monolayers were treated with vascular endothelial growth factor (50 ng/ml) or its vehicle for 1 h. Some cell monolayers were treated with geldanamycin (10 ng/ml) or its vehicle for 8 h before exposure to vascular endothelial growth factor or its vehicle. Some cell monolayers were pretreated with heat (43°C for 60 min), then recovered overnight at 37°C before exposure to vascular endothelial growth factor or its vehicle. Control cell monolayers were maintained at 37°C. Paracellular protein permeability was measured with 14C-albumin. Data are shown as percent of controls; results are shown as means ± SEM; *P < 0.05 from controls; **P < 0.05 from VEGF-treated cell monolayers. C and D) SPR activation with geldanamycin or hyperthermia leads to increased expression of Hsp72 protein. Bovine macrovascular lung endothelial cells were cultured for 4 d, as described in Materials and Methods. After 96 h, some cell monolayers were treated with geldanamycin (10 ng/ml) or its vehicle for 0–8 h before being harvested. Some cell monolayers were stress preconditioned with heat (43°C for 30–120 min), then recovered overnight at 37°C before being harvested. Control cell monolayers were maintained at 37°C. The expression of Hsp72 protein was determined by Western blot analysis. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments.


Figure 5
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  		Figure 5. Stress preconditioning with geldanamycin attenuates VEGF-mediated VE- phosphorylation and actin stress fiber formation in primary bovine macrovascular lung endothelial cells. A) SPR activation with geldanamycin prevents VEGF-mediated VE-cadherin tyrosine phosphorylation. Macrovascular bovine lung endothelial cells were cultured for 4 d, as described in the Materials and Methods section. After 96 h, confluent cell monolayers were treated with VEGF (50 ng/ml) or its vehicle for 5 min. Some cell monolayers were pretreated with geldanamycin (10 ng/ml) or its vehicle for 8 h, before exposure to VEGF or its vehicle. Cells were harvested, and cell extracts were subjected to immunoprecipitation with an Ab against VE-cadherin and immunoblotted with an Ab to phosphotyrosine. The same blots were then reprobed with an Ab to VE-cadherin. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments. B) SPR activation with geldanamycin prevents the VEGF-mediated actin stress fiber formation. Bovine macrovascular lung endothelial cells were grown on collagen-coated round glass coverslips to confluence, serum-starved for 8 h, stimulated with VEGF (50 ng/ml) or its vehicle for 10 min, fixed with 3.7% formaldehyde, washed twice in PBS, permeabilized with 0.5% Triton X-100, and incubated with rhodamine-phalloidin. Some of the coverslips were pretreated with geldanamycin (10 ng/ml) or its vehicle for 6 h before exposure to VEGF or its vehicle. One representative image is shown. Three additional experiments gave comparable results.

A recently published study has reported that VE-cadherin is not expressed in lung microvascular endothelial cells (17) . In fact, VE-cadherin is replaced by E-cadherin as part of the adherens junction protein complex in these cells (17) . Thus, the final objective of these studies was to determine whether VEGF would cause an increase in protein permeability across bovine microvascular endothelial cell monolayers and whether prior SPR activation would prevent the effect of VEGF on the paracellular transport of proteins across these monolayers. The results indicated that VEGF significantly increased protein permeability across these cell monolayers (Fig. 6 A, B). SPR activation (documented by a significant increase in the expression of Hsp72) (Fig. 6C, D ) prevented the VEGF-dependent increase in protein permeability across these cell monolayers (Fig. 6A, B ). Furthermore, the VEGF-mediated increase in protein permeability was associated with phosphorylation of the adherens junction protein E-cadherin (Fig. 7 A) and the formation of actin stress fibers (Fig. 7B ). The VEGF-dependent E-cadherin phosphorylation and actin stress fiber formation were also inhibited in stress preconditioned cells (Fig. 7A, B ).


Figure 6
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  		Figure 6. SPR activation with geldanamycin or hyperthermia significantly attenuates VEGF-mediated increase in protein permeability and increases Hsp72 expression. A and B) SPR activation with geldanamycin or hyperthermia significantly attenuates VEGF-mediated increase in protein permeability across primary cultures of bovine microvascular lung endothelial cells. Bovine microvascular lung endothelial cells were cultured for 4 d, as described in the Method section. After 96 h, confluent cell monolayers were treated with VEGF (50 ng/ml) or its vehicle for 1 h. Some cell monolayers were treated with geldanamycin (10 ng/ml) or its vehicle for 8 h before exposure to VEGF or its vehicle. Some cell monolayers were pretreated with heat (43°C for 60 min), then recovered overnight at 37°C before exposure to VEGF or its vehicle. Control cell monolayers were maintained at 37°C. Paracellular protein permeability was measured with 14C-albumin. Data are shown as a percent of controls; results are shown as means ± SEM; *P < 0.05 from controls; **P < 0.05 from VEGF-treated cell monolayers. C and D) SPR activation with geldanamycin or hyperthermia leads to increased expression of Hsp72 protein in primary cultures of bovine microvascular lung endothelial cells. Bovine microvascular lung endothelial cells were cultured for 4 d, as described in Materials and Methods. After 96 h, some cell monolayers were treated with geldanamycin (10 ng/ml) or its vehicle for 0–8 h before being harvested. Some cell monolayers were stress preconditioned with heat (43°C for 30–120 min), then recovered overnight at 37°C before being harvested. Control cell monolayers were maintained at 37°C. The expression of Hsp72 protein was determined by Western blot analysis. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments.


Figure 7
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  		Figure 7. Stress preconditioning with geldanamycin attenuates VEGF-mediated E-cadherin phosphorylation and actin stress fiber formation in bovine microvascular lung endothelial cells. A) SPR activation with geldanamycin prevents VEGF-mediated E-cadherin tyrosine phosphorylation. Bovine microvascular lung endothelial cells were cultured for 4 d, as described in Materials and Methods. After 96 h, confluent cell monolayers were treated with VEGF (50 ng/ml) or its vehicle for 5 min. Some cell monolayers were pretreated with geldanamycin (10 ng/ml) or its vehicle for 8 h before exposure to VEGF or its vehicle. Control cell monolayers were maintained at 37°C. Cells were harvested and cell extracts were subjected to immunoprecipitation with an Ab against E-cadherin and immunoblotted with an Ab to phosphotyrosine. The same blots were then reprobed with an Ab to E-cadherin. One representative blot is shown. For all experiments, densitometry analysis results are the mean ± SEM of 4 experiments; *P < 0.05 from control experiments. B) Stress preconditioning with geldanamycin prevents the VEGF-mediated actin stress fiber formation. Bovine microvascular lung endothelial cells were grown on collagen-coated round glass coverslips to confluence, serum-starved for 8 h, stimulated with VEGF (50 ng/ml), or its vehicle for 10 min, fixed with 3.7% formaldehyde, washed twice in PBS, permeabilized with 0.5% Triton X-100, and incubated with rhodamine-phalloidin. Some of the coverslips were pretreated with geldanamycin (10 ng/ml) or its vehicle for 6 h before exposure to VEGF or its vehicle. One representative image is shown. Three additional experiments gave comparable results.

DISCUSSION

The main results of these studies indicate that 1) VEGF cell signaling mediates the early increase in lung vascular permeability associated with I/R lung injury in rats; 2) prior SPR activation prevents the development of pulmonary edema associated with this syndrome; and 3) directly inhibits VEGF-dependent cell signaling in lung endothelium by preventing VEGF-mediated increase in protein permeability, phosphorylation of VE- and E-cadherins and actin stress fiber formation in these cell monolayers. I/R-mediated injury plays an important role in the lung injury associated with severe hemorrhagic shock, pulmonary embolism, or lung transplantation (1) . Previous studies have reported that the zinc-finger transcription factor EGR-1 and HIF-1{alpha} are critical for coordinating the up-regulation of divergent gene families after a hypoxemic and ischemic stress in the lung. HIF-1{alpha} has been shown to be important in up-regulating a series of hypoxia-responsive genes, including VEGF after global hypoxia (18) . Furthermore, EGR-1 has been shown to trigger expression of pivotal regulators of inflammation, coagulation, and vascular hyperpermeability, including VEGF (2) . Finally, one recent study has demonstrated a key role for PKCIIß activation in I/R lung injury by regulating the expression of proinflammatory/prothrombotic mediators, at least in part through EGR-1 (3) . However, none of these factors has been shown to have a direct effect on the vascular permeability in the lung. In contrast, VEGF expression is up-regulated after I/R in the lung (6) , suggesting that VEGF, a mediator known to increase vascular permeability (5) , could play an important role in the development of pulmonary edema associated with I/R in the lung. Therefore, the first objective of these studies was to test the hypothesis that VEGF cell signaling mediates the increase in lung endothelial permeability early after onset of I/R in rats.

To answer this question, we developed an experimental model of I/R lung injury in rats by clamping the hilum of the left lung for 30 min and reperfusing it for 3 h after pretreating some of the rats with an adenovirus encoding a soluble VEGF receptor type II (Ad-Flk1-Fc) that has previously been shown to block the effect of VEGF in different organs (14) . The experimental model that we chose was based on preliminary studies showing that there was a direct correlation between the time of lung ischemia and the accumulation of pulmonary edema in the lungs. Furthermore, the ventilation of the ischemic lung was interrupted during the ischemic phase of the protocol, because we have previously shown that the maintenance of lung ventilation during the ischemic phase was protective against changes in lung endothelial permeability induced by I/R (19) . Our results indicated that pretreatment with the Ad-Flk1-Fc adenovirus completely prevented the development of pulmonary edema associated with ischemia and reperfusion in rats. These results are the first to demonstrate that VEGF cell signaling is directly implicated in the changes in vascular permeability associated with I/R in the lung. They are also in accordance with our previously published work that demonstrated that the overexpression of the VEGF gene in the lung caused pulmonary edema (5) . Furthermore, in other organs, such as the brain, VEGF antagonism has been shown to reduce edema formation and tissue damage after I/R injury in mice (20) . Taken together, our results and those of previous studies provide experimental evidence for a role of VEGF cell signaling in early vascular hyperpermeability associated with I/R injury in the lung.

To further understand the role of VEGF in I/R-mediated lung injury, we measured VEGF protein expression in the lungs of rats that underwent I/R lung injury. The results showed that there was no increase in the expression of lung tissue VEGF immunoreactive protein after 30 min of ischemia and 3 h of reperfusion compared to the values measured in normal lungs. VEGF gene and protein expression have previously been shown to be increased after I/R in the lung, but only after longer periods of ischemia. For example, Kazi et al. (6) reported that VEGF immunoreactive protein was significantly increased after 4 h of lung ischemia and without any reperfusion time. In another study, the same research group reported that the expression of VEGF protein was up-regulated after 3 h of ventilated ischemia and that this effect was oxygen-independent (21) . Finally, Serraf et al. (22) reported that lung edema after cardio-pulmonary bypass (90 min ischemia, 2 h reperfusion) is largely due to the VEGF accumulation on the lung, a phenonmenon not seen in nonischemic territories.

Because there was no increase in VEGF protein expression in the lung after ischemia in our model, what is the mechanism of I/R injury involving VEGF? There are several mechanisms that could explain our results. First, I/R may be associated with an increased activation of the VEGF receptor type II without a change in the total lung concentration of VEGF. In fact, we found that I/R was associated with a sustained phosphorylation of the VEGF receptor type II 3 hours after reperfusion that was not observed in sham rats or in rats that were pretreated with an adenovirus encoding a soluble VEGF receptor type II. Interestingly, I/R has already been shown to be associated with an increase in vascular permeability and in activation of VEGF receptor type II without a parallel increase in VEGF protein expression in the kidney (23) . The mechanism of activation of the VEGF receptor type II could be unrelated to the binding of VEGF to its receptor but involves ligand-independent stimulation of the receptor tyrosine kinase activity of VEGF receptor type II by oxidant radicals, such as H2O2 (24) . A second mechanism could be the release of biologically active VEGF from cells without any new induction of VEGF transcription. For example, matrix metalloproteases (MMP), such as MMP-2 and MMP-9, have been shown to cause the release of active VEGF from tumors (25) and from normal tissues (26) . Furthermore, the expression of MMP-9 is increased in I/R lung injury and prior MMP-9 inhibition decreased the severity of I/R injury after lung transplantation (27) . Thus, it is possible that biologically active VEGF could be released from cells after onset of ischemia and/or reperfusion due to increased activity of MMPs. Third, VEGF has been shown to be compartmentalized in the lung with alveolar levels of VEGF protein 500 times higher than plasma (28) . Under physiological conditions, the high levels of VEGF protein in the alveolar epithelial lining fluid are mostly restricted to the alveolar space. However, VEGF might move onto the nearby lung endothelium if the tightness of the epithelial barrier is decreased, as is the case after I/R lung injury, thus increasing vascular permeability and facilitating the development of pulmonary edema (28) . Furthermore, the compartmentalization of VEGF in the normal lung that is associated with high VEGF levels in the distal airspaces may explain why we did not observe any increase in VEGF protein content in the lung after I/R-mediated lung injury. In fact, recent clinical studies have reported that patients with acute lung injury have reduced levels of VEGF in the nondiluted alveolar edema fluid (29) . Taken together, these studies and our data indicate first that the lung tissue measurement of the VEGF antigen alone is an inadequate method to assess its pathogenetic role in acute lung injury. Secondly, VEGF cell signaling responsible for changes in vascular permeability early after onset of I/R can be activated without new transcription or translation of VEGF in the lung.

The present results do not exclude that other mechanisms may be implicated in the development of pulmonary edema after onset of I/R injury. In particular, neutrophil activation and infiltration into the lung parenchyma has been shown to be an important mechanism that mediates lung injury after onset of I/R (30) . However, in animal models, lung vascular injury associated with I/R has been defined as bimodal (31) . The early phase is neutrophil-independent and occurs within 15–30 min of reperfusion, whereas the late phase is dependent on neutrophil sequestration in the lung (30) . This sequence of events correlates with the fact that alpha-chemokines (MP-1, CINC-1/interleukin-8) that are critical for the diapedesis of neutrophils into the lung parenchyma are only expressed 2–3 h after onset of reperfusion (32) . Interestingly, VEGF cell signaling not only appears to be important in the early phase of I/R-mediated lung injury but may also play a role in the neutrophil diapedesis into the lung by causing the translocation of P-selectin to the cell membrane of endothelial cells (33) .

The second objective was to determine whether SPR activation would affect the I/R-mediated increase in lung vascular permeability induced by VEGF cell signaling. SPR activation was induced by either geldanamycin, a benzoquinone ansamycin or PDTC, an antioxidant. Both compounds have previously been shown to induce a stress protein response (34 , 35) . The results showed that SPR activation with either drug resulted in a significant decrease in the accumulation of pulmonary edema and in the phosphorylation of the VEGF receptor type II associated with I/R lung injury. The present study adds to a growing number of studies, including ours (16) , that show that SPR activation mediates cytoprotection in cell and animal models of I/R lung injury (10 , 11 , 36 37 38) . Our in vivo data in the present study suggest that prior SPR activation either prevented the release of biologically active VEGF from cells and/or affected the VEGF-dependent cell signaling pathway responsible for its effect on endothelial permeability.

To answer this question, we developed an in vitro cell model to determine the effect of VEGF on protein permeability across primary cultures of bovine macrovascular lung endothelial cell monolayers. We found that exposure to vascular endothelial growth factor caused a significant increase in the protein permeability across these monolayers that was associated with phosphorylation of VE-cadherin and actin stress fiber formation. It has been shown that the extracellular domain of VE-cadherin mediates homophilic binding and adhesion between endothelial cells (39) . Furthermore, vascular endothelial growth factor-mediated tyrosine phosphorylation of VE-cadherin is followed by increase in junction permeation (40) and VE-cadherin redistribution from intercellular junctions (41) . When the cell monolayers were stress preconditioned either with geldanamycin or with hyperthermia, the vascular endothelial growth factor-mediated increase in protein permeability across these cell monolayers, as well as VE-cadherin phosphorylation and actin stress fiber formation, were inhibited. Because a recently published study has reported that VE-cadherin is not expressed in microvascular lung endothelial cells and that E-cadherin is part of the adherens junction protein complex in these cells (17) , we also examined the effect of stress preconditioning of vascular endothelial growth factor-mediated permeability across these cell monolayers. We found that stress preconditioning also prevented vascular endothelial growth factor-mediated protein permeability, tyrosine phosphorylation of E-cadherin and actin stress fiber formation in bovine microvascular lung endothelial cell monolayers. Overall, these data support the hypothesis that the activation of the stress protein response inhibits vascular endothelial growth factor-mediated cell signaling responsible for the increase in lung vascular permeability.

The present results do not exclude that SPR activation could also negatively regulate hypoxia-induced vascular endothelial growth factor gene and protein expression in experimental models of prolonged ischemia. Increased vascular endothelial growth factor gene expression has been reported in animal models of prolonged hypoxic lung ischemia secondary to the activation of HIF-1{alpha} (6) . Furthermore, Hsp90 has been shown to be a critical regulator of oxygen-independent HIF-1{alpha} activity, because geldanamycin, a Hsp90 inhibitor and an inducer of the heat shock response, caused a reduction of HIF-1{alpha} levels and its downstream transcriptional activity by accelerating protein degradation independent of O2 tension (42) . Because SPR activation may affect the binding of Hsp90 to its client proteins (43) , further studies will be needed to determine whether SPR activation could inhibit hypoxia-induced and HIF-1 {alpha}-dependent vascular endothelial growth factor transcription in the lung.

In summary, these results provide the first in vivo evidence that vascular endothelial growth factor cell signaling mediates the early development of pulmonary edema caused by lung ischemia and reperfusion in rats, an effect prevented by prior SPR activation, in part, by inhibiting the vascular endothelial growth factor-mediated actin stress fiber formation and phosphorylation of VE- and E-cadherins, crucial components of the adherens junction protein complex controlling paracellular permeability in lung endothelial cells. Although these results indicate that vascular endothelial growth factor functions primarily as proinjurious molecule in the lung after ischemia and reperfusion, recent data have suggested that vascular endothelial growth factor release is compartmentalized in the lung (28) and that vascular endothelial growth factor might promote repair of the alveolar epithelium during recovery phase from acute lung injury (44) . Thus, additional studies will be needed to understand the role of vascular endothelial growth factor cell signaling in this disease process in order to develop new therapeutic strategies for acute lung injury.

ACKNOWLEDGMENTS

This work was primarily supported by National Institutes of Health Grants GM-62188 (J.F.P.) and HL-51854 (M.A.M.) and P50HL74005 (J.F.P. and M.A.M.).

FOOTNOTES

1 These authors equally contributed to this work.

Received for publication September 1, 2005. Accepted for publication February 21, 2006.

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